Articles

Safety, tolerability, and immunogenicity of a recombinantadenovirus type-5 vectored COVID-19 vaccine:a dose-escalation, open-label, non-randomised,
first-in-human trial
Feng-Cai Zhu*, Yu-Hua Li*, Xu-Hua Guan, Li-Hua Hou, Wen-Juan Wang, Jing-Xin Li, Shi-Po Wu, Bu-Sen Wang, Zhao Wang, Lei Wang, Si-Yue Jia,
Hu-Dachuan Jiang, Ling Wang, Tao Jiang, Yi Hu, Jin-Bo Gou, Sha-Bei Xu, Jun-Jie Xu, Xue-Wen Wang, Wei Wang, Wei Chen

Summary

Background A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, andimmunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spikeglycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain.
Methods We did a dose-escalation, single-centre, open-label, non-randomised, phase 1 trial of an Ad5 vectored
COVID-19 vaccine in Wuhan, China. Healthy adults aged between 18 and 60 years were sequentially enrolled andallocated to one of three dose groups (5 × 10¹⁰, 1 × 10¹¹, and 1·5 × 10¹¹ viral particles) to receive an intramuscular
injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over
28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralising antibody responses
induced by vaccination were detected with SARS-CoV-2 virus neutralisation and pseudovirus neutralisation tests.
T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registeredwith ClinicalTrials.gov, NCT04313127.
Findings Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants(51% male, 49% female; mean age 36·3 years) were recruited and received the low dose (n=36), middle dose
(n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the analysis. At least
one adverse reaction within the first 7 days after the vaccination was reported in 30 (83%) par­ticipants in the lowdose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The
most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, andthe most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache(42 [39%]), and muscle pain (18 [17%]. Most adverse reactions that were reported in all dose groups were mild or
moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies andneutralising antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell
response peaked at day 14 post-vaccination.
Interpretation The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination.
Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell
responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine
warrants further investigation.
Funding National Key R&D Program of China, National Science and Technology Major Project, and CanSino Biologics.
Copyright © 2020 Elsevier Ltd. All rights reserved.

Introduction
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) was first reported in January, 2020. The
virus is highly transmissible between humans and has
spread rapidly, causing the COVID-19 pandemic.1,2
Patients infected with SARS-CoV-2, especially older
patients and those with pre-existing respiratory or
cardiovascular conditions are at greater risk for severe
complications, including severe pneumonia, acuterespiratory distress syndrome, multiple organ failure,and in some cases, death.3,4 By May 20, 2020, SARS-CoV-2
www.thelancet.com Vol 395 June 13, 2020

had infected more than 4·7 million people across
215 countries or territories and killed more than
316 000 worldwide.5
In the absence of effective prevention measures, current
management to control the epidemic is the enforcementof quarantine, isolation, and physical distancing.6,7 Effec­
tive vaccines against COVID-19 are urgently needed toreduce the enormous burden of mortality and morbidity
associated with SARS-CoV-2 infection.8 There are morethan 100 candidate vaccines in development worldwide,9among them at least eight have started or will soon

Lancet 2020; 395: 1845–54
Published Online
May 22, 2020
https://doi.org/10.1016/
S0140-6736(20)31208-3
See Comment page 1815
*Contributed equally
Beijing Institute of
Biotechnology, Beijing, China(Prof W Chen PhD,Prof J-J Xu PhD,Prof L-H Hou PhD, S-P Wu PhD,
B-S Wang PhD); NHC Key
Laboratory of EntericPathogenic Microbiology,
Jiangsu Provincial Center for
Disease Control andPrevention, Nanjing, China(Prof F-C Zhu MSc,
W-J Wang MSc, Prof J-X Li PhD,
S-Y Jia MPH, H-D Jiang BSc);
China National Institute forFood and Drug Control, Beijing,
China (Prof Y-H Li PhD,
Li Wang PhD); Hubei Provincial
Center for Disease Control andPrevention, Wuhan, China(Prof X-H Guan PhD,
Z Wang MPH, Le Wang MPH);
Beijing Institute ofMicrobiology andEpidemiology, State Key
Laboratory of Pathogen and
Biosecurity, Beijing, China(Prof T Jiang PhD, Y Hu PhD);
CanSino Biologics, Tianjin,
China (J-B Gou PhD); ClinicalResearch Center, Tongji
Hospital, Tongji Medical
College, Huazhong Universityof Science and Technology,
Wuhan, China (Prof W Wang
PhD, Prof S-B Xu PhD); andShanghai Canming Medical
Technology, Shanghai, China(X-W Wang MD)
Correspondence to:Prof Wei Chen, Beijing Instituteof Biotechnology,
Beijing 100071, China
cw0226@foxmail.com
or

1845

Articles

Prof Wei Wang, Clinical ResearchCenter, Tongji Hospital, Tongji
Medical College, Huazhong
University of Science andTechnology, Wuhan 430032,
China
wwang@vip.126.com
orProf Feng-Cai Zhu, NHC Key
Laboratory of Enteric Pathogenic
Microbiology, Jiangsu Provincial
Center for Disease Control andPrevention, Nanjing 210009,
China
jszfc@vip.sina.com

Research in context
Evidence before this study
We searched PubMed on May 8, 2020, for clinical trial reports
with the terms “COVID-19” or “SARS-CoV-2”, “vaccine”,
and “clinical trial” and no date or language restrictions; no otherdata from a human clinical trial of COVID-19 vaccine have beenreported thus far, to our knowledge. We also searched theClinicalTrials.gov registry for unpublished trials of COVID-19
vaccines, up to May 6, 2020. In addition to the adenovirus
type-5 (Ad5) vectored COVID-19 vaccine reported here, sevencandidate COVID-19 vaccines are in ongoing clinical trials,
including Moderna’s mRNA COVID-19 vaccine, Inovio
Pharmaceuticals’ DNA vaccine, Sinovac, Wuhan and Beijing
Institute of Biological Products’ inactive COVID-19 vaccines,
University of Oxford’s chimpanzee adenovirus-vectored vaccine,and BioNTech’s mRNA COVID-19 vaccine.
Added value of this study
This ﬁrst-in-human trial showed that the Ad5 vectored
COVID-19 vaccine was tolerable and immunogenic in healthy
adults. One dose of the vaccine at all dose concentrations(5 × 10¹⁰, 1 × 10¹¹, and 1·5 × 10¹¹ viral particles) tested induced

start clinical trials. These include Moderna’s mRNA
COVID-19 vaccine and CanSino’s non-replicating adeno­
virus type-5 (Ad5) vectored COVID-19 vaccine, which both
entered phase 1 clinical trials on March 16, 2020; Inovio
Pharmaceuticals’ DNA vaccine for COVID-19, which
entered trials on April 3, 2020; three inactive COVID-19
vaccines man­ufactured by Sinovac, Wuhan Institute of
Biological Products, and Beijing Institute of Biological
Products entered clinical trials in April, 2020, successively;
University of Oxford’s non-replicating chimpanzee adeno­
virus vectored vaccine ChAdOx1 nCoV-19, and BioNTech’smRNA COVID-19 vaccine also started trials in recent
months.
Here, we report the preliminary assessment at 28 days
post-vaccination of the safety, tolerability, and immu­
nogenicity of CanSino’s non-replicating Ad5 vectored
COVID-19 vaccine in healthy adults in China.

MethodStudy design and participants
We did a single-centre, open-label, non-randomised,dose-escalation phase 1 trial of an Ad5 vectored COVID-19
vaccine candidate in a rehabilitation centre in Wuhan,
Hubei province, China. Eligible participants were healthy
adults aged between 18 and 60 years, who did not have
SARS-CoV-2 infection, confirmed by negative results ofserum specific IgM and IgG antibodies with a commercial
SARS-CoV-2 rapid test kit (Jinwofu, Beijing, China),
negative nucleic acid for SARS-CoV-2 in pharyngeal
swabs or sputum and anal swabs detected with a nucleic
acid diagnostic kit (PCR-fluorescence probing, Sansure
Biotech, Changsha, China), and a clear chest CT image
with no evidence of lesions in the lungs at the time of1846

both speciﬁc antibody and T-cell responses in most participants.
Rapid specific T-cell responses were noted at day 14 and specifichumoral responses against severe acute respiratory syndrome
coronavirus 2 peaked at day 28 post-vaccination. Although wefound that the high dose vaccine tended to be moreimmunogenic than the middle dose and low dose vaccines,
it was also associated with a higher reactogenicity. Severe fever,fatigue, dyspnoea, muscle pain, and joint pain were reported
in some of the recipients in the high dose group.
Implications of all the available evidence
Many vaccine candidates are in rapid development, including
recombinant-protein based vaccines, replicating or
non-replicating viral vector-based vaccines, DNA vaccines, andmRNA vaccines (which mostly have focused on the spikeglycoprotein or receptor binding domain), live attenuatedvaccines, and inactivated virus vaccines. All of these vaccine
platforms have advantages and disadvantages, and it is too
soon to predict which will be more successful. Our study
suggests that there is potential for further investigation of theAd5 vectored COVID-19 vaccine for prevention of COVID-19.

screening. Exclusion criteria were a history of seizures ormental illness; allergy to any ingredient included in thevaccine; acute febrile disease on the day of enrolment;
receipt of any blood products in the past 4 months;
receipt of any research medicines or vaccine in the pastmonth; and being unable to comply with the studyschedule. Further details are outlined in the protocol.10
Participants were sequentially enrolled to receive a single
intramuscular injection in a dose-escalating manner:the first group of participants were allocated to receive
the low dose, followed up for a minimum of 3 days before
proceeding to recruit further participants to receive themiddle dose, and then after safety observation for 3 days,
the last group of participants were recruited and allocatedto receive the high dose. The administration of higher
dose injections and new enrolment were paused if any
criteria for pausing dose escalation were met.
The protocol and informed consent were approved by
the institutional review board of the Jiangsu Provincial
Center of Disease Control and Prevention. Written
informed consent from all participants was obtained
before screening. This study was undertaken by Jiangsu
Provincial Center for Disease Control and Prevention,
Hubei Provincial Center for Disease Control andPrevention, and Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology inaccordance with the Declaration of Helsinki and Good
Clinical Practice.

Procedures
The Ad5 vectored COVID-19 vaccine was developed by
Beijing Institute of Biotechnology (Beijing, China) and
CanSino Biologics (Tianjin, China). The vaccine is a
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Articles

replication defective Ad5 vectored vaccine expressing
the spike glycoprotein of SARS-CoV-2. We cloned an
optimised full-length spike gene based on Wuhan-Hu-1(GenBank accession number YP_009724390) with thetissue plasminogen activator signal peptide gene into an
E1 and E3 deleted Ad5 vector, and constructed the Ad5vectored COVID-19 vaccine using the Admax systemfrom Microbix Biosystem (Toronto, ON, Canada). The
Ad5 vectored COVID-19 vaccine was manufactured as aliquid for­mulation containing 5 × 10¹⁰ viral particles per
0·5 mL in a vial.
A single shot was allocated intramuscularly in the armof the participants in the low dose group, with one vialof the Ad5 vectored COVID-19 vaccine (5 × 10¹⁰ viralparticles per 0·5 mL). The participants in the middle
dose group received one shot intramuscularly in the armwith two vials of the Ad5 vectored COVID-19 vaccine(1 × 10¹¹ viral particles per mL). Participants in the highdose group received a double-shot regimen with one vialof the Ad5 vectored COVID-19 vaccine in one arm andtwo vials of the Ad5 vectored COVID-19 vaccine in theother arm (1·5 × 10¹¹ viral particles per 1·5 mL).
Adverse events were self-reported by the participants,
but verified by investigators daily during the first 14 days
after vaccination. Subsequently, adverse events wererecorded by the participants on diary cards in thefollowing weeks. Laboratory safety tests including whiteblood cell count, lymphocyte count, neutrophils, plate­lets, haemoglobin, alanine aminotransferase, aspartate
aminotransferase, total bilirubin, fasting blood glucose,and creatinine were measured on day 7 to assess any
toxic effects post-vaccination. We graded adverse eventsand abnormal changes in laboratory tests according to
the scale issued by the China State Food and Drug
Administration (version 2019).11
Blood samples were taken from participants for serology
tests at the scheduled site visits before the vaccination, andon days 14 and 28 after the vaccination. We assessedbinding antibody responses against the receptor binding
domain (RBD) and spike glycoprotein with ELISA kits
manufactured by Beijing Wantai BioPharm (Beijing,
China). A dilution of 1:40 was the positivity cutoff value
for ELISA. We also measured the neutralising anti­
body responses induced by vaccination using both live
SARS-CoV-2 virus neutra­lisation (virus strain SARS-CoV-2/
human/CHN/Wuhan_IME-BJ01/2020, GenBank number
MT291831.1) and pseudovirus neutralisation tests (avesicular stoma­titis virus pseudovirus system expressing
the spike glycoprotein).12 Peripheral blood mononuclear
cells were isolated from whole blood before the vaccination
and at days 14 and 28 post-vaccination. Specific T-cell
responses were quantified with an interferon (IFN)γ enzyme-linked immunospot (ELISpot) assay using fresh
peripheral blood mononuclear cells stimulated withoverlapping spike glycoprotein peptide pools for about
12–24 h before detection, and expressed as the number ofspot-forming cells per 100 000 cells. All measurements
www.thelancet.com Vol 395 June 13, 2020

were subtracted from the unstimulated control values, andminus values were corrected to zero. The results wereconsidered positive if there was at least a two-times
increase in the number of IFNγ secreting T cells postvaccination. We also assessed the CD4+ and CD8+ T-cell
responses to vaccination according to the secretion of
IFNγ, interleukin-2 (IL-2), and tumour necrosis factor α(TNFα), which were measured by intracellular cytokine
staining assays in peripheral blood mononuclear cells after
the stimulation with overlapping spike glyco­
proteinpeptide pools for about 6 h and detected by flow cytometry.
Similar methods to measure T-cell responses by intra­
cellular cytokine staining assays have been reported
previously.13,14 The pre-vaccination and post-vaccination
anti-Ad5 neutralising antibody titres were detected with aserum neutralisation assay.15

Outcomes
We analysed all outcomes in the intention-to-treat cohort.
The primary endpoint for safety was the occurrence ofadverse reactions within 7 days after the vaccination. Any
abnormal changes in laboratory measures at 7 days postvaccination, and adverse events within 28 days across thetreatment groups were also analysed as secondary safety
endpoints. The specific ELISA antibody titres to RBD andthe spike glycoprotein, and the neutralising antibody
amounts against live SARS-CoV-2 and a pseudovirus were
measured as humoral immu­
nogenicity endpoints. We
defined a positive antibody response (seroconversion) as
at least a four-fold increase in post-vaccination titre frombaseline. ELISpot IFNγ and positive T-cell responses
measured by intracellular cytokine staining assays werecompared across the groups as endpoints for cell-mediatedresponses. Stratified ana­lyses of the immune responses
were done based on the pre-existing Ad5 neutralising
antibody titres among the participants as low or negative(≤1:200) or high (>1:200).

Statistical analysis
The sample size was not determined on the basis ofstatistical power calculations; however, a minimum
sample size of 20–30 participants for a pilot vaccine trial
has been recommended by the National Medical Products
Administration, China. We assessed the number andproportion of participants with adverse reactions postvaccination and compared safety profiles across thedose groups. The antibodies against SARS-CoV-2 were
presented as geometric mean titres with 95% CIs and thecellular responses were shown as a proportion of positive
responders. We used the χ² test or Fisher’s exact test to
analyse categorical data, ANOVA to analyse the logtransformed antibody titres, and Wilcoxon rank-sum testfor data that were not normally distributed. When theoverall difference across the three groups was significant,pairwise comparisons were made and the differences
between groups were estimated with 95% CIs. Multi­
variable analysis was used to establish the possible effects
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Articleson the immunogenicity and safety pro­file of the vaccine
candidates. Hypothesis testing was two-sided with anα value of 0·05. Statistical analyses were done by a statis­
tician using SAS (version 9.4) or GraphPad Prism 8.0.1.
SPICE (version 6.0) was used for the analysis of datafrom multicolour flow cytometry experiments.
An independent data and safety monitoring committee,
with one independent statistician, one clinician, and one
epidemiologist, was established before the start of thetrial. Safety data for the first 3 days post-vaccination wereassessed and reviewed by the committee to ensure that
there was sufficient holding time between dose esca­
lation. This study is registered with ClinicalTrials.gov,
NCT04313127.

Role of the funding source
The sponsors of the study participated in study design,
but had no role in data collection, data analysis, datainterpretation, or writing of the report. All authors
had full access to all the data in the study and had
final responsibility for the decision to submit for
publication.

Results

See Online for appendix

Between March 16 and March 27, 2020, we screened
195 individuals for eligibility. Of them, 108 were sequen­
tially enrolled and assigned to receive the low dose
(n=36 [33%]), middle dose (n=36 [33%]), or high dose
(n=36 [33%]) of the Ad5 vectored COVID-19 vaccine(appendix p 2). All participants completed the vaccinationand the scheduled visits within 28 days. Baseline
Low dosegroup (n=36)

Middle dosegroup (n=36)

High dosegroup (n=36)

Age, years
18–29

9 (25%)

12 (33%)10 (28%)30–39

13 (36%)

14 (39%)15 (42%)40–49

8 (22%)

3 (8%)

7 (19%)

50–60

6 (17%)

7 (19%)

4 (11%)

37·2 (10·7)

36·3 (11·5)

35·5 (10·1)Male

18 (50%)19 (53%)18 (50%)Female18 (50%)17 (47%)18 (50%)23·3 (2·7)

23·9 (2·7)

24·1 (3·1)

Mean age, years
Sex

Mean body-massindex, kg/m²
Underlying diseases*
Yes

1 (3%)

2 (6%)

4 (11%)

No

35 (97%)34 (94%)32 (89%)Pre-existing adenovirus type-5 neutralising antibody
Mean GMT

168·9 (13·9)

149·5 (10·5)

115·0 (13·4)≤200, titre

16 (44%)17 (47%)20 (56%)>200, titre

20 (56%)19 (53%)16 (44%)

Data are n (%) or mean (SD). GMT=geometric mean titre. *Seven participants had
hypertension, chronic bronchitis, gout, or were a carrier of hepatitis B virus.

Table 1: Baseline characteristics

1848

characteristics of the participants were similar across thetreatment groups (table 1).
87 (81%) of 108 participants reported at least one adverse
reaction within the first 7 days after the vaccination:
30 (83%) in the low dose group, 30 (83%) in the middle
dose group, and 27 (75%) in the high dose group (table 2).
No significant difference in the overall number of adverse
reactions across the treatment groups was observed. The
most common injection site adverse reaction was pain,
which was reported in 58 (54%) vaccine recipients. Pain
was reported in 17 (47%) participants in the low dosegroup, 20 (56%) participants in the middle dose group,and 21 (58%) participants in the high dose group. The
most commonly reported systematic adverse reactions
overall were fever (50 [46%]), fatigue (47 [44%]), headache(42 [39%]), and muscle pain (18 [17%]). Fever was reported
in 15 (42%) participants in the low dose group,
15 (42%) participants in the middle dose group, and20 (56%) participants in the high dose group. Headache
was reported in 14 (39%) participants in the low dosegroup, 11 (31%) participants in the middle dose group, and
17 (47%) participants in the high dose group. Muscle pain
was reported in seven (19%) participants in the low dosegroup, three (8%) participants in the middle dose group,and eight (22%) participants in the high dose group. Most
adverse reactions were mild or moderate in severity.
Nine participants (two [6%] in the low dose group,
two [6%] in the middle dose group, and five [14%] in thehigh dose group) had an episode of severe fever (grade 3)
with axillary temperature greater than 38·5°C. Of them,one (3%) from the high dose group reported severe feveralong with severe symptoms of fatigue, dyspnoea, andmuscle pain. One participant in the high dose groupreported severe fatigue and joint pain (appendix p 3).
These reactions occurred within 24 h post-vaccination, andpersisted for no more than 48 h. We found no significant
difference in the incidences of adverse reactions or overall
adverse events among the dose groups. High pre-existingAd5 immunity (titre of >1:200 vs ≤1:200) was associatedwith significantly fewer occurrences of fever post-vacci­
nation (odds ratio 0·3, 95% CI 0·1–0·6; appendix p 4).
No serious adverse event was reported within 28 days.
At day 7 after vaccination, nine (8%) participants had mild
to moderate total bilirubin increase, ten (9%) had alanine
aminotransferase increase, and four (4%) had fasting
hyperglycaemia (appendix p 5), but no instances wereconsidered as clinically significant.
Rapid binding antibody responses to RBD were observedin all three dose groups from day 14 (table 3). At day 28, therecipients in the high dose group tended to have a higher
binding antibody geometric mean titre of 1445·8 (95% CI
935·5–2234·5), followed by 806·0 (528·2–1229·9) in the
middle dose group, and 615·8 (405·4–935·5) in the lowdose group (high dose vs low dose 1611·5, 531·5–2691·5).
At least a four-fold increase of anti-RBD antibodies wasnoted in 35 (97%) of 36 participants in the low dose group,
34 (94%) of 36 in the middle dose group, and 36 (100%) of
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36 in the high dose group. Neutralising antibodies against
live SARS-CoV-2 were all negative at day 0, and increased
moderately at day 14, peaking at 28 days post-vaccination.
Neutralising antibody titre with a geometric mean titre of
34·0 (95% CI 22·6–50·1) was noted in the high dosegroup, which was significantly higher compared with
16·2 (10·4–25·2) in the middle dose group and 14·5
(9·6–21·8) in the low dose group, with an estimateddifference of 27·7 (1·0–54·4) between the high dose groupand the middle dose group and 33·2 (6·5–59·9) between
the high dose group and the low dose group at day 28.
Meanwhile, 18 (50%) participants in the low dose group,
18 (50%) in the middle dose group, and 27 (75%) in thehigh dose group had at least a four-fold increase inneutralising antibody titres by day 28. Similar patterns of
the binding antibody to spike glycoprotein and neutra­l­ising antibody titre to pseudovirus post-vaccination acrossthe dose groups were also noted (appendix p 6). The
association between the ELISA antibodies to RBD andneutralising antibody titres against live virus showed amoderate positive correlation of 0·749, and that between
the ELISA antibodies to spike glycoprotein and neutra­l­ising antibody titres against live virus was 0·753, at thepeak antibody response (p<0·0001). The neutralising
antibody titres measured using a pseudovirus were also
correlated well with those measured by live SARS-CoV-2(appendix p 7).
Before vaccination, 20 (56%) participants in the lowdose group, 19 (53%) participants in the middle dosegroup, and 16 (44%) participants in the high dose group
had a high pre-existing Ad5 neutralising antibody titre(>1:200). Only five (25%) participants of 20 in the lowdose group, seven (37%) participants of 19 in the middle
dose group, and ten (63%) participants of 16 in the highdose group, who had high pre-existing Ad5 immunity,
had at least a four-fold increase in neutralising antibody
titre at day 28 post-vaccination (appendix pp 8–10).
Multivariable analysis showed that high pre-existingAd5 neutralising antibody titres compromised theseroconversion of neu­tralising antibody post-vaccination,
regardless of the vaccine doses, and recipients aged
45–60 years seemed to have lower seroconversion ofneutralising antibody compared with the younger reci­
pients (appendix p 11). The Ad5 neutralising anti­bodies
were significantly boosted post-vaccination (appendix
p 12).
ELISpot responses at baseline were undetectable withspot-forming cells below the level of detection of the assay
in all participants, but peaked at day 14 post-vaccination.
The proportions of positive responders ranged from
83–97% across the dose groups, with a mean number ofspot-forming cells per 100 000 cells of 20·8 (95% CI
12·7–34·0) in the low dose group, 40·8 (27·6–60·3) in the
middle dose group, and 58·0 (39·1–85·9) in the highdose group (figure 1). T-cell responses in the high dosegroup were significantly higher than that in the low dosegroup (p<0·0010), but not significant compared with thatwww.thelancet.com Vol 395 June 13, 2020

Low dosegroup (n=36)

Middle dosegroup (n=36)

High dosegroup (n=36)

Total(N=108)All adverse reactions within 0–7 daysAny

30 (83%)30 (83%)27 (75%)87 (81%)

Grade 3

2 (6%)

2 (6%)

6 (17%)

10 (9%)
58 (54%)

Injection site adverse reactions within 0–7 daysPain

17 (47%)20 (56%)21 (58%)Induration

2 (6%)

1 (3%)

1 (3%)

4 (4%)

Redness

2 (6%)

1 (3%)

1 (3%)

4 (4%)

Swelling

4 (11%)

4 (11%)0

8 (7%)Itch

2 (6%)

3 (8%)0

5 (5%)

Muscular weakness

00

1 (3%)

1 (1%)

15 (42%)15 (42%)20 (56%)50 (46%)2 (6%)

2 (6%)

5 (14%)

9 (8%)Headache14 (39%)11 (31%)17 (47%)42 (39%)Fatigue

17 (47%)14 (39%)16 (44%)47 (44%)00

2 (6%)

2 (2%)

Vomiting

1 (3%)0

1 (3%)

2 (2%)

Diarrhoea

3 (8%)

4 (11%)

5 (14%)

12 (11%)18 (17%)

Systemic adverse reactions within 0–7 days
Fever
Grade 3 fever

Grade 3 fatigue

Muscle pain

7 (19%)

3 (8%)

8 (22%)00

1 (3%)

1 (1%)

2 (6%)

2 (6%)

5 (14%)

9 (8%)00

1 (3%)

1 (1%)

1 (3%)

3 (8%)

4 (11%)

8 (7%)Cough

1 (3%)

2 (6%)

3 (8%)

6 (6%)

Nausea2 (6%)

1 (3%)

3 (8%)

6 (6%)

Grade 3 muscle pain
Joint pain
Grade 3 joint pain
Throat pain

Functional GI disorder

1 (3%)00

1 (1%)

Dyspnoea00

2 (6%)

2 (2%)

Grade 3 dyspnoea

00

1 (3%)

1 (1%)

6 (17%)

5 (14%)

6 (17%)

17 (16%)

Dizziness

1 (3%)0

1 (3%)

2 (2%)

Mucosal abnormality00

1 (3%)

1 (1%)

Pruritus

1 (3%)

1 (3%)

1 (3%)

3 (3%)

Appetite impaired

Overall adverse events within 0–28 daysAny

31 (86%)30 (83%)27 (75%)88 (81%)

Grade 3

2 (6%)

2 (6%)

6 (17%)

10 (9%)

Data are n (%). Any refers to all the participants with any grade adverse reactions or events. Adverse reactions andevents were graded according to the scale issued by the China State Food and Drug Administration. Grade 3=severe(ie, prevented activity). GI=gastrointestinal.

Table 2: Adverse reactions within 7 days and overall adverse events within 28 days after vaccinationin the middle dose group. A slight decrease of the T-cell
responses across the dose groups was noted at day 28.
High levels of baseline Ad5 neutralising antibody titre
reduced the peak of post-vaccination T-cell responses inall the dose groups, particularly for the low dose group.
Despite the effect of high pre-existing Ad5 immunity,
positive responders were identified in 15 (75%) of
20 participants in the low dose group, 18 (95%) of
19 participants in the middle dose group, and 15 (94%)
of 16 participants in the high dose group at day 14, and
12 (60%) of 20 participants in the low dose group, 16 (84%)
of 19 participants in the middle dose group, and 16 (100%)of 16 participants in the high dose group at day 28.
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Day 14

Day 28Low dosegroup (n=36)

Middle dosegroup (n=36)

High dosegroup (n=36)

p valueLow dosegroup (n=36)

Middle dosegroup (n=36)

High dosegroup (n=36)

p value

ELISA antibodies to the receptor binding domain
GMT

76·5
(44·3–132·0)

91·2(55·9–148·7)

132·6(80·7–218·0)

0·29

615·8
(405·4–935·5)806·0(528·2–1229·9)

1445·8(935·5–2234·5)

0·016≥4-fold increase

16 (44%)18 (50%)22 (61%)0·35

35 (97%)34 (94%)36 (100%)

0·77

Neutralising antibodies to live SARS-CoV-2
GMT

8·2(5·8–11·5)

9·6(6·6–14·1)

12·7(8·5–19·0)

0·24

14·5
(9·6–21·8)

16·2(10·4–25·2)

34·0(22·6–50·1)

0·0082≥4-fold increase

10 (28%)11 (31%)15 (42%)0·42

18 (50%)18 (50%)27 (75%)0·046

Data are mean (95% CI) or n (%). The p values are the result of comparison across the three dose groups. If the difference was significant across the three groups, the differences
between groups were estimated with 95% CIs. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. GMT=geometric mean titre.

Table 3: Specific antibody responses to the receptor binding domain, and neutralising antibodies to live SARS-CoV-2

A OverallPre-existing Ad5 neutralising antibody titres≤1:200p=0·021p=0·0029

IFNγ expressing cells per 105 PBMCs

p<0·0010
103

p=0·012

p=0·039
Low dose
Middle doseHigh dose

p=0·026

p=0·021

102

101

100

Day 0

Day 14
Time since vaccination

Day 28B Overall
Proportion of positive responders (%)

Pre-existing Ad5 neutralising antibody titres>1:200p=0·037

100

Day 0

Day 14
Day 28Time since vaccination

Pre-existing Ad5 neutralising antibody titres≤1:200

Low dose

Middle dose

Day 0

Day 28Day 14
Time since vaccination

Pre-existing Ad5 neutralising antibody titres>1:200High dose

80
60
40
20
0

Day 14

Day 28Time since vaccination

Day 14

Day 28Time since vaccination

Day 14

Day 28Time since vaccination

Figure 1: Speciﬁc T-cell response measured by ELISpot(A) The number of specific T cells with secretion of IFNγ at days 0, 14, and 28 in all participants, and stratified by pre-existing Ad5 neutralising antibody titres.
(B) The proportion of positive ELISpot responders at days 14 and 28 post-vaccination in all participants, and stratified by pre-existing Ad5 neutralising antibody
titres. IFN=interferon. PBMCs=peripheral blood mononuclear cells. Ad5=adenovirus type-5. ELISpot=enzyme-linked immunospot.

IFNγ was detected from CD4+ and CD8+ T cells after thevaccination at day 14 and 28, in all dose groups (figure 2,
appendix p 13). The TNFα expression from CD4+ T cells
tended to be significantly lower in the low dose group thanthat in the high dose (p<0·0001) and middle dose groups(p=0·0032), on day 14. The TNFα expression from CD8+
T cells showed an overall p value of less than 0·0001 acrossthe three groups on day 14. And the TNFα expression
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from CD8+ T cells tended to be higher in the high dosegroup than that in both the middle dose group (p=0·016)
and the low dose group (p<0·0001). The p values arefor the pairwise comparisons between groups. Amountsof IL-2 detected from CD4+ T cells were higher than that
detected from CD8+ cells. The proportions of polyfunc­
tional phenotypes detected from memory CD4+ T cells
were higher than those from CD8+ T cells. Higher
www.thelancet.com Vol 395 June 13, 2020

ArticlesA
p<0·0001
p<0·0001
Low dose
Middle doseHigh dose

p=0·0032

10–1

10–2

10–3

100
CD4+ IL-2 proportion (%)

100
CD4+ TNFα proportion (%)

CD4+ IFNγ proportion (%)

100

10–1

10–2

10–3

10–1

10–2

10–3

B
p<0·0001
p<0·0001

10–2

Day 0

Day 14

Day 28Time since vaccination

100
CD8+ IL-2 proportion (%)

CD8+ TNFα proportion (%)

CD8+ IFNγ proportion (%)

10–1

10–3

p=0·016 p=0·016

100

100

10–1

10–2

10–3

Day 0

Day 14

Day 28

10–1

10–2

10–3

Day 0

Time since vaccination

Day 14

Day 28Time since vaccination

C CD8+ T cells, day 14
Low dose

Middle doseHigh dose

Middle doseHigh doseCD4+ T cells, day 14
Low dose

IFNγ
+
+
+
+
–
–
–

TNFα
+
+
–
–
+
+
–

IL-2
+
–
+
–
+
–
+

www.thelancet.com Vol 395 June 13, 2020

Figure 2: Flow cytometry
with intracellular cytokine
staining before and after
vaccination(A) Percentage of cells secreting
IFNγ, TNFα, and IL-2 from CD4+
T cells. (B) Percentage of cells
secreting IFNγ, TNFα, and IL-2
from CD8+ T cells. (C) The
proportion of CD4+ T cells andCD8+ T cells producing any
combination of IFNγ, TNFα,and IL-2. The analyses are for
108 participants, with 36 ineach dose group.
IFN=interferon. TNF=tumour
necrosis factor. IL=interleukin.

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proportions of polyfunctional phenotypes were noted withthe higher vaccine doses. We also noted that pre-existingAd5 neutralising antibody had a negative effect on thepattern of T-cell responses (appendix pp 14–16). A post-hoc
analysis showed that 28 (78%) participants in the lowdose group, 33 (92%) participants in the middle dosegroup, and 36 (100%) participants in the high dose group
showed either positive T-cell responses to spike glycopro­tein or seroconversion of neutralising antibody to live
SARS-CoV-2, at day 28 post-vaccination (appendix p 17).
To exclude any possible SARS-CoV-2 exposure during
the study period, we tested the serum antibodies tonucleocapsid protein of SARS-CoV-2 in participants at
day 28 using a special IgG/IgM rapid test kit (Vazyme
Biotech, number CD101, Nanjing, China), but none ofthe participants were positive.

Discussion
To our knowledge, this is the first report on a first-inhuman clinical trial of a novel Ad5 vectored COVID-19
vaccine. The Ad5 vectored COVID-19 vaccine wastolerated in healthy adults in all three dose groups. The
most common adverse reactions were fever, fatigue,headache, and muscle pain with no significant differencein the incidence of adverse reactions across the groups.
Most adverse events reported were mild or moderate inseverity. We noticed a higher reactogenicity profile of thehigh dose at 1·5 × 10¹¹ viral particles, presenting as severe
fever, fatigue, muscle pain, or joint pain, which might beassociated with viraemia caused by Ad5 vector infection.
However, the severe adverse reactions were transientand self-limiting. Additionally, no abnormal changes inlaboratory measurements were clinically significant or
considered to be related to the vaccine. The profile ofadverse events reported in this trial is similar to thatof another Ad5 vector-based Ebola vaccine expressing
glycoprotein.16 To accelerate the process of clinicalevaluation of the candidate COVID-19 vaccine, we
selected doses for the phase 2 study mainly on the basisof the safety profile of the candidate vaccines shownin the participants within 7 days and 14 days postvaccination. We chose the low dose (5 × 10¹⁰ viral particles)
and middle dose (1 × 10¹¹ viral particles) to be further
assessed in a phase 2 clinical trial.
The Ad5 vectored COVID-19 vaccine was immunogenic,
inducing humoral and T-cell responses rapidly in mostparticipants. Onset of detectable immune responses was
rapid, with T-cell responses peaking at day 14 after
vaccination and antibodies peaking at day 28. The
antibody response to the vaccine in the high dose groupwas slightly greater than that in the middle dose and low
dose groups. A single dose of Ad5 vectored COVID-19
vaccine was able to elicit a four-fold increase in binding
antibodies to RBD in 94–100% of participants, and afour-fold increase to live virus in 50–75% of participants.
Despite differences in magnitudes of the antibodies
measured through different methods, there was a strong1852

positive correlation between binding antibodies andneutralising antibody titres to the live virus. Highproportions of participants with positive T-cell responses
were noted across the all dose groups post-vaccination.
The activation of both CD4+ T cells and CD8+ T cells wasobserved in vaccine recipients, particularly for antigenspecific CD4+ T cells and CD8+ T cells. However, both thespecific antibody response and T-cell response inducedby vaccination were partly diminished by the presence ofhigh pre-existing anti-Ad5 immunity.
Currently, correlates of protection for a vaccine against
COVID-19 are unknown, and the roles of the specificantibodies or T cells in building effective protection are
not yet defined. Therefore, we are unable to predict theprotection of the Ad5 vectored COVID-19 vaccine on
the basis of the vaccine-elicited immune responses inthis study. However, previous studies investigating SARS
and Middle East respiratory syndrome (MERS) foundthat the increases in specific antibodies were temporary,and declined quickly in patients after recovery, whereas
the specific CD4+ and CD8+ T-cell responses played anessential role in immunity.17,18 A similar rapid decline of
the speciﬁc antibody amounts in patients with COVID-19
after recovery was also noted,19,20 suggesting that both
speciﬁc cellular and humoral immunity are potentially
important for a successful COVID-19 vaccine. Here, weonly report the data within 28 days after the vaccination,
but we are going to follow up the vaccine recipients for atleast 6 months, so more data will be obtained.
This study was done in Wuhan, Hubei province, which
was the centre of the COVID-19 epidemic in China.21
People living in the city of Wuhan had a much higher
risk of SARS-CoV-2 infection compared with those living
in other cities outside of Hubei province, even thoughwhen we initiated this trial, the city had already begun
lockdown and implemented mandatory home isolation
for residents. Therefore, we did serological screening,
nucleic acid testing, and chest CT to exclude partici­
pants who had been previously exposed to SARS-CoV-2
during recruitment. In addition, we arranged for allparticipants in our study to stay in a designated hotel for
14 days post-vaccination. This arrangement facilitated theobservation of adverse events after the immunisation of
the participants, and reduced the risk of SARS-CoV-2
exposure during the following 2 weeks. These measures
allowed the study to be done successfully without
interference by the circulation of SARS-CoV-2, which isespecially important in the absence of a placebo control.
Interpretation of the results of this study is limited by
the small size of the cohort, the short duration of followup, and the absence of a randomised control group. As it
was a first-in-human study of the Ad5 vectored COVID-19
vaccine, it was not designed to measure the vaccine
efficacy. However, in preclinical studies, seven out ofeight ferrets were protected from having detectable virus
copies when challenged by SARS-CoV-2 through nasal
dripping 21 days after immunisation with the vaccine,www.thelancet.com Vol 395 June 13, 2020

Articles

whereas only one out of eight ferrets in the control group
was negative for virus copies (Wei C, unpublished). We
aimed to evaluate the safety and tolerability of thecandidate vac­cine in healthy adults, with no interference
by underlying diseases or medicines. However, results ofour study indicated that older age could have a negative
effect on the vaccine-elicited responses to SARS-CoV-2.
In this trial, no participants were older than 60 years
and only 16% of the participants were older than 50 years,
providing limited information on the capability ofgenerating a potent cellular and humoral response in theolder population. Since age has also been identified as anindependent risk factor for severe disease associated with
SARS-CoV-2 infection,4,22 and there is a possibility that aneven lower immune response might be found in theolder population, we are going to include participants
who are older than 60 years in the phase 2 study
considering this population as an important target
population for a COVID-19 vaccine. Additionally,
experience with vaccine candidates for SARS and MERS
have raised concerns about the antibody-dependentenhancement in participants who are infected witha circulating SARS-CoV-2 post-vaccination.23 However,this study was not statistically powered to measure any
safety outcome, especially for the con­cerns around
immunopathology and antibody-dependent enhance­
ment events associated with the full-length spikeglycoprotein vaccine antigen.24 Our study found that thepre-existing Ad5 immunity could slow down the rapid
immune responses to SARS-CoV-2 and also lower the
peak of the responses, particularly for humoral immunity.
The high pre-existing Ad5 immunity might also have anegative effect on the persistence of the vaccine-elicitedimmune responses. In previous studies, heterologous
prime-boost combinations or homologous prime-boost
regimens with Ad5 vectored vaccines were shown to beable to induce more strong and durable immunogenic
responses in populations with high pre-existing Ad5
immunity.25–27 Nevertheless, limited infor­mation is avail­able for the effects of multiple doses of the candidate Ad5vectored COVID-19 vaccine in humans, which warrants
further investigation.
Over the past decade, the vaccine industry and clinicalresearch centres have been asked to provide urgent
responses to epidemics of emerging infectious diseases,
such as H1N1 influenza, Ebola virus, Zika, MERS, andnow SARS-CoV-2.23 The risk of COVID-19 caused by
SARS-CoV-2 is ongoing, making the need for effective
vaccines even more urgent.28 We started the develop­
ment of this candidate vaccine in January, 2020, when
SARS-CoV-2 was first isolated and sequenced. The fulllength spike glycoprotein was selected as the vaccine
antigen, mainly on the basis of previous experience
with SARS and MERS vaccines. Previous findings
suggested that those vaccines expressing full-length
spike glycoprotein can induce good immune responses
and protective efficacy.29 Although the RBD comprises
www.thelancet.com Vol 395 June 13, 2020

the crit­ical neutralising domains for the coronaviruses,
the neutralising epitopes located outside the RBD werealso identified.30,31 The full-length spike was chosen in
most of the viral vectored, mRNA, or DNA COVID-19
vaccines in development.9 The Ad5 vector vaccine
platform is highly efficient and well established as a
vaccine antigen delivery system. In addition to ourcandidate Ad5 vectored COVID-19 vaccine, there areseveral other Ad5-based vaccines against COVID-19
listed in the WHO draft landscape of COVID-19 can­didate vaccines, including Ad5 S (GREVAXTM platform)in the USA, and Oral Ad5 S (Stabilitech Biopharma)in the UK.9 However, aside from pre-existing anti-Ad5
immunity, there is a concern about the increased riskof HIV-1 acquisition associated with Ad5 activated CD4+
T cells.32 Although the association between HIV-1 acqui­sition risk and Ad5 vectored vaccine is controversial andits mechanism is unclear, the potential risks should betaken into account in studies with this viral vector
delivery platform. We plan to monitor the participants in
our upcoming phase 2 and phase 3 studies to assess theindication for any such acquisition.
In conclusion, we found that the Ad5 vectored COVID-19
vaccine is tolerable and immunogenic in healthy adults.
Specific humoral responses against SARS-CoV-2 peakedat day 28 post-vaccination, and rapid, specific T-cell
responses were noted from day 14 after one shot ofthe vaccine. There is potential for further investigation of
the Ad5 vectored COVID-19 vaccine for the control of the
COVID-19 outbreak. An ongoing phase 2 trial in China(NCT04341389) will provide more information on thesafety and immunogenicity of the Ad5 vectored COVID-19
vaccine.
Contributors
F-CZ and Y-HL were co-first authors. F-CZ, WW, and WC were joint
corresponding authors. F-CZ is the principal investigator of this trial.
X-HG and WW worked as co-principal investigators of this trial.
F-CZ, WC, X-HG, L-HH, Y-HL, W-JW, J-XL, and S-YJ designed the trial
and the study protocol. J-XL drafted the manuscript. WC contributed to
critical review and revision of the manuscript. F-CZ, W-JW, J-XL,
and S-YJ contributed to the data interpretation and revision of themanuscript. X-WW was responsible for the statistical analysis. J-JX and
B-SW contributed to study supervision. WW, W-JW, ZW, LeW, S-YJ,
and H-DJ led and participated in the site work, including therecruitment, follow-up, and data collection. Y-HL, TJ, YH, LiW, and S-BX
were responsible for laboratory analyses. W-JW, J-XL, and S-YJ
contributed to the literature search. J-BG and S-PW monitored the trial.
Declaration of interests
WC reports grants from the National Key R&D Program of China(2020YFC10841400), and grants from the National Science andTechnology Major Project (2016ZX10004001, 2018ZX09201005). J-BG isan employee of CanSino Biologics. All other authors declare nocompeting interests.
Data sharing
We support data sharing of the individual participant data. The individual
participant data that underlie the results reported in this Article, after
de-identiﬁcation (text, tables, figures, and appendix) will be shared.
Individual participant data will be available beginning 3 months andending 1 year after publication. Supporting clinical documents including
the study protocol, statistical analysis plan, and the informed consent
form will be available immediately following publication for at least
1 year. Researchers who provide a scientifically sound proposal will be

For supporting clinicaldocuments see http://www.
jscdc.cn/jkfw/kygz/202005/
t20200517_68894.html

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allowed access to the individual participant data. Proposals should bedirected to jszfc@vip.sina.com or cw0226@foxmail.com. These
proposals will be reviewed and approved by the sponsor, investigator,and collaborators on the basis of scientific merit. To gain access,data requesters will need to sign a data access agreement.
Acknowledgments
We thank Peng Deng, Qiong Li, and Xiaoai Qian from Hubei Provincial
Center for Disease Control and Prevention for participant recruitment
and sample collection. We thank Miao Xu and Jingjing Liu from the
National Institute for Food and Drug Control (China), Yansong Sun,
Sen Zhang, and Yuchang Li from the Beijing Institute of Microbiology
and Epidemiology, and Feng Wang, Hongyan Hou, Hanxiong Guan,
and Bo Liu from Tongji Hospital for laboratory analysis. We thank
Ke Zhang from the Academy of Military Medical Sciences, Kun Liu fromthe General Hospital of Central Theater Command, and Changlong Fu
from Wuhan Rest Center, Chinese People’s Armed Police Force for the
management of the clinical trial site.
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