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2a study
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Safety and immunogenicity of a chimpanzee adenovirus-vectored Ebola vaccine in healthy adults: a randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a study Olga De Santis, Régine Audran, Emilie Pothin, Loane Warpelin-Decrausaz, Laure Vallotton, Grégoire Wuerzner, Camille Cochet, Daniel Estoppey, Viviane Steiner-Monard, Sophie Lonchampt, Anne-Christine Thierry, Carole Mayor, Robert T Bailer, Olivier Tshiani Mbaya, Yan Zhou, Aurélie Ploquin, Nancy J Sullivan, Barney S Graham, François Roman, Iris De Ryck, W Ripley Ballou, Marie Paule Kieny, Vasee Moorthy, François Spertini, Blaise Genton
Summary Background The ongoing Ebola outbreak led to accelerated efforts to test vaccine candidates. On the basis of a request by WHO, we aimed to assess the safety and immunogenicity of the monovalent, recombinant, chimpanzee adenovirus type-3 vector-based Ebola Zaire vaccine (ChAd3-EBO-Z). Methods We did this randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a trial at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Participants (aged 18–65 years) were randomly assigned (2:2:1), via two computer-generated randomisation lists for individuals potentially deployed in endemic areas and those not deployed, to receive a single intramuscular dose of high-dose vaccine (5 × 10¹⁰ viral particles), low-dose vaccine (2·5 × 10¹⁰ viral particles), or placebo. Deployed participants were allocated to only the vaccine groups. Group allocation was concealed from non-deployed participants, investigators, and outcome assessors. The safety evaluation was not masked for potentially deployed participants, who were therefore not included in the safety analysis for comparison between the vaccine doses and placebo, but were pooled with the non-deployed group to compare immunogenicity. The main objectives were safety and immunogenicity of ChAd3-EBO-Z. We did analysis by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT02289027. Findings Between Oct 24, 2014, and June 22, 2015, we randomly assigned 120 participants, of whom 18 (15%) were potentially deployed and 102 (85%) were non-deployed, to receive high-dose vaccine (n=49), low-dose vaccine (n=51), or placebo (n=20). Participants were followed up for 6 months. No vaccine-related serious adverse events were reported. We recorded local adverse events in 30 (75%) of 40 participants in the high-dose group, 33 (79%) of 42 participants in the lowdose group, and five (25%) of 20 participants in the placebo group. Fatigue or malaise was the most common systemic adverse event, reported in 25 (62%) participants in the high-dose group, 25 (60%) participants in the low-dose group, and five (25%) participants in the placebo group, followed by headache, reported in 23 (57%), 25 (60%), and three (15%) participants, respectively. Fever occurred 24 h after injection in 12 (30%) participants in the high-dose group and 11 (26%) participants in the low-dose group versus one (5%) participant in the placebo group. Geometric mean concentrations of IgG antibodies against Ebola glycoprotein peaked on day 28 at 51 μg/mL (95% CI 41·1–63·3) in the high-dose group, 44·9 μg/mL (25·8–56·3) in the low-dose group, and 5·2 μg/mL (3·5–7·6) in the placebo group, with respective response rates of 96% (95% CI 85·7–99·5), 96% (86·5–99·5), and 5% (0·1–24·9). Geometric mean concentrations decreased by day 180 to 25·5 μg/mL (95% CI 20·6–31·5) in the high-dose group, 22·1 μg/mL (19·3–28·6) in the low-dose group, and 3·2 μg/mL (2·4–4·9) in the placebo group. 28 (57%) participants given high-dose vaccine and 31 (61%) participants given low-dose vaccine developed glycoprotein-specific CD4 cell responses, and 33 (67%) and 35 (69%), respectively, developed CD8 responses. Interpretation ChAd3-EBO-Z was safe and well tolerated, although mild to moderate systemic adverse events were common. A single dose was immunogenic in almost all vaccine recipients. Antibody responses were still significantly present at 6 months. There was no significant difference between doses for safety and immunogenicity outcomes. This acceptable safety profile provides a reliable basis to proceed with phase 2 and phase 3 efficacy trials in Africa. Funding Swiss State Secretariat for Education, Research and Innovation (SERI), through the EU Horizon 2020 Research and Innovation Programme.
Introduction Ebola virus causes a severe, often fatal, illness, and several outbreaks have occurred since the virus was first reported in 1976. The largest recorded outbreak of Ebola virus
Lancet Infect Dis 2015 Published Online December 22, 2015 http://dx.doi.org/10.1016/ S1473-3099(15)00486-7 See Online/Comment http://dx.doi.org/10.1016/ S1473-3099(15)00534-4 Policlinique Médicale Universitaire, Lausanne, Switzerland (O De Santis MRes, C Cochet MD, D Estoppey MD, S Lonchampt MSc, Prof B Genton MD); Division of Immunology and Allergy (R Audran PhD, V Steiner-Monard MD, A-C Thierry BS, C Mayor BS, F Spertini MD), Clinical Trial Unit (L Warpelin-Decrausaz PhD, L Vallotton MD, G Wuerzner MD), and Infectious Diseases Service, Department of Medicine (Prof B Genton), Lausanne University Hospital, Switzerland; Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland (E Pothin PhD, Prof B Genton); Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA (R T Bailer PhD, O T Mbaya MD, Y Zhou PhD, A Ploquin PhD, N J Sullivan PhD, B S Graham MD); GSK Vaccines, Rixensart, Belgium (F Roman MD, I De Ryck MD, W R Ballou MD); and WHO, Geneva, Switzerland (M P Kieny PhD, V Moorthy MD) Correspondence to: Olga De Santis, Policlinique Médicale Universitaire, Lausanne 1011, Switzerland [email protected]
disease is ongoing, and more than 28 000 cases and more than 11 000 deaths in three countries in west Africa had been reported by September, 2015.1 WHO has declared the current outbreak an international public health emergency.
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Research in context Evidence before this study We searched PubMed for clinical trials up to Aug 17, 2015, with the search terms “Ebola” AND “vaccine”, with no language or date restrictions. Two DNA vaccines and one recombinant adenovirus serotype 5 using different versions of the Ebola virus or Marburg virus glycoprotein protein have been tested in the past 10 years. Chimpanzee adenovirus 3 (ChAd3)-vectored vaccines using monovalent and bivalent formulations of the Ebola virus glycoprotein were tested in late 2014 in US and UK phase 1 clinical trials with small sample sizes. A recombinant vesicular stomatitis virus (rVSV)-vectored Ebola vaccine was simultaneously tested in a multisite phase 1 trial. More recently, investigators of a phase 1 trial in China used a recombinant adenovirus serotype 5 vector-based Ebola vaccine expressing the glycoprotein of the 2014 epidemic strain. No safety issues arose from these trials, except for cases of arthritis and rash with rVSV, mainly reported at one site. All these trials done simultaneously to ours were published as preliminary reports including safety and immunogenicity data up to day 28 after injection. Added value of this study Our study provides the most comprehensive results of a phase 1/2 trial with ChAd3 vector-based vaccine expressing the Ebola virus glycoprotein. This trial was the only one that was placebocontrolled, allowing for the most accurate assessment of safety and reactogenicity. Among all Ebola vaccine trials, this is the only one that provides safety and immunogenicity results up to 6 months after injection, which provides some insight into the value of the vaccine over the course of an epidemic. No safety signal was recorded in our trial. All vaccine recipients had humoral immune responses that peaked at day 28, and then decreased by about half 6 months after injection. Interferon-γ mononuclear cell responses were still present at that time.
As a result of large multilateral public health interventions, the case incidence of Ebola virus disease had declined to less than ten cases per week by the end of July, 2015; however, no approved treatment or vaccine is yet available. Current efforts to develop a vaccine are focused on the viral glycoprotein encoded by the virus. The most advanced vaccine candidates tested so far are based on the glycoprotein from either the Zaire strain of Ebola virus (responsible for the current outbreak) or the Sudan strain. Candidates in which viral glycoprotein is expressed in either chimpanzee adenovirus, human adenovirus, or vesicular stomatitis virus have shown promise in nonhuman primate models of Ebola virus disease, and in initial clinical trials.2–7 Moreover, preliminary results of a phase 3 clinical trial with the replication-competent recombinant vesicular stomatitis virus (rVSV)-vectored vaccine showed encouraging efficacy results in Guinea.8,9 Rationale for development of this vaccine is based on previous human experience with other investigational filovirus vaccines and the development of non-human 2
Implications of all the available evidence When compared with results of the rVSV-vectored Ebola vaccine at 2×10⁷ or 5×10⁷ plaque-forming units, we can conclude that the safety profile of the ChAd3 Ebola vaccine (ChAd3-EBO-Z) at doses of 10¹⁰ viral particles is slightly better, but the humoral responses slightly lower, 1 month after injection. In view of the good safety profile of ChAd3-EBO-Z at doses of 10¹⁰ viral particles in the present trial, the 10¹¹ viral particle dose would seem appropriate to use when proceeding to phase 2 and 3 trials in Africa as planned, especially because the few available safety data with ChAd3-EBO-Z at 10¹¹ viral particles show an acceptable adverse events profile and, more importantly, similar antibody responses, as those obtained with the 2×10⁷ plaque-forming units dose of the rVSV-vectored vaccine. Assuming that the anti-glycoprotein antibody concentration is correlated with protection (even if the antibodies are not themselves protective), the promising efficacy results reported in the preliminary report of the rVSV-vectored vaccine in the phase 3 trial in Guinea could also be obtained with the ChAd3-EBO-Z vaccine at a dose of 10¹¹ viral particles. The persistence of antibodies at month 6, although at a reduced concentration, might suggest that some protection remains. However, this theory needs to be confirmed in a thorough phase 3 trial. Detailed correlation of immunological data and protection in non-human primate studies might also give some insight into efficacy, if a phase 3 trial becomes impossible to do because of an insufficient number of new cases of Ebola virus disease.
adenovirus vectors with low seroprevalence in human beings.3,10–14 Previous phase 1 clinical trials investigated the bivalent and monovalent vaccines encoding wild-type glycoprotein from Zaire and Sudan species of Ebola virus4 or Zaire species only.15 In response to a request from WHO in September, 2014, we undertook the present study to assess the safety and immunogenicity of the monovalent, recombinant, chimpanzee adenovirus type-3 vector-based Ebola Zaire (ChAd3-EBO-Z) vaccine construct. We aimed to build on and extend the clinical development plan for a chimpanzee adenovirus 3 (ChAd3)-vectored vaccine encoding Ebola glycoproteins that has been developed by the US National Institutes of Health in collaboration with GSK–Okairos, WHO, and the University of Oxford.
Methods Study design and participants We did this randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a study at the Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland.
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All participants in Lausanne were recruited by use of advertisements in the hospital and university halls. Because the study was largely published in the press, many people contacted the team directly to ask for their participation. Eligible volunteers had to be healthy, aged 18–65 years, and had to practise continuous contraception throughout the study. Exclusion criteria included previous participation in a trial of investigational Ebola or Marburg vaccine, or a chimpanzee adenovirus-vectored vaccine; receipt of any other live vaccine within 28 days or killed vaccine within 14 days before the trial; and presence of any immunodeficiency state or any acute or chronic disease not well controlled, which could increase the risk for serious adverse events or could impair interpretation of the data. The appendix provides full details of the inclusion and exclusion criteria. The study was reviewed and approved by the local ethics review board (Commission cantonale d’éthique de la recherche sur l’être humain), by the WHO Research Ethics Review Committee, and by the Swiss regulatory authorities (Swissmedic). All participants provided written informed consent.
Randomisation and masking We randomly assigned participants (2:2:1), via two computer-generated randomisation lists for individuals deployed in epidemic areas and those not deployed, to receive a single intramuscular dose of ChAd3-EBO-Z containing either 5×10¹⁰ (high dose) or 2·5 × 10¹⁰ (low dose) viral particles, or a single dose of placebo (diluent only). Deployed participants were allocated to only the vaccine groups. The randomisation lists were kept confidential in the central pharmacy at CHUV. The rationale to choose the two doses with only a two-fold difference was based on previous safety experience in clinical trials with chimpanzee adenovirus vectors.16 Because 100% of the non-human primates were protected 1 month after vaccination, there was a clear signal that this vaccine could be effective in human beings. Therefore, participants deployed from non-epidemic to epidemic areas could be the first beneficiaries of the vaccine, which is why WHO requested these individuals were not allocated to receive placebo. The safety evaluation was not masked for all potentially deployed participants, but group allocation was concealed from deployed participants, investigators, and outcome assessors.
Procedures ChAd3-EBO-Z consists of a recombinant replicationdeficient adenovirus chimpanzee serotype 3 vector expressing wild-type Ebola virus glycoprotein from the Zaire Mayinga strain. The appendix provides details about the composition of vaccine and diluent. We assessed local and systemic adverse events 1 h after injection and at follow-up visits on days 1, 7, 14, and 28. Additionally, participants recorded adverse events on a daily notification sheet for the first week. Solicited adverse events
were events that arose at any time from injection to day 7 (plus or minus 1 day), and included both local (pain, erythema and swelling at injection site, plus axillary lymphnode enlargement) and systemic (fever, fatigue or malaise, musculo-articular pain, headache, chills, and nausea) adverse events. Unsolicited adverse events were all other events not listed above, and all events that arose after the 7 day follow-up and up to day 28 (plus or minus 7 days). Grading of adverse events for severity and assignment of causal association of unsolicited adverse events was assessed by clinicians in charge of monitoring participants during the whole study, according to predefined criteria in the study protocol (appendix). Safety biological monitoring was done on blood samples taken on days 0, 1, 7, 14, 28, and 180 after injection, and included a full blood count, electrolytes, liver and renal function tests, C-reactive protein, and activated partial thromboplastin time. We did this assay because an asymptomatic prolongation of activated partial thromboplastin time has been reported in the 2 weeks after vaccination in previous adenovirus vaccine trials.3,15 This finding was due to the induction of a non-specific antiphospholipid antibody, and not to coagulopathy. This effect is actually an artifact of the activated partial thromboplastin time test, because this test measures the clotting cascade and the assay requires the presence of phospholipid as a reagent.3,15 Anti-EBO-Z immunoglobulins were measured with ELISA and antigen-specific T-cell responses were assessed with ex-vivo ELISPOT. The appendix details methods of antibody measurement and assessment of cell-mediated immunity. 3 months after injection, a follow-up assessment was done via phone call or email to record the occurrence of serious adverse events or relevant adverse events possibly related to injection. 6 months after injection, a last followup visit was done to obtain information about serious or relevant unsolicited adverse events, and about laboratory samples.
See Online for appendix
Outcomes The primary objective was the safety and reactogenicity of ChAd3-EBO-Z. The secondary objective was vaccine immunogenicity. The main exploratory outcome measure was intracellular cytokine staining assays.
Statistical analysis A sample size of 100 vaccinated participants was calculated to achieve a total of 250 vaccinated participants, taking into account all three concurrent phase 1 trials of the ChAd3-EBO-Z vaccine (Lausanne, Oxford, and Mali).15,17 This sample size was expected to produce reliable data for the incidence of frequent adverse events. Because safety evaluation for potentially deployed participants was not masked, and because too few participants had gone to epidemic regions after vaccination to expect potential immunological boost after hypothetical exposure, only results from the
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150 volunteers screened for eligibility
30 excluded 28 ineligible 1 withdrew consent 1 because accrual goal had been met
120 enrolled
18 potentially deployed
9 randomly assigned to low-dose vaccine
9 randomly assigned to high-dose vaccine
1 missed day 14 visit 1 missed day 90 visit
7 completed 6 month follow-up 7 completed safety and immunogenicity analysis
102 non-deployed
42 randomly assigned to low-dose vaccine
40 randomly assigned to high-dose vaccine
20 randomly assigned to placebo
1 missed day 28 visit
7 completed 6 month follow-up 7 completed safety and immunogenicity analysis
42 completed 6 month follow-up 42 completed safety and immunogenicity analysis
40 completed 6 month follow-up 40 completed safety and immunogenicity analysis
20 completed 6 month follow-up 20 completed safety and immunogenicity analysis
Figure 1: Trial profile
non-deployed group were used to compare safety between the high-dose, low-dose, and placebo groups; results from both the deployed and non-deployed groups were pooled to compare immunogenicity because the laboratory team assessing antibody or cellular responses was masked to group assignment. We present anti-Ebola virus glycoprotein IgG concentrations as geometric mean concentrations with 95% CIs. We compared groups with Fisher’s exact test for safety and the Mann–Whitney test for immunogenicity. The lower dose was compared with the higher dose, and the two doses were pooled and termed “vaccinated” for comparison with placebo. For each participant, we defined a positive antibody response as a significant increase in post-vaccination titre from baseline (t test assuming non-equal variance), with use of the anti-glycoprotein antibody titres assessed by ELISA done in the Vaccine Research Center (VRC; National Institutes of Health, Bethesda, MD, USA).11 We used Friedman or Kruskal–Wallis with Dunn’s post tests to compare magnitude of T-cell responses before and after vaccination with GraphPrism (version 6.07). A Data Safety Monitoring Board was established before the start of the trial, and included two independent clinicians and one epidemiologist. The Board reviewed the safety data from days 0–7 for the first 20 participants vaccinated to ensure that holding rules were not met. This trial is registered with ClinicalTrials.gov, number NCT02289027. 4
Role of the funding source The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and shared final responsibility for the decision to submit for publication with the principal investigator.
Results Between Oct 24, 2014, and June 22, 2015, we randomly assigned 120 participants, of whom 18 (15%) were potentially deployed and 102 (85%) were non-deployed, to receive low-dose vaccine (n=51), high-dose vaccine (n=49), or placebo (n=20; figure 1). All participants, except three deployed individuals who missed one visit each, completed the six visits after injection (figure 1). Baseline characteristics were similar between groups (table 1). No vaccine-related serious adverse events were noted. Most of the adverse events reported were mild and selflimiting, arising during the first 24 h after injection and lasting less than 48 h. We recorded seven grade 3 adverse events, all of which resolved within 3 days with no residual effect. Only the placebo-controlled results for the nondeployed participants are shown below. The most common solicited local adverse event was grade 1 pain, which differed significantly between the vaccine and placebo groups, but not between vaccine dose groups (table 2). At least one solicited systemic adverse event was reported in 71 (87%) participants in the vaccine groups (n=37 in the highdose group and n=34 in the low-dose group) and ten (50%)
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of those in the placebo group (p=0·0015; figure 2). The most common solicited systemic adverse events were fatigue or malaise and headache (table 2). Musculo-articular pains were also frequently reported (table 2). Most solicited adverse events were mild and resolved within 24 h after injection (figure 2). 28% of non-deployed vaccine recipients developed fever versus 5% of placebo recipients, with no significant difference between the dose groups (table 2); however, the highest vaccine-related temperatures were in participants given high-dose vaccine (appendix p 14). One relevant unsolicited adverse event possibly related to the vaccine was an episode of macroscopic haematuria associated with pain and burning with urination and mild left costovertebral angle tenderness at percussion that arose within 24 h after injection with low-dose vaccine. Results from the medical investigations (urinary sediment and culture, renal ultrasound, blood count, coagulation assays) were normal and the episode spontaneously resolved 48 h after injection. Because no biological cause was identified for this episode, and because the participant had not previously had a similar episode, the adverse event was considered to be possibly related to the vaccine. A second relevant unsolicited adverse event possibly related to the high-dose vaccine was a herpetiform dermatitis that arose at day 15 after injection and lasted for 2 weeks. Located in the L2 dermatome, this adverse event was clinically diagnosed as shingles, although not confirmed by PCR. None of the laboratory abnormal values were clinically significant (appendix pp 15–17). At day 1, we recorded 51 grade 1 lymphopenias (<1·5–0·8 × 10⁹ lymphocytes per L) in 23 (58%) participants in the high-dose vaccine group, 22 (52%) participants in the low-dose group, and six (30%) participants in the placebo group; and four grade 2 (<0·8–0·5 × 10⁹/L) transient lymphopenias in one (3%), three (7%), and no participants, respectively (appendix). One transient grade 1 thrombocytopenia (<150–75 × 10⁹ platelets per L) arose in one (2%) participant in the low-dose group (appendix). During month 1 of follow-up, we recorded six transient grade 1 anaemias (haemoglobin <117–100 g/L) in one (3%) participant in the high-dose group and five (12%) participants in the low-dose group. We also recorded six transient neutropenias of mild intensity (<1·8–1·5 × 10⁹ neutrophils per L; three [8%] participants in the high-dose group, two [5%] in the low-dose group, and one [5%] in the placebo group) and moderate intensity (<1·5–1 × 10⁹/L; three [7%] in the low-dose group), and two neutropenias of severe intensity (<1 × 10⁹/L; one [2%] in the high-dose group and one [5%] in the placebo group; appendix). We recorded one case of asymptomatic grade 1 prolonged activated partial thromboplastin time at day 14 in the high-dose group (appendix). Investigation showed no coagulopathy. The antiphospholipid screening was positive for a lupus anticoagulant and doubtful for an anticardiolipin IgM. The prolonged activated partial thromboplastin time and presence of anticardiolipin IgM had resolved by month 3
Non-deployed participants (N=102)
Potentially deployed participants (N=18)
Placebo Low-dose group (n=20) group (n=42)
Low-dose group (n=9)
High-dose group (n=40)
High-dose group (n=9)
Sex Male
11 (55%)
22 (52%)
19 (48%)
4 (44%)
3 (33%)
9 (45%)
20 (48%)
21 (52%)
5 (56%)
6 (67%)
White
16 (80%)
40 (95%)
35 (88%)
9 (100%)
6 (67%)
Black
1 (5%)
1 (2%)
1 (2%)
0
2 (22%)
Hispanic
0
0
1 (2%)
0
0
Other
3 (15%)
1 (2%)
3 (8%)
0
1 (11%)
Mean (SD)
37·2 (13·4)
30·7 (11·1)
33·2 (13·1)
42 (12·4)
46 (10·8)
Median (min–max range)
37 (19–61)
27·5 (19–63)
27 (19–63)
39 (28–62)
43 (32–64)
23·7 (3·3)
24·2 (2·9)
23·4 (4·1)
26·6 (3·9)
23·8 (19·4–31·2)
23·4 (18·3–30·3)
27·4 (20·3–33·2)
Female Race or ethnic origin
Age (years)
Body-mass index (kg/m²) Mean (SD) Median (min–max range)
23·5 (3·6) 22·3 (18·9–33·9)
23·3 (17·6–32)
Data are n (%), unless otherwise specified.
Table 1: Baseline characteristics
and the presence of lupus anticoagulant by month 9. No associated clinical sign of hypercoagulability was present. Of the grade 3 adverse events, one was an unsolicited local event, four were solicited systemic events, and two were laboratory adverse events. The one local grade 3 adverse event was an erythema at injection site of 11 cm in diameter with presence of redness and warmth but no pain, which arose at day 9 in a participant in the high-dose group and lasted for less than 24 h. Of the four solicited systemic grade 3 adverse events, two were sudden and strong headaches that arose during the 24 h after injection and resolved in less than 2 h with paracetamol. The other two events were fevers with temperatures exceeding 39°C, one arose during the night after injection of high-dose vaccine and lasted less than 24 h, and the other arose at day 4 after injection of low-dose vaccine, but was associated with a streptococcus angina and therefore not related to the vaccine. We recorded two grade 3 neutropenias, one at day 1 in the high-dose group and the other at day 14 in the placebo group. None were associated with symptom or clinical sign and both were resolved at the following visit 3 days later. At month 3 of follow-up, all volunteers except one were reached by telephone or email to assess safety. Three mild to moderate adverse events were possibly related to the injection. One was a second episode of an axillary lymphnode enlargement at day 63 after injection of high-dose vaccine, and lasted 2 days (first episode previously described at day 1 and lasted 2 days). The two other events were mild fatigue at day 34 in the high-dose group, and lasted 1 week, and moderate fatigue with several episodes of frontal headache at day 34 in the low-dose group, which lasted for roughly 3 weeks. At the last visit at 6 months,
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Non-deployed (N=102)
p value
Potentially deployed (N=18)
Placebo group (n=20)
Low-dose group (n=42)
High-dose group (n=40)
Grade 1
5 (25%)
32 (76%)
25 (62%)
6 (67%)
7 (78%)
Grade 2
0
1 (2%)
5 (12%)
1 (11%)
0
Grade 1
0
0
2 (5%)
0
0
Grade 2
0
0
1 (2%)
0
0
0
0
0
0
Placebo vs vaccinated
Low dose vs high dose
Low-dose group (n=9)
High-dose group (n=9)
Local Pain
<0·0001
Swelling
1
Erythema Grade 1
0
1 (2%)
0
0
0·11
1
0·61
0·58
0·05
2 (5%)
Axillary lymphatic-node enlargement Grade 1
0·80
4 (10%)
Systemic Fatigue or malaise
0·01
Grade 1
5 (25%)
Grade 2
0
20 (50%)
4 (44%)
4 (44%)
2 (5%)
5 (12%)
1 (11%)
1 (11%)
3 (33%)
2 (22%)
0
1 (11%)
Musculo-articular pain
0·08
Grade 1
5 (25%)
Grade 2
0
0·82
23 (55%)
17 (40%)
16 (40%)
1 (2%)
6 (15%)
Chills
0·02
0·38
0·60
Grade 1
0
6 (14%)
8 (20%)
3 (33%)
3 (33%)
Grade 2
0
2 (5%)
2 (5%)
0
0
Grade 1
4 (20%)
5 (12%)
2 (5%)
2 (22%)
1 (11%)
Grade 2
0
0
1 (2%)
0
0
Grade 1
0
6 (14%)
7 (18%)
2 (22%)
1 (11%)
Grade 2
1 (5%)
5 (12%)
4 (10%)
0
1 (11%)
Grade 3
0
0
1 (2%)
0
0 2 (22%)
Nausea
0·22
Fever
0·04
Headache
0·0043
0·43
0·81
1
Grade 1
2 (10%)
15 (36%)
14 (35%)
1 (11%)
Grade 2
1 (5%)
9 (21%)
8 (20%)
1 (11%)
2 (22%)
Grade 3
0
1 (2%)
1 (2%)
0
0
0
1 (2%)
1 (2%)
0
1 (11%)
1 (11%)
0
0
1 (11%)
Unsolicited related Abdominal pain Grade 1 Conjunctivitis Grade 1
0
0
0
1 (2%)
1
1
0·49
1
1
1
1
1 (2%)
Rhinitis Grade 1
1
0
Sweating Grade 1
0
0
0
0
0
Grade 2
0
1 (2%)
0
0
0
Other*
0·13
0·83
Grade 1
2 (10%)
14 (33%)
14 (35%)
4 (44%)
2 (22%)
Grade 2
2 (10%)
6 (14%)
2 (5%)
1 (11%)
0
Grade 3
1 (5%)†
0
2 (5%)†‡
0
0
Data are n (%), unless otherwise specified. Events are presented in frequency of participants presenting at least one adverse event of the category; if many, the maximum intensity is retained. *Includes (injection-site) erythema, headache, fatigue, and fever occurring after day 8, and grade 3 neutropenia. †One grade 3 neutropenia at day 14 in one participant from the placebo group, and one grade 3 neutropenia at day 1 in one participant in the high-dose vaccine group. ‡Erythema at injection site of 11 cm in diameter at day 9 after injection (p value from Fisher’s exact test).
Table 2: Solicited local and systemic adverse events occurring up to day 7 (plus or minus 1 day) and unsolicited related adverse events up to day 28 (plus or minus 7 days)
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A
B Local
100
Grade 1 Grade 2 Grade 3
Solicited systemic
Any related
Any
Unsolicited related
Unsolicited
Proportion (%)
75
50
25
Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se
0
Figure 2: Proportion of individuals affected and severity of adverse events in non-deployed participants (A) Adverse events reported up to day 7 (plus 1 day, up to 8 days). (B) Adverse events reported up to day 28 (plus 7 days, up to 35 days). Only the maximum grade per adverse event was reported for each individual.
A
p<0·0001
Anti-glycoprotein IgG (μg/mL)
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only one adverse event was reported as possibly related to the vaccine. The participant reported mild arthralgia in the distal interphalangeal joints of the fifth fingers on both hands, lasting 1 month. Neither swelling nor warmth was reported. Mobility was normal, but a light red macula of 2–3 mm was observed on the dorsal face of each joint. This participant had received the placebo and was sent for a specialised consultation for further investigations. From day 28 to day 180, three serious adverse events were reported, none of which were related to the injection, and all of which were due to trauma—namely, an elective hospital admission for a dislocated shoulder surgery in a participant given placebo, an emergency admission and surgery for a broken radius in a participant given placebo, and a persistent disability due to a broken anterior cruciate knee ligament in participant given low-dose vaccine. Finally, at month 3 of follow-up, a participant in the lowdose group reported the pregnancy of his wife. At this time the pregnancy was in the first trimester. The date of conception was difficult to determine because this was an unexpected pregnancy arising despite oral contraception, but it was estimated at 2 weeks after vaccination. The pregnancy was terminated 3 weeks later because of trisomy 21 diagnosed by the gynaecologist. There is no biological plausibility that this diagnosis could be related to the vaccination of the partner. Figure 3 summarises results for anti-Ebola virus glycoprotein IgG responses, including all data from deployed and non-deployed vaccine recipients. Antibody response was detected from day 14 onwards and peaked at day 28 up to a geometric mean concentration of 51 μg/mL (95% CI 41·1–63·3) in the high-dose group and of 44·9 μg/mL (25·8–56·3) in the low-dose group (figure 3). Antibody concentration did not differ between the two vaccine groups. The proportion of responders was 96% (95% CI 85·7–99·5) in the high-dose group, 96% (86·5–99·5) in the low-dose group, and 5% (0·1–24·9) in the placebo group (appendix). Antibody response decreased by roughly half from day 28 to day 180, with geometric mean concentrations of 25·5 μg/mL (95% CI 20·6–31·5) in the high-dose group, 22·1 μg/mL (19·3–28·6) in the lowdose group, and 3·2 μg/mL (2·4–4·9) in the placebo group 6 months after injection (figure 3). At day 28, geometric means of the VRC titres were 434·7 (min–max range 77·7–5576·3) in the high-dose group, 467·3 (41·5–4265·3) in the low-dose group, and 33·0 (6·9–198·0) in the placebo group (figure 3). Mononuclear cell responses to vaccination were evaluated by interferon (IFN)-γ ELISPOT on days 0, 7, 14, 28, and 180. Responses had already increased by day 7 in the high-dose group and, similar to antibody response, peaked at day 14, with a significant median response of 182·7 (range 91·3–345·3) spot-forming units per million peripheral blood mononuclear cells (PBMCs) in the high-dose group and 180 (66·95–382·0) spot-forming units per million PBMCs in the low-dose group (appendix). Although still significantly higher than at day 0 (within group analysis
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Figure 3: Antibody responses to the Ebola Zaire glycoprotein (A) Kinetics of responses as assessed by commercial ELISA. Boxes show median values and IQRs; whiskers are 5th and 95th percentiles, and geometric mean concentrations (μg/mL) are compared between groups. P values from the Mann–Whitney test. (B) Individual Vaccine Research Centre (VRC)-assessed EC90 titres at day 28. Squares show geometric mean concentrations and vertical lines show 95% CIs. (C) Spearman’s correlation between the two ELISA assays (Lausanne and VRC). EC90=90% effective concentration (the dilution of serum at which there is a 90% decrease in antigen binding).
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Friedman test p=0·001), responses at day 180 declined in most participants, and did not differ significantly from those in the placebo group (Dunn’s post tests p>0·05; appendix). T-cell-specific response was measured by flow cytometry at days 0, 14, and 28 and was expressed as frequencies of CD4 and CD8 cells producing IFN-γ, interleukin 2, or tumour necrosis factor (TNF)α after stimulation with Ebola Zaire glycoprotein peptides (appendix p 17). Significant glycoprotein-specific CD4 and CD8 cell responses were obtained from day 14 in both vaccine groups (appendix p 18), with no significant difference between doses. On the basis of positive responses for at least one of the three cytokines, 28 (57%) participants from the high-dose group and 31 (61%) participants from the low-dose group developed glycoprotein-specific CD4 cell responses, and 33 (67%) and 35 (69%) participants, respectively, developed glycoproteinA
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Figure 4: Memory T-cell responses to the Ebola virus Zaire glycoprotein Kinetics of individual CD4 (A) and CD8 (B) responses expressed as frequencies of subsets expressing at least one cytokine, IFN-γ, IL-2, or TNFα. Boxes show median values and IQRs; whiskers are 5% percentiles. We used the Kruskal–Wallis test to assess statistical significance of vaccine versus placebo. Proportions of glycoprotein-specific memory CD4 (C) and CD8 (D) cells that produce any combination of the three cytokines, at day 14 and day 28, in the vaccine groups. TNF=tumour necrosis factor. IL=interleukin. IFN=interferon.
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specific CD8 cell responses. Vaccine-specific memory T-cell responses showed the same kinetics and were equally distributed between CD4 and CD8 T cells (figure 4). Both memory CD4 and CD8 T cells presented polyfunctional and monofunctional phenotypes (figure 4). The CD8 cell response consisted mainly of IFN-γ-producing cells among which the IFN-γ TNFα co-producing subsets represented 40% of the response (figure 4). ChAd3-neutralising antibodies were measured in all participants at days 0 and 28 (appendix p 22). Notably, the concentration of neutralising antibodies at day 0 negatively correlated with anti-glycoprotein antibody responses, and with CD8 IFN-γ responses at day 28 (appendix).
Discussion This is the largest phase 1/2a clinical trial reported so far with an experimental Ebola vaccine, and the first to report data with a 6 month follow-up. No vaccine-related serious adverse events were reported during the 6 month follow-up. The ChAd3-EBO-Z vaccine led to more local and systemic adverse events than did placebo alone. Most adverse events were mild and all resolved with no sequelae, mostly within the first 24 h after injections. These results are in line with those shown in other trials of adenovirus-vectored vaccine.3,4,15,18,19 More precisely, the reactogenicity was similar to that shown in previous phase 1 trials16,18.19 using chimpanzee adenovirus vectors and expression proteins from other pathogens, showing that adverse events were more likely to be induced by the vector rather than by the Ebola virus glycoprotein. Inclusion of a placebo group allowed us to show that local pain and fatigue or malaise, musculo-articular pain, chills, fever, and headache—all components of reactogenicity— were due to the vaccine. Moreover, rates of unsolicited adverse events did not differ significantly between the vaccine and placebo groups, inferring that larger trials are needed to investigate a potential association with the vaccine. Incidence of local reactogenicity was similar to that reported after routine vaccinations (eg, influenza; hepatitis B; DTPa; or measles, mumps, and rubella vaccinations20–24), with the exception of pain at injection site, which was more common, but almost always mild and with little erythema or swelling. By contrast, the incidence of systemic adverse events was substantially higher, especially for headache, musculo-articular pains, and fever. Although the safety data were roughly similar to those reported in Rampling and colleagues’ phase 1 trial,15 with headache, fatigue, and malaise being the most common adverse events, the frequency of adverse events was higher in our study. Only 5% (n=2) of participants had objective fever in Rampling and colleagues’ study, compared with 28% of participants in our study. This difference might be explained by measurement technique, because feverishness in general was present in 30% of participants in Rampling and colleagues’ study. Although more common, adverse events in our study were of mild intensity, short-lived, and selflimited, which makes them acceptable in a risk–benefit
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balance in relation to such a severe disease as Ebola. Moreover, 81% of the fevers induced by the vaccine resolved within 24 h after injection. This rapid resolution makes them manageable, even during an outbreak, by preventing confusion with early onset of a new Ebola case. Frequencies and intensities of adverse events were similar between the two vaccine dose groups, although fevers of higher temperatures and four of the seven grade 3 adverse events were reported in the high-dose group. The absence of a significant dose effect might be because the two doses differed by only a factor of two. The slight increase of fever in the high-dose group might become clinically relevant when the dose of 1 × 10¹¹ viral particles is used—the dose currently deployed in Africa. Indeed, in the clinical trial of the bivalent ChAd3-EBO (Zaire and Sudan) vaccine,4 the dose of 2 × 10¹¹ viral particles was more reactogenic than the dose of 2 × 10¹⁰ viral particles, with two (20%) of the ten vaccine recipients reporting fever compared with none of the recipients given the lower dose. These data might suggest that the 10¹¹ dose will be more reactogenic. The published short-term safety results of the rVSV vaccine trial,5 the other major promising Ebola vaccine, showed a similar early reactogenicity profile. Although no vaccine-related serious adverse events have been reported with either vaccine, cases of arthritis or arthralgia with maculopapular rash or vesicular dermatitis did arise in some participants 2 weeks after vaccination with rVSV. These findings were reported at differing rates in different trials, with the highest reported rate being 22% (11 of 52 recipients) in Geneva.5 ChAd3 vaccine recipients only complained of transient musculo-articular pain within 3 days after vaccination as part of general influenzalike symptoms, but with no clinical evidence of arthritis. Notably, a higher frequency of adverse events was reported in both phase 1 trials of Ebola vaccine (the rVSV Ebola vaccine trial25 and our study), both done in Switzerland, than in other trials with the same vaccine. This difference is unlikely to be due to specific genetic traits, because participants in our study were of many different origins, and is probably related to the reporting mode. A single vaccination with ChAd3-EBO-Z induced antibody responses in 96% of participants, independently of the dose. The anti-Ebola Zaire virus glycoprotein titres obtained at day 28 confirmed the responses obtained with doses of 5×10¹⁰ and 2·5×10¹⁰ viral particles of ChAd3-EBO-Z reported in a previous study.15 There was no dose effect in our trial, probably because the two doses were close. The 6 month follow-up results showed for the first time that antibody titres in these groups were maintained at a significantly increased concentration. Notably, the presence of ChAd3 neutralising antibodies at day 0 correlated negatively with both the concentration of anti-glycoprotein antibodies, and the CD8 IFN-γ T-cell responses, at day 28. This finding was in line with similar observations in a previous preliminary report,4 although the result reached significance in the present study with
a larger sample size. With regards to the durability of the T-cell response, the IFN-γ mononuclear cell responses by ELISPOT decreased, but were still present, at month 6, despite not differing significantly from the response with placebo at this later timepoint. Remarkably, the presence of an Ebola-specific CD8 T-cell response with an IFN-γ and TNFα co-producer reinforces the potential for protection of the current vaccine formulation, because these markers are associated with vaccine-mediated protection in non-human primates.2 The proportion of IFN-γ and TNFα co-producers in this study was similar to that in a previous study.15 IFN-γ polyfunctional CD8 T cells were also found in similar proportions in Ledgerwood and colleagues’ phase 1 trial4 when doses of 2×10¹⁰ viral particles were used, but seemed to further expend with the highest dose of 2×10¹¹ viral particles. The promising efficacy provided by the VSV-vectored vaccine in Guinea8 gives hope that other vaccines based on the Ebola virus glycoprotein could be protective. Although correlates of immunity in human vaccination against Ebola virus are unknown, it is interesting to see that anti-glycoprotein titres observed with bivalent ChAd3-EBO at a dose of 2×10¹¹ viral particles were equivalent to those obtained with the rVSV-vectored vaccine assessed in the Guinea phase 3 trial. Available anti-EBO-Z glycoprotein ELISA data suggest that the humoral immune responses induced by the dose of 1 × 10¹¹ viral particles (for the monovalent form) are higher than those induced by the lower doses, which is why the 1 × 10¹¹ dose was selected for phase 2 and phase 3 studies (ClinicalTrials.gov, number NCT02485301). A key limitation of our study is that we could not test a higher vaccine dose because of the risk of inducing too many adverse events and therefore jeopardising the course of the study. Indeed, in the present emergency situation, results of early phases of vaccine development were crucial to proceed with further development in an epidemic area. Our study complements the clinical development plan for a ChAd3-vectored Ebola vaccine in several ways. First, our study is the only one of the existing ChAd3-vectored Ebola vaccine studies to include a placebo group, which enables precise assessment of the vaccine reactogenicity. Second, with the relatively large sample size and balanced inclusion of men and women, our findings substantially increase the data already obtained in previous studies and allow for a better assessment of safety and a valid comparison between doses, increasing the likelihood of identification of an optimum dose that balances both immunogenicity and reactogenicity, and enabling detection of a possible dose–response effect. Finally, this is the first report to provide safety and immunogenicity data at 6 months. Altogether, our results, showing an acceptable safety profile linked to Ebola virus-specific antibody response and polyfunctional CD8-specific T-cell response, provide a reliable basis for proceeding with efficacy
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trials in Africa and have greatly assisted in decisionmaking for the initiation of further phase 2b and 3 trials. This acceptable safety profile linked to Ebola virus-specific antibody response and polyfunctional CD8-specific T-cell response provides a reliable basis to proceed with efficacy trials in Africa.
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Contributors ODS, LW-D, LV, GW, FS, MPK, VM, IDR, FR, and BG contributed to the conception and design of the study. ODS, CC, DE, VS-M, SL, RA, A-CT, CM, and BG collected the data. ODS, EP, LW-D, LV, GW, SL, IDR, WRB, FS, and BG analysed and interpreted safety data. RA, A-CT, CM, EP, ODS, BSG, NJS, OTM, YZ, AP, RTB, BG, and FS analysed and interpreted immunogenicity data. ODS, RA, EP, FS, and BG wrote the manuscript. All authors contributed to revision of the manuscript.
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Declaration of interests FR, IDR, WRB, are employees of GSK. All other authors declare no competing interests. Acknowledgments We thank all persons interested, but eventually not screened, and all volunteers for their generous and reliable participation in this trial; Loredana Otgon, Emmanuelle Paccou, Christiane Pellet, Sylvie Poget, and Françoise Secretan, the clinical research nurses of the Clinic Trial Unit of the Centre Hospitalier Universitaire Vaudois (CHUV) for their exceptional expertise and commitment; Stéphanie Dartevelle and Isabelle Angelstorf from the CHUV pharmacy for the randomisation and daily preparation of the vaccine doses; Claudia Rochat and all laboratory members for the accurate handling and analyses of a large amount of blood samples; Kristina Moemi Geiger and Tom Stornetta for their generous assistance in blood sample preparations; Fady Fares and Ali Maghraoui from the Clinical Trial Unit of the CHUV for data management; Pascal Savary and Alexia Kaeser from the Legal Unit of the CHUV for contracts revision; Richard Pink for his contribution to manuscript writing; Patrick Francioli (Chairman of the Commission cantonale d’éthique de la recherche sur l’être humain) and his team, and the WHO Research Ethics Review Committee for their rapid review and approval; Eric Huber and Marek Sochor from the Swiss Tropical and Public Health Institute for monitoring; Peter Smith, Pierre-Alexandre Bart, and Pierre Landry—members of the Data and Safety Monitoring Board; the Vaccine Research Centre team for their support in development of study documentation, for making the vaccine doses available, and for doing the intracellular cytokine staining analysis; Adrian Hill (Oxford University, London, UK) with his team for providing documentation and assistance in initiating a simian adenoviral vectored vaccine trial. This work was supported by the Swiss State Secretariat for Education, Research, and Innovation (SERI; contract number 14.0001) through the EU Horizon 2020 Research and Innovation Programme (EbolaVac project) under grant agreement number 666085. The opinions expressed and arguments employed herein do not necessarily reflect the official views of the Swiss Government or the World Health Organization. References 1 WHO. Ebola situation report–30 September 2015. http://apps.who. int/ebola/current-situation/ebola-situation-report-30september-2015 (accessed Oct 31, 2015). 2 Stanley DA, Honko AN, Asiedu C, et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge. Nat Med 2014; 20: 1126–29. 3 Ledgerwood JE, Costner P, Desai N, et al. A replication defective recombinant Ad5 vaccine expressing Ebola virus GP is safe and immunogenic in healthy adults. Vaccine 2010; 29: 304–13. 4 Ledgerwood JE, DeZure AD, Stanley DA, et al. Chimpanzee adenovirus vector Ebola vaccine—preliminary report. N Engl J Med 2014; 373: 776. 5 Agnandji ST, Huttner A, Zinser ME, et al. Phase 1 trials of rVSV Ebola vaccine in Africa and Europe—preliminary report. N Engl J Med 2015; published online April 1. DOI:10.1056/ NEJMoa1502924.
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Geisbert TW, Bailey M, Hensley L, et al. Recombinant adenovirus serotype 26 (Ad26) and Ad35 vaccine vectors bypass immunity to ad5 and protect nonhuman primates against Ebolavirus challenge. J Virol 2011; 85: 4222–33. Zhu F-C, Hou L-H, Li J-X, et al. Safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in China: preliminary report of a randomised, doubleblind, placebo-controlled, phase 1 trial. Lancet 2015; 385: 2272–79. Henao-Restrepo AM, Longini IM, Egger M, et al. Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial. Lancet 2015; 386: 857–66. Krause PR. Interim results from a phase 3 Ebola vaccine study in Guinea. Lancet 2015; 386: 831–33. Colloca S, Folgori A, Ammendola V, et al. Generation and screening of a large collection of novel simian adenovirus allows the identification of vaccine vectors inducing potent cellular immunity in humans. Sci Transl Med 2012; 4: 115ra2. Martin JE, Sullivan NJ, Enama ME, et al. A DNA vaccine for Ebola virus is safe and immunogenic in a phase I clinical trial. Clin Vaccine Immunol 2006; 13: 1267–77. Sarwar UN, Costner P, Enama ME, et al. Safety and immunogenicity of DNA vaccines encoding Ebolavirus and Marburgvirus wild-type glycoproteins in a phase I clinical trial. J Infect Dis 2015; 211: 549–57. Sullivan NJ, Sanchez A, Rollin PE, Yang Z, Nabel GJ. Development of a preventive vaccine for Ebola virus infection in primates. Nature 2000; 408: 605–09. Sullivan NJ, Geisbert TW, Geisbert JB, et al. Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates. Nature 2003; 424: 681–84. Rampling T, Ewer K, Bowyer G, et al. A monovalent chimpanzee adenovirus Ebola vaccine—preliminary report. N Engl J Med 2015; published online Jan 28. DOI:10.1056/NEJMoa1411627. O’Hara GA, Duncan CJA, Ewer KJ, et al. Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector. J Infect Dis 2012; 205: 772–81. Tapia MD, Sow SO, Lyke KE, et al. Use of ChAd3-EBO-Z Ebola virus vaccine in Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial. Lancet Infect Dis 2015; published online Nov 3. http://dx.doi. org/10.1016/S1473-3099(15)00362-X. Barnes E, Folgori A, Capone S, et al. Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man. Sci Transl Med 2012; 4: 115ra1. Sheehy SH, Duncan CJA, Elias SC, et al. Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors. PLoS One 2012; 7: e31208. Compendium Suiss des Médicaments. Engerix®-B 20/Engerix®-B 10. 2012. http://compendium.ch/mpro/mnr/2546/html/fr (accessed April 15, 2015). Compendium Suisse des Médicaments. Boostrix. 2014. http://compendium.ch/mpro/mnr/9423/html/fr (accessed April 15, 2015). Compendium Suisse des Médicaments. Priorix®. 2013. http://compendium.ch/mpro/mnr/8773/html/fr (accessed April 15, 2015). Compendium Suisse des Médicaments. Tetanol® pur. 2012. http://compendium.ch/mpro/mnr/15386/html/fr (accessed April 15, 2015). Centers for Disease Control and Prevention. Vaccines and Immunizations. Possible side-effects from vaccines. 2015. http://www.cdc.gov/vaccines/vac-gen/side-effects.htm (accessed April 13, 2015). Huttner A, Dayer J-A, Yerly S, et al. The effect of dose on the safety and immunogenicity of the VSV Ebola candidate vaccine: a randomised double-blind, placebo-controlled phase 1/2 trial. Lancet Infect Dis 2015; 15: 1156–66.
www.thelancet.com/infection Published online December 22, 2015 http://dx.doi.org/10.1016/S1473-3099(15)00486-7
Safety and immunogenicity of a chimpanzee adenovirus-vectored Ebola vaccine in healthy adults: a randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a study Olga De Santis, Régine Audran, Emilie Pothin, Loane Warpelin-Decrausaz, Laure Vallotton, Grégoire Wuerzner, Camille Cochet, Daniel Estoppey, Viviane Steiner-Monard, Sophie Lonchampt, Anne-Christine Thierry, Carole Mayor, Robert T Bailer, Olivier Tshiani Mbaya, Yan Zhou, Aurélie Ploquin, Nancy J Sullivan, Barney S Graham, François Roman, Iris De Ryck, W Ripley Ballou, Marie Paule Kieny, Vasee Moorthy, François Spertini, Blaise Genton
Summary Background The ongoing Ebola outbreak led to accelerated efforts to test vaccine candidates. On the basis of a request by WHO, we aimed to assess the safety and immunogenicity of the monovalent, recombinant, chimpanzee adenovirus type-3 vector-based Ebola Zaire vaccine (ChAd3-EBO-Z). Methods We did this randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a trial at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Participants (aged 18–65 years) were randomly assigned (2:2:1), via two computer-generated randomisation lists for individuals potentially deployed in endemic areas and those not deployed, to receive a single intramuscular dose of high-dose vaccine (5 × 10¹⁰ viral particles), low-dose vaccine (2·5 × 10¹⁰ viral particles), or placebo. Deployed participants were allocated to only the vaccine groups. Group allocation was concealed from non-deployed participants, investigators, and outcome assessors. The safety evaluation was not masked for potentially deployed participants, who were therefore not included in the safety analysis for comparison between the vaccine doses and placebo, but were pooled with the non-deployed group to compare immunogenicity. The main objectives were safety and immunogenicity of ChAd3-EBO-Z. We did analysis by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT02289027. Findings Between Oct 24, 2014, and June 22, 2015, we randomly assigned 120 participants, of whom 18 (15%) were potentially deployed and 102 (85%) were non-deployed, to receive high-dose vaccine (n=49), low-dose vaccine (n=51), or placebo (n=20). Participants were followed up for 6 months. No vaccine-related serious adverse events were reported. We recorded local adverse events in 30 (75%) of 40 participants in the high-dose group, 33 (79%) of 42 participants in the lowdose group, and five (25%) of 20 participants in the placebo group. Fatigue or malaise was the most common systemic adverse event, reported in 25 (62%) participants in the high-dose group, 25 (60%) participants in the low-dose group, and five (25%) participants in the placebo group, followed by headache, reported in 23 (57%), 25 (60%), and three (15%) participants, respectively. Fever occurred 24 h after injection in 12 (30%) participants in the high-dose group and 11 (26%) participants in the low-dose group versus one (5%) participant in the placebo group. Geometric mean concentrations of IgG antibodies against Ebola glycoprotein peaked on day 28 at 51 μg/mL (95% CI 41·1–63·3) in the high-dose group, 44·9 μg/mL (25·8–56·3) in the low-dose group, and 5·2 μg/mL (3·5–7·6) in the placebo group, with respective response rates of 96% (95% CI 85·7–99·5), 96% (86·5–99·5), and 5% (0·1–24·9). Geometric mean concentrations decreased by day 180 to 25·5 μg/mL (95% CI 20·6–31·5) in the high-dose group, 22·1 μg/mL (19·3–28·6) in the low-dose group, and 3·2 μg/mL (2·4–4·9) in the placebo group. 28 (57%) participants given high-dose vaccine and 31 (61%) participants given low-dose vaccine developed glycoprotein-specific CD4 cell responses, and 33 (67%) and 35 (69%), respectively, developed CD8 responses. Interpretation ChAd3-EBO-Z was safe and well tolerated, although mild to moderate systemic adverse events were common. A single dose was immunogenic in almost all vaccine recipients. Antibody responses were still significantly present at 6 months. There was no significant difference between doses for safety and immunogenicity outcomes. This acceptable safety profile provides a reliable basis to proceed with phase 2 and phase 3 efficacy trials in Africa. Funding Swiss State Secretariat for Education, Research and Innovation (SERI), through the EU Horizon 2020 Research and Innovation Programme.
Introduction Ebola virus causes a severe, often fatal, illness, and several outbreaks have occurred since the virus was first reported in 1976. The largest recorded outbreak of Ebola virus
Lancet Infect Dis 2015 Published Online December 22, 2015 http://dx.doi.org/10.1016/ S1473-3099(15)00486-7 See Online/Comment http://dx.doi.org/10.1016/ S1473-3099(15)00534-4 Policlinique Médicale Universitaire, Lausanne, Switzerland (O De Santis MRes, C Cochet MD, D Estoppey MD, S Lonchampt MSc, Prof B Genton MD); Division of Immunology and Allergy (R Audran PhD, V Steiner-Monard MD, A-C Thierry BS, C Mayor BS, F Spertini MD), Clinical Trial Unit (L Warpelin-Decrausaz PhD, L Vallotton MD, G Wuerzner MD), and Infectious Diseases Service, Department of Medicine (Prof B Genton), Lausanne University Hospital, Switzerland; Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland (E Pothin PhD, Prof B Genton); Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA (R T Bailer PhD, O T Mbaya MD, Y Zhou PhD, A Ploquin PhD, N J Sullivan PhD, B S Graham MD); GSK Vaccines, Rixensart, Belgium (F Roman MD, I De Ryck MD, W R Ballou MD); and WHO, Geneva, Switzerland (M P Kieny PhD, V Moorthy MD) Correspondence to: Olga De Santis, Policlinique Médicale Universitaire, Lausanne 1011, Switzerland [email protected]
disease is ongoing, and more than 28 000 cases and more than 11 000 deaths in three countries in west Africa had been reported by September, 2015.1 WHO has declared the current outbreak an international public health emergency.
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Research in context Evidence before this study We searched PubMed for clinical trials up to Aug 17, 2015, with the search terms “Ebola” AND “vaccine”, with no language or date restrictions. Two DNA vaccines and one recombinant adenovirus serotype 5 using different versions of the Ebola virus or Marburg virus glycoprotein protein have been tested in the past 10 years. Chimpanzee adenovirus 3 (ChAd3)-vectored vaccines using monovalent and bivalent formulations of the Ebola virus glycoprotein were tested in late 2014 in US and UK phase 1 clinical trials with small sample sizes. A recombinant vesicular stomatitis virus (rVSV)-vectored Ebola vaccine was simultaneously tested in a multisite phase 1 trial. More recently, investigators of a phase 1 trial in China used a recombinant adenovirus serotype 5 vector-based Ebola vaccine expressing the glycoprotein of the 2014 epidemic strain. No safety issues arose from these trials, except for cases of arthritis and rash with rVSV, mainly reported at one site. All these trials done simultaneously to ours were published as preliminary reports including safety and immunogenicity data up to day 28 after injection. Added value of this study Our study provides the most comprehensive results of a phase 1/2 trial with ChAd3 vector-based vaccine expressing the Ebola virus glycoprotein. This trial was the only one that was placebocontrolled, allowing for the most accurate assessment of safety and reactogenicity. Among all Ebola vaccine trials, this is the only one that provides safety and immunogenicity results up to 6 months after injection, which provides some insight into the value of the vaccine over the course of an epidemic. No safety signal was recorded in our trial. All vaccine recipients had humoral immune responses that peaked at day 28, and then decreased by about half 6 months after injection. Interferon-γ mononuclear cell responses were still present at that time.
As a result of large multilateral public health interventions, the case incidence of Ebola virus disease had declined to less than ten cases per week by the end of July, 2015; however, no approved treatment or vaccine is yet available. Current efforts to develop a vaccine are focused on the viral glycoprotein encoded by the virus. The most advanced vaccine candidates tested so far are based on the glycoprotein from either the Zaire strain of Ebola virus (responsible for the current outbreak) or the Sudan strain. Candidates in which viral glycoprotein is expressed in either chimpanzee adenovirus, human adenovirus, or vesicular stomatitis virus have shown promise in nonhuman primate models of Ebola virus disease, and in initial clinical trials.2–7 Moreover, preliminary results of a phase 3 clinical trial with the replication-competent recombinant vesicular stomatitis virus (rVSV)-vectored vaccine showed encouraging efficacy results in Guinea.8,9 Rationale for development of this vaccine is based on previous human experience with other investigational filovirus vaccines and the development of non-human 2
Implications of all the available evidence When compared with results of the rVSV-vectored Ebola vaccine at 2×10⁷ or 5×10⁷ plaque-forming units, we can conclude that the safety profile of the ChAd3 Ebola vaccine (ChAd3-EBO-Z) at doses of 10¹⁰ viral particles is slightly better, but the humoral responses slightly lower, 1 month after injection. In view of the good safety profile of ChAd3-EBO-Z at doses of 10¹⁰ viral particles in the present trial, the 10¹¹ viral particle dose would seem appropriate to use when proceeding to phase 2 and 3 trials in Africa as planned, especially because the few available safety data with ChAd3-EBO-Z at 10¹¹ viral particles show an acceptable adverse events profile and, more importantly, similar antibody responses, as those obtained with the 2×10⁷ plaque-forming units dose of the rVSV-vectored vaccine. Assuming that the anti-glycoprotein antibody concentration is correlated with protection (even if the antibodies are not themselves protective), the promising efficacy results reported in the preliminary report of the rVSV-vectored vaccine in the phase 3 trial in Guinea could also be obtained with the ChAd3-EBO-Z vaccine at a dose of 10¹¹ viral particles. The persistence of antibodies at month 6, although at a reduced concentration, might suggest that some protection remains. However, this theory needs to be confirmed in a thorough phase 3 trial. Detailed correlation of immunological data and protection in non-human primate studies might also give some insight into efficacy, if a phase 3 trial becomes impossible to do because of an insufficient number of new cases of Ebola virus disease.
adenovirus vectors with low seroprevalence in human beings.3,10–14 Previous phase 1 clinical trials investigated the bivalent and monovalent vaccines encoding wild-type glycoprotein from Zaire and Sudan species of Ebola virus4 or Zaire species only.15 In response to a request from WHO in September, 2014, we undertook the present study to assess the safety and immunogenicity of the monovalent, recombinant, chimpanzee adenovirus type-3 vector-based Ebola Zaire (ChAd3-EBO-Z) vaccine construct. We aimed to build on and extend the clinical development plan for a chimpanzee adenovirus 3 (ChAd3)-vectored vaccine encoding Ebola glycoproteins that has been developed by the US National Institutes of Health in collaboration with GSK–Okairos, WHO, and the University of Oxford.
Methods Study design and participants We did this randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a study at the Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland.
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All participants in Lausanne were recruited by use of advertisements in the hospital and university halls. Because the study was largely published in the press, many people contacted the team directly to ask for their participation. Eligible volunteers had to be healthy, aged 18–65 years, and had to practise continuous contraception throughout the study. Exclusion criteria included previous participation in a trial of investigational Ebola or Marburg vaccine, or a chimpanzee adenovirus-vectored vaccine; receipt of any other live vaccine within 28 days or killed vaccine within 14 days before the trial; and presence of any immunodeficiency state or any acute or chronic disease not well controlled, which could increase the risk for serious adverse events or could impair interpretation of the data. The appendix provides full details of the inclusion and exclusion criteria. The study was reviewed and approved by the local ethics review board (Commission cantonale d’éthique de la recherche sur l’être humain), by the WHO Research Ethics Review Committee, and by the Swiss regulatory authorities (Swissmedic). All participants provided written informed consent.
Randomisation and masking We randomly assigned participants (2:2:1), via two computer-generated randomisation lists for individuals deployed in epidemic areas and those not deployed, to receive a single intramuscular dose of ChAd3-EBO-Z containing either 5×10¹⁰ (high dose) or 2·5 × 10¹⁰ (low dose) viral particles, or a single dose of placebo (diluent only). Deployed participants were allocated to only the vaccine groups. The randomisation lists were kept confidential in the central pharmacy at CHUV. The rationale to choose the two doses with only a two-fold difference was based on previous safety experience in clinical trials with chimpanzee adenovirus vectors.16 Because 100% of the non-human primates were protected 1 month after vaccination, there was a clear signal that this vaccine could be effective in human beings. Therefore, participants deployed from non-epidemic to epidemic areas could be the first beneficiaries of the vaccine, which is why WHO requested these individuals were not allocated to receive placebo. The safety evaluation was not masked for all potentially deployed participants, but group allocation was concealed from deployed participants, investigators, and outcome assessors.
Procedures ChAd3-EBO-Z consists of a recombinant replicationdeficient adenovirus chimpanzee serotype 3 vector expressing wild-type Ebola virus glycoprotein from the Zaire Mayinga strain. The appendix provides details about the composition of vaccine and diluent. We assessed local and systemic adverse events 1 h after injection and at follow-up visits on days 1, 7, 14, and 28. Additionally, participants recorded adverse events on a daily notification sheet for the first week. Solicited adverse events
were events that arose at any time from injection to day 7 (plus or minus 1 day), and included both local (pain, erythema and swelling at injection site, plus axillary lymphnode enlargement) and systemic (fever, fatigue or malaise, musculo-articular pain, headache, chills, and nausea) adverse events. Unsolicited adverse events were all other events not listed above, and all events that arose after the 7 day follow-up and up to day 28 (plus or minus 7 days). Grading of adverse events for severity and assignment of causal association of unsolicited adverse events was assessed by clinicians in charge of monitoring participants during the whole study, according to predefined criteria in the study protocol (appendix). Safety biological monitoring was done on blood samples taken on days 0, 1, 7, 14, 28, and 180 after injection, and included a full blood count, electrolytes, liver and renal function tests, C-reactive protein, and activated partial thromboplastin time. We did this assay because an asymptomatic prolongation of activated partial thromboplastin time has been reported in the 2 weeks after vaccination in previous adenovirus vaccine trials.3,15 This finding was due to the induction of a non-specific antiphospholipid antibody, and not to coagulopathy. This effect is actually an artifact of the activated partial thromboplastin time test, because this test measures the clotting cascade and the assay requires the presence of phospholipid as a reagent.3,15 Anti-EBO-Z immunoglobulins were measured with ELISA and antigen-specific T-cell responses were assessed with ex-vivo ELISPOT. The appendix details methods of antibody measurement and assessment of cell-mediated immunity. 3 months after injection, a follow-up assessment was done via phone call or email to record the occurrence of serious adverse events or relevant adverse events possibly related to injection. 6 months after injection, a last followup visit was done to obtain information about serious or relevant unsolicited adverse events, and about laboratory samples.
See Online for appendix
Outcomes The primary objective was the safety and reactogenicity of ChAd3-EBO-Z. The secondary objective was vaccine immunogenicity. The main exploratory outcome measure was intracellular cytokine staining assays.
Statistical analysis A sample size of 100 vaccinated participants was calculated to achieve a total of 250 vaccinated participants, taking into account all three concurrent phase 1 trials of the ChAd3-EBO-Z vaccine (Lausanne, Oxford, and Mali).15,17 This sample size was expected to produce reliable data for the incidence of frequent adverse events. Because safety evaluation for potentially deployed participants was not masked, and because too few participants had gone to epidemic regions after vaccination to expect potential immunological boost after hypothetical exposure, only results from the
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150 volunteers screened for eligibility
30 excluded 28 ineligible 1 withdrew consent 1 because accrual goal had been met
120 enrolled
18 potentially deployed
9 randomly assigned to low-dose vaccine
9 randomly assigned to high-dose vaccine
1 missed day 14 visit 1 missed day 90 visit
7 completed 6 month follow-up 7 completed safety and immunogenicity analysis
102 non-deployed
42 randomly assigned to low-dose vaccine
40 randomly assigned to high-dose vaccine
20 randomly assigned to placebo
1 missed day 28 visit
7 completed 6 month follow-up 7 completed safety and immunogenicity analysis
42 completed 6 month follow-up 42 completed safety and immunogenicity analysis
40 completed 6 month follow-up 40 completed safety and immunogenicity analysis
20 completed 6 month follow-up 20 completed safety and immunogenicity analysis
Figure 1: Trial profile
non-deployed group were used to compare safety between the high-dose, low-dose, and placebo groups; results from both the deployed and non-deployed groups were pooled to compare immunogenicity because the laboratory team assessing antibody or cellular responses was masked to group assignment. We present anti-Ebola virus glycoprotein IgG concentrations as geometric mean concentrations with 95% CIs. We compared groups with Fisher’s exact test for safety and the Mann–Whitney test for immunogenicity. The lower dose was compared with the higher dose, and the two doses were pooled and termed “vaccinated” for comparison with placebo. For each participant, we defined a positive antibody response as a significant increase in post-vaccination titre from baseline (t test assuming non-equal variance), with use of the anti-glycoprotein antibody titres assessed by ELISA done in the Vaccine Research Center (VRC; National Institutes of Health, Bethesda, MD, USA).11 We used Friedman or Kruskal–Wallis with Dunn’s post tests to compare magnitude of T-cell responses before and after vaccination with GraphPrism (version 6.07). A Data Safety Monitoring Board was established before the start of the trial, and included two independent clinicians and one epidemiologist. The Board reviewed the safety data from days 0–7 for the first 20 participants vaccinated to ensure that holding rules were not met. This trial is registered with ClinicalTrials.gov, number NCT02289027. 4
Role of the funding source The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and shared final responsibility for the decision to submit for publication with the principal investigator.
Results Between Oct 24, 2014, and June 22, 2015, we randomly assigned 120 participants, of whom 18 (15%) were potentially deployed and 102 (85%) were non-deployed, to receive low-dose vaccine (n=51), high-dose vaccine (n=49), or placebo (n=20; figure 1). All participants, except three deployed individuals who missed one visit each, completed the six visits after injection (figure 1). Baseline characteristics were similar between groups (table 1). No vaccine-related serious adverse events were noted. Most of the adverse events reported were mild and selflimiting, arising during the first 24 h after injection and lasting less than 48 h. We recorded seven grade 3 adverse events, all of which resolved within 3 days with no residual effect. Only the placebo-controlled results for the nondeployed participants are shown below. The most common solicited local adverse event was grade 1 pain, which differed significantly between the vaccine and placebo groups, but not between vaccine dose groups (table 2). At least one solicited systemic adverse event was reported in 71 (87%) participants in the vaccine groups (n=37 in the highdose group and n=34 in the low-dose group) and ten (50%)
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of those in the placebo group (p=0·0015; figure 2). The most common solicited systemic adverse events were fatigue or malaise and headache (table 2). Musculo-articular pains were also frequently reported (table 2). Most solicited adverse events were mild and resolved within 24 h after injection (figure 2). 28% of non-deployed vaccine recipients developed fever versus 5% of placebo recipients, with no significant difference between the dose groups (table 2); however, the highest vaccine-related temperatures were in participants given high-dose vaccine (appendix p 14). One relevant unsolicited adverse event possibly related to the vaccine was an episode of macroscopic haematuria associated with pain and burning with urination and mild left costovertebral angle tenderness at percussion that arose within 24 h after injection with low-dose vaccine. Results from the medical investigations (urinary sediment and culture, renal ultrasound, blood count, coagulation assays) were normal and the episode spontaneously resolved 48 h after injection. Because no biological cause was identified for this episode, and because the participant had not previously had a similar episode, the adverse event was considered to be possibly related to the vaccine. A second relevant unsolicited adverse event possibly related to the high-dose vaccine was a herpetiform dermatitis that arose at day 15 after injection and lasted for 2 weeks. Located in the L2 dermatome, this adverse event was clinically diagnosed as shingles, although not confirmed by PCR. None of the laboratory abnormal values were clinically significant (appendix pp 15–17). At day 1, we recorded 51 grade 1 lymphopenias (<1·5–0·8 × 10⁹ lymphocytes per L) in 23 (58%) participants in the high-dose vaccine group, 22 (52%) participants in the low-dose group, and six (30%) participants in the placebo group; and four grade 2 (<0·8–0·5 × 10⁹/L) transient lymphopenias in one (3%), three (7%), and no participants, respectively (appendix). One transient grade 1 thrombocytopenia (<150–75 × 10⁹ platelets per L) arose in one (2%) participant in the low-dose group (appendix). During month 1 of follow-up, we recorded six transient grade 1 anaemias (haemoglobin <117–100 g/L) in one (3%) participant in the high-dose group and five (12%) participants in the low-dose group. We also recorded six transient neutropenias of mild intensity (<1·8–1·5 × 10⁹ neutrophils per L; three [8%] participants in the high-dose group, two [5%] in the low-dose group, and one [5%] in the placebo group) and moderate intensity (<1·5–1 × 10⁹/L; three [7%] in the low-dose group), and two neutropenias of severe intensity (<1 × 10⁹/L; one [2%] in the high-dose group and one [5%] in the placebo group; appendix). We recorded one case of asymptomatic grade 1 prolonged activated partial thromboplastin time at day 14 in the high-dose group (appendix). Investigation showed no coagulopathy. The antiphospholipid screening was positive for a lupus anticoagulant and doubtful for an anticardiolipin IgM. The prolonged activated partial thromboplastin time and presence of anticardiolipin IgM had resolved by month 3
Non-deployed participants (N=102)
Potentially deployed participants (N=18)
Placebo Low-dose group (n=20) group (n=42)
Low-dose group (n=9)
High-dose group (n=40)
High-dose group (n=9)
Sex Male
11 (55%)
22 (52%)
19 (48%)
4 (44%)
3 (33%)
9 (45%)
20 (48%)
21 (52%)
5 (56%)
6 (67%)
White
16 (80%)
40 (95%)
35 (88%)
9 (100%)
6 (67%)
Black
1 (5%)
1 (2%)
1 (2%)
0
2 (22%)
Hispanic
0
0
1 (2%)
0
0
Other
3 (15%)
1 (2%)
3 (8%)
0
1 (11%)
Mean (SD)
37·2 (13·4)
30·7 (11·1)
33·2 (13·1)
42 (12·4)
46 (10·8)
Median (min–max range)
37 (19–61)
27·5 (19–63)
27 (19–63)
39 (28–62)
43 (32–64)
23·7 (3·3)
24·2 (2·9)
23·4 (4·1)
26·6 (3·9)
23·8 (19·4–31·2)
23·4 (18·3–30·3)
27·4 (20·3–33·2)
Female Race or ethnic origin
Age (years)
Body-mass index (kg/m²) Mean (SD) Median (min–max range)
23·5 (3·6) 22·3 (18·9–33·9)
23·3 (17·6–32)
Data are n (%), unless otherwise specified.
Table 1: Baseline characteristics
and the presence of lupus anticoagulant by month 9. No associated clinical sign of hypercoagulability was present. Of the grade 3 adverse events, one was an unsolicited local event, four were solicited systemic events, and two were laboratory adverse events. The one local grade 3 adverse event was an erythema at injection site of 11 cm in diameter with presence of redness and warmth but no pain, which arose at day 9 in a participant in the high-dose group and lasted for less than 24 h. Of the four solicited systemic grade 3 adverse events, two were sudden and strong headaches that arose during the 24 h after injection and resolved in less than 2 h with paracetamol. The other two events were fevers with temperatures exceeding 39°C, one arose during the night after injection of high-dose vaccine and lasted less than 24 h, and the other arose at day 4 after injection of low-dose vaccine, but was associated with a streptococcus angina and therefore not related to the vaccine. We recorded two grade 3 neutropenias, one at day 1 in the high-dose group and the other at day 14 in the placebo group. None were associated with symptom or clinical sign and both were resolved at the following visit 3 days later. At month 3 of follow-up, all volunteers except one were reached by telephone or email to assess safety. Three mild to moderate adverse events were possibly related to the injection. One was a second episode of an axillary lymphnode enlargement at day 63 after injection of high-dose vaccine, and lasted 2 days (first episode previously described at day 1 and lasted 2 days). The two other events were mild fatigue at day 34 in the high-dose group, and lasted 1 week, and moderate fatigue with several episodes of frontal headache at day 34 in the low-dose group, which lasted for roughly 3 weeks. At the last visit at 6 months,
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Non-deployed (N=102)
p value
Potentially deployed (N=18)
Placebo group (n=20)
Low-dose group (n=42)
High-dose group (n=40)
Grade 1
5 (25%)
32 (76%)
25 (62%)
6 (67%)
7 (78%)
Grade 2
0
1 (2%)
5 (12%)
1 (11%)
0
Grade 1
0
0
2 (5%)
0
0
Grade 2
0
0
1 (2%)
0
0
0
0
0
0
Placebo vs vaccinated
Low dose vs high dose
Low-dose group (n=9)
High-dose group (n=9)
Local Pain
<0·0001
Swelling
1
Erythema Grade 1
0
1 (2%)
0
0
0·11
1
0·61
0·58
0·05
2 (5%)
Axillary lymphatic-node enlargement Grade 1
0·80
4 (10%)
Systemic Fatigue or malaise
0·01
Grade 1
5 (25%)
Grade 2
0
20 (50%)
4 (44%)
4 (44%)
2 (5%)
5 (12%)
1 (11%)
1 (11%)
3 (33%)
2 (22%)
0
1 (11%)
Musculo-articular pain
0·08
Grade 1
5 (25%)
Grade 2
0
0·82
23 (55%)
17 (40%)
16 (40%)
1 (2%)
6 (15%)
Chills
0·02
0·38
0·60
Grade 1
0
6 (14%)
8 (20%)
3 (33%)
3 (33%)
Grade 2
0
2 (5%)
2 (5%)
0
0
Grade 1
4 (20%)
5 (12%)
2 (5%)
2 (22%)
1 (11%)
Grade 2
0
0
1 (2%)
0
0
Grade 1
0
6 (14%)
7 (18%)
2 (22%)
1 (11%)
Grade 2
1 (5%)
5 (12%)
4 (10%)
0
1 (11%)
Grade 3
0
0
1 (2%)
0
0 2 (22%)
Nausea
0·22
Fever
0·04
Headache
0·0043
0·43
0·81
1
Grade 1
2 (10%)
15 (36%)
14 (35%)
1 (11%)
Grade 2
1 (5%)
9 (21%)
8 (20%)
1 (11%)
2 (22%)
Grade 3
0
1 (2%)
1 (2%)
0
0
0
1 (2%)
1 (2%)
0
1 (11%)
1 (11%)
0
0
1 (11%)
Unsolicited related Abdominal pain Grade 1 Conjunctivitis Grade 1
0
0
0
1 (2%)
1
1
0·49
1
1
1
1
1 (2%)
Rhinitis Grade 1
1
0
Sweating Grade 1
0
0
0
0
0
Grade 2
0
1 (2%)
0
0
0
Other*
0·13
0·83
Grade 1
2 (10%)
14 (33%)
14 (35%)
4 (44%)
2 (22%)
Grade 2
2 (10%)
6 (14%)
2 (5%)
1 (11%)
0
Grade 3
1 (5%)†
0
2 (5%)†‡
0
0
Data are n (%), unless otherwise specified. Events are presented in frequency of participants presenting at least one adverse event of the category; if many, the maximum intensity is retained. *Includes (injection-site) erythema, headache, fatigue, and fever occurring after day 8, and grade 3 neutropenia. †One grade 3 neutropenia at day 14 in one participant from the placebo group, and one grade 3 neutropenia at day 1 in one participant in the high-dose vaccine group. ‡Erythema at injection site of 11 cm in diameter at day 9 after injection (p value from Fisher’s exact test).
Table 2: Solicited local and systemic adverse events occurring up to day 7 (plus or minus 1 day) and unsolicited related adverse events up to day 28 (plus or minus 7 days)
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A
B Local
100
Grade 1 Grade 2 Grade 3
Solicited systemic
Any related
Any
Unsolicited related
Unsolicited
Proportion (%)
75
50
25
Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se Pla ce Lo bo w d Hi ose gh do se
0
Figure 2: Proportion of individuals affected and severity of adverse events in non-deployed participants (A) Adverse events reported up to day 7 (plus 1 day, up to 8 days). (B) Adverse events reported up to day 28 (plus 7 days, up to 35 days). Only the maximum grade per adverse event was reported for each individual.
A
p<0·0001
Anti-glycoprotein IgG (μg/mL)
1000
p<0·0001
p<0·0001
Placebo Low dose High dose 100
10
1 0
7
14
28
180
Day
B
C
10 000
1000
Placebo Low dose High dose Anti-glycoprotein IgG (μg/mL)
Not seroconverted Seroconverted Anti-glycoprotein IgG EC90 titre
only one adverse event was reported as possibly related to the vaccine. The participant reported mild arthralgia in the distal interphalangeal joints of the fifth fingers on both hands, lasting 1 month. Neither swelling nor warmth was reported. Mobility was normal, but a light red macula of 2–3 mm was observed on the dorsal face of each joint. This participant had received the placebo and was sent for a specialised consultation for further investigations. From day 28 to day 180, three serious adverse events were reported, none of which were related to the injection, and all of which were due to trauma—namely, an elective hospital admission for a dislocated shoulder surgery in a participant given placebo, an emergency admission and surgery for a broken radius in a participant given placebo, and a persistent disability due to a broken anterior cruciate knee ligament in participant given low-dose vaccine. Finally, at month 3 of follow-up, a participant in the lowdose group reported the pregnancy of his wife. At this time the pregnancy was in the first trimester. The date of conception was difficult to determine because this was an unexpected pregnancy arising despite oral contraception, but it was estimated at 2 weeks after vaccination. The pregnancy was terminated 3 weeks later because of trisomy 21 diagnosed by the gynaecologist. There is no biological plausibility that this diagnosis could be related to the vaccination of the partner. Figure 3 summarises results for anti-Ebola virus glycoprotein IgG responses, including all data from deployed and non-deployed vaccine recipients. Antibody response was detected from day 14 onwards and peaked at day 28 up to a geometric mean concentration of 51 μg/mL (95% CI 41·1–63·3) in the high-dose group and of 44·9 μg/mL (25·8–56·3) in the low-dose group (figure 3). Antibody concentration did not differ between the two vaccine groups. The proportion of responders was 96% (95% CI 85·7–99·5) in the high-dose group, 96% (86·5–99·5) in the low-dose group, and 5% (0·1–24·9) in the placebo group (appendix). Antibody response decreased by roughly half from day 28 to day 180, with geometric mean concentrations of 25·5 μg/mL (95% CI 20·6–31·5) in the high-dose group, 22·1 μg/mL (19·3–28·6) in the lowdose group, and 3·2 μg/mL (2·4–4·9) in the placebo group 6 months after injection (figure 3). At day 28, geometric means of the VRC titres were 434·7 (min–max range 77·7–5576·3) in the high-dose group, 467·3 (41·5–4265·3) in the low-dose group, and 33·0 (6·9–198·0) in the placebo group (figure 3). Mononuclear cell responses to vaccination were evaluated by interferon (IFN)-γ ELISPOT on days 0, 7, 14, 28, and 180. Responses had already increased by day 7 in the high-dose group and, similar to antibody response, peaked at day 14, with a significant median response of 182·7 (range 91·3–345·3) spot-forming units per million peripheral blood mononuclear cells (PBMCs) in the high-dose group and 180 (66·95–382·0) spot-forming units per million PBMCs in the low-dose group (appendix). Although still significantly higher than at day 0 (within group analysis
1000
100
100
10
r=0·3 p<0·0001
10 1
Placebo
Low dose
High dose
10
100 1000 10 000 Anti-glycoprotein IgG EC90 titre
Figure 3: Antibody responses to the Ebola Zaire glycoprotein (A) Kinetics of responses as assessed by commercial ELISA. Boxes show median values and IQRs; whiskers are 5th and 95th percentiles, and geometric mean concentrations (μg/mL) are compared between groups. P values from the Mann–Whitney test. (B) Individual Vaccine Research Centre (VRC)-assessed EC90 titres at day 28. Squares show geometric mean concentrations and vertical lines show 95% CIs. (C) Spearman’s correlation between the two ELISA assays (Lausanne and VRC). EC90=90% effective concentration (the dilution of serum at which there is a 90% decrease in antigen binding).
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Friedman test p=0·001), responses at day 180 declined in most participants, and did not differ significantly from those in the placebo group (Dunn’s post tests p>0·05; appendix). T-cell-specific response was measured by flow cytometry at days 0, 14, and 28 and was expressed as frequencies of CD4 and CD8 cells producing IFN-γ, interleukin 2, or tumour necrosis factor (TNF)α after stimulation with Ebola Zaire glycoprotein peptides (appendix p 17). Significant glycoprotein-specific CD4 and CD8 cell responses were obtained from day 14 in both vaccine groups (appendix p 18), with no significant difference between doses. On the basis of positive responses for at least one of the three cytokines, 28 (57%) participants from the high-dose group and 31 (61%) participants from the low-dose group developed glycoprotein-specific CD4 cell responses, and 33 (67%) and 35 (69%) participants, respectively, developed glycoproteinA
0·0317
2·0 Total cytokine responses (% of subsets)
B
CD4 memory T cells
CD8 memory T cells <0·0001 <0·0001
<0·0001
0·0046 0·0007
<0·0001
1·0 0·50
0·25
0·00
–0·25
Placebo (n=20) Low dose (n=51) High dose (n=49)
–0·50 0
14
28
0
Day
C
14
28
Day
CD4 cells
Day 14
Day 28 TNF-γ IL-2 TNFα
+ + +
Three cytokines
+ + – Low dose
D
High dose
Low dose
High dose
+ – +
Two cytokines
– + + CD8 cells
+ – –
Day 14
Day 28
– + –
One cytokine
– – +
Low dose
High dose
Low dose
High dose
Figure 4: Memory T-cell responses to the Ebola virus Zaire glycoprotein Kinetics of individual CD4 (A) and CD8 (B) responses expressed as frequencies of subsets expressing at least one cytokine, IFN-γ, IL-2, or TNFα. Boxes show median values and IQRs; whiskers are 5% percentiles. We used the Kruskal–Wallis test to assess statistical significance of vaccine versus placebo. Proportions of glycoprotein-specific memory CD4 (C) and CD8 (D) cells that produce any combination of the three cytokines, at day 14 and day 28, in the vaccine groups. TNF=tumour necrosis factor. IL=interleukin. IFN=interferon.
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specific CD8 cell responses. Vaccine-specific memory T-cell responses showed the same kinetics and were equally distributed between CD4 and CD8 T cells (figure 4). Both memory CD4 and CD8 T cells presented polyfunctional and monofunctional phenotypes (figure 4). The CD8 cell response consisted mainly of IFN-γ-producing cells among which the IFN-γ TNFα co-producing subsets represented 40% of the response (figure 4). ChAd3-neutralising antibodies were measured in all participants at days 0 and 28 (appendix p 22). Notably, the concentration of neutralising antibodies at day 0 negatively correlated with anti-glycoprotein antibody responses, and with CD8 IFN-γ responses at day 28 (appendix).
Discussion This is the largest phase 1/2a clinical trial reported so far with an experimental Ebola vaccine, and the first to report data with a 6 month follow-up. No vaccine-related serious adverse events were reported during the 6 month follow-up. The ChAd3-EBO-Z vaccine led to more local and systemic adverse events than did placebo alone. Most adverse events were mild and all resolved with no sequelae, mostly within the first 24 h after injections. These results are in line with those shown in other trials of adenovirus-vectored vaccine.3,4,15,18,19 More precisely, the reactogenicity was similar to that shown in previous phase 1 trials16,18.19 using chimpanzee adenovirus vectors and expression proteins from other pathogens, showing that adverse events were more likely to be induced by the vector rather than by the Ebola virus glycoprotein. Inclusion of a placebo group allowed us to show that local pain and fatigue or malaise, musculo-articular pain, chills, fever, and headache—all components of reactogenicity— were due to the vaccine. Moreover, rates of unsolicited adverse events did not differ significantly between the vaccine and placebo groups, inferring that larger trials are needed to investigate a potential association with the vaccine. Incidence of local reactogenicity was similar to that reported after routine vaccinations (eg, influenza; hepatitis B; DTPa; or measles, mumps, and rubella vaccinations20–24), with the exception of pain at injection site, which was more common, but almost always mild and with little erythema or swelling. By contrast, the incidence of systemic adverse events was substantially higher, especially for headache, musculo-articular pains, and fever. Although the safety data were roughly similar to those reported in Rampling and colleagues’ phase 1 trial,15 with headache, fatigue, and malaise being the most common adverse events, the frequency of adverse events was higher in our study. Only 5% (n=2) of participants had objective fever in Rampling and colleagues’ study, compared with 28% of participants in our study. This difference might be explained by measurement technique, because feverishness in general was present in 30% of participants in Rampling and colleagues’ study. Although more common, adverse events in our study were of mild intensity, short-lived, and selflimited, which makes them acceptable in a risk–benefit
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balance in relation to such a severe disease as Ebola. Moreover, 81% of the fevers induced by the vaccine resolved within 24 h after injection. This rapid resolution makes them manageable, even during an outbreak, by preventing confusion with early onset of a new Ebola case. Frequencies and intensities of adverse events were similar between the two vaccine dose groups, although fevers of higher temperatures and four of the seven grade 3 adverse events were reported in the high-dose group. The absence of a significant dose effect might be because the two doses differed by only a factor of two. The slight increase of fever in the high-dose group might become clinically relevant when the dose of 1 × 10¹¹ viral particles is used—the dose currently deployed in Africa. Indeed, in the clinical trial of the bivalent ChAd3-EBO (Zaire and Sudan) vaccine,4 the dose of 2 × 10¹¹ viral particles was more reactogenic than the dose of 2 × 10¹⁰ viral particles, with two (20%) of the ten vaccine recipients reporting fever compared with none of the recipients given the lower dose. These data might suggest that the 10¹¹ dose will be more reactogenic. The published short-term safety results of the rVSV vaccine trial,5 the other major promising Ebola vaccine, showed a similar early reactogenicity profile. Although no vaccine-related serious adverse events have been reported with either vaccine, cases of arthritis or arthralgia with maculopapular rash or vesicular dermatitis did arise in some participants 2 weeks after vaccination with rVSV. These findings were reported at differing rates in different trials, with the highest reported rate being 22% (11 of 52 recipients) in Geneva.5 ChAd3 vaccine recipients only complained of transient musculo-articular pain within 3 days after vaccination as part of general influenzalike symptoms, but with no clinical evidence of arthritis. Notably, a higher frequency of adverse events was reported in both phase 1 trials of Ebola vaccine (the rVSV Ebola vaccine trial25 and our study), both done in Switzerland, than in other trials with the same vaccine. This difference is unlikely to be due to specific genetic traits, because participants in our study were of many different origins, and is probably related to the reporting mode. A single vaccination with ChAd3-EBO-Z induced antibody responses in 96% of participants, independently of the dose. The anti-Ebola Zaire virus glycoprotein titres obtained at day 28 confirmed the responses obtained with doses of 5×10¹⁰ and 2·5×10¹⁰ viral particles of ChAd3-EBO-Z reported in a previous study.15 There was no dose effect in our trial, probably because the two doses were close. The 6 month follow-up results showed for the first time that antibody titres in these groups were maintained at a significantly increased concentration. Notably, the presence of ChAd3 neutralising antibodies at day 0 correlated negatively with both the concentration of anti-glycoprotein antibodies, and the CD8 IFN-γ T-cell responses, at day 28. This finding was in line with similar observations in a previous preliminary report,4 although the result reached significance in the present study with
a larger sample size. With regards to the durability of the T-cell response, the IFN-γ mononuclear cell responses by ELISPOT decreased, but were still present, at month 6, despite not differing significantly from the response with placebo at this later timepoint. Remarkably, the presence of an Ebola-specific CD8 T-cell response with an IFN-γ and TNFα co-producer reinforces the potential for protection of the current vaccine formulation, because these markers are associated with vaccine-mediated protection in non-human primates.2 The proportion of IFN-γ and TNFα co-producers in this study was similar to that in a previous study.15 IFN-γ polyfunctional CD8 T cells were also found in similar proportions in Ledgerwood and colleagues’ phase 1 trial4 when doses of 2×10¹⁰ viral particles were used, but seemed to further expend with the highest dose of 2×10¹¹ viral particles. The promising efficacy provided by the VSV-vectored vaccine in Guinea8 gives hope that other vaccines based on the Ebola virus glycoprotein could be protective. Although correlates of immunity in human vaccination against Ebola virus are unknown, it is interesting to see that anti-glycoprotein titres observed with bivalent ChAd3-EBO at a dose of 2×10¹¹ viral particles were equivalent to those obtained with the rVSV-vectored vaccine assessed in the Guinea phase 3 trial. Available anti-EBO-Z glycoprotein ELISA data suggest that the humoral immune responses induced by the dose of 1 × 10¹¹ viral particles (for the monovalent form) are higher than those induced by the lower doses, which is why the 1 × 10¹¹ dose was selected for phase 2 and phase 3 studies (ClinicalTrials.gov, number NCT02485301). A key limitation of our study is that we could not test a higher vaccine dose because of the risk of inducing too many adverse events and therefore jeopardising the course of the study. Indeed, in the present emergency situation, results of early phases of vaccine development were crucial to proceed with further development in an epidemic area. Our study complements the clinical development plan for a ChAd3-vectored Ebola vaccine in several ways. First, our study is the only one of the existing ChAd3-vectored Ebola vaccine studies to include a placebo group, which enables precise assessment of the vaccine reactogenicity. Second, with the relatively large sample size and balanced inclusion of men and women, our findings substantially increase the data already obtained in previous studies and allow for a better assessment of safety and a valid comparison between doses, increasing the likelihood of identification of an optimum dose that balances both immunogenicity and reactogenicity, and enabling detection of a possible dose–response effect. Finally, this is the first report to provide safety and immunogenicity data at 6 months. Altogether, our results, showing an acceptable safety profile linked to Ebola virus-specific antibody response and polyfunctional CD8-specific T-cell response, provide a reliable basis for proceeding with efficacy
www.thelancet.com/infection Published online December 22, 2015 http://dx.doi.org/10.1016/S1473-3099(15)00486-7
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trials in Africa and have greatly assisted in decisionmaking for the initiation of further phase 2b and 3 trials. This acceptable safety profile linked to Ebola virus-specific antibody response and polyfunctional CD8-specific T-cell response provides a reliable basis to proceed with efficacy trials in Africa.
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Contributors ODS, LW-D, LV, GW, FS, MPK, VM, IDR, FR, and BG contributed to the conception and design of the study. ODS, CC, DE, VS-M, SL, RA, A-CT, CM, and BG collected the data. ODS, EP, LW-D, LV, GW, SL, IDR, WRB, FS, and BG analysed and interpreted safety data. RA, A-CT, CM, EP, ODS, BSG, NJS, OTM, YZ, AP, RTB, BG, and FS analysed and interpreted immunogenicity data. ODS, RA, EP, FS, and BG wrote the manuscript. All authors contributed to revision of the manuscript.
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Declaration of interests FR, IDR, WRB, are employees of GSK. All other authors declare no competing interests. Acknowledgments We thank all persons interested, but eventually not screened, and all volunteers for their generous and reliable participation in this trial; Loredana Otgon, Emmanuelle Paccou, Christiane Pellet, Sylvie Poget, and Françoise Secretan, the clinical research nurses of the Clinic Trial Unit of the Centre Hospitalier Universitaire Vaudois (CHUV) for their exceptional expertise and commitment; Stéphanie Dartevelle and Isabelle Angelstorf from the CHUV pharmacy for the randomisation and daily preparation of the vaccine doses; Claudia Rochat and all laboratory members for the accurate handling and analyses of a large amount of blood samples; Kristina Moemi Geiger and Tom Stornetta for their generous assistance in blood sample preparations; Fady Fares and Ali Maghraoui from the Clinical Trial Unit of the CHUV for data management; Pascal Savary and Alexia Kaeser from the Legal Unit of the CHUV for contracts revision; Richard Pink for his contribution to manuscript writing; Patrick Francioli (Chairman of the Commission cantonale d’éthique de la recherche sur l’être humain) and his team, and the WHO Research Ethics Review Committee for their rapid review and approval; Eric Huber and Marek Sochor from the Swiss Tropical and Public Health Institute for monitoring; Peter Smith, Pierre-Alexandre Bart, and Pierre Landry—members of the Data and Safety Monitoring Board; the Vaccine Research Centre team for their support in development of study documentation, for making the vaccine doses available, and for doing the intracellular cytokine staining analysis; Adrian Hill (Oxford University, London, UK) with his team for providing documentation and assistance in initiating a simian adenoviral vectored vaccine trial. This work was supported by the Swiss State Secretariat for Education, Research, and Innovation (SERI; contract number 14.0001) through the EU Horizon 2020 Research and Innovation Programme (EbolaVac project) under grant agreement number 666085. The opinions expressed and arguments employed herein do not necessarily reflect the official views of the Swiss Government or the World Health Organization. References 1 WHO. Ebola situation report–30 September 2015. http://apps.who. int/ebola/current-situation/ebola-situation-report-30september-2015 (accessed Oct 31, 2015). 2 Stanley DA, Honko AN, Asiedu C, et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge. Nat Med 2014; 20: 1126–29. 3 Ledgerwood JE, Costner P, Desai N, et al. A replication defective recombinant Ad5 vaccine expressing Ebola virus GP is safe and immunogenic in healthy adults. Vaccine 2010; 29: 304–13. 4 Ledgerwood JE, DeZure AD, Stanley DA, et al. Chimpanzee adenovirus vector Ebola vaccine—preliminary report. N Engl J Med 2014; 373: 776. 5 Agnandji ST, Huttner A, Zinser ME, et al. Phase 1 trials of rVSV Ebola vaccine in Africa and Europe—preliminary report. N Engl J Med 2015; published online April 1. DOI:10.1056/ NEJMoa1502924.
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www.thelancet.com/infection Published online December 22, 2015 http://dx.doi.org/10.1016/S1473-3099(15)00486-7