Safety and Immunogenicity of a DNA SARS-CoV-2 vaccine (ZyCoV-D): Results of an open-label, non-randomized phase I part of phase I/II clinical study by intradermal route in healthy subjects in India

Abstract

Background:
ZyCoV-D is a DNA vaccine candidate, which comprises a plasmid DNA carrying spike-S gene of SARS-CoV-2 virus along with gene coding for signal peptide. The spike(S) region includes the receptor-binding domain (RBD), which binds to the human angiotensin converting Enzyme (ACE)-2 receptor and mediates the entry of virus inside the cell.

Methods:
We conducted a single-center, open-label, non-randomized, Phase 1 trial in India between July 2020 and October 2020. Healthy adults aged between 18 and 55 years were sequentially enrolled and allocated to one of four treatment arms in a dose escalation manner. Three doses of vaccine were administered 28 days apart and each subject was followed up for 28 days post third dose to evaluate safety and immunogenicity.

Findings:
Out of 126 individuals screened for eligibility. Forty-eight subjects (mean age 34·9 years) were enrolled and vaccinated in the Phase 1 study Overall, 12/48 (25%) subjects reported at least one AE (i.e. combined solicited and unsolicited) during the study. There were no deaths or serious adverse events reported in Phase 1 of the study. The proportion of subjects who seroconverted based on IgG titers on day 84 was 4/11 (36·36%), 4/12 (33·33%), 10/10 (100·00%) and 8/10 (80·00%) in the treatment Arm 1 (1 mg: Needle), Arm 2 (1 mg: NFIS), Arm 3 (2 mg: Needle) and Arm 4 (2 mg: NFIS), respectively.

Interpretation:
ZyCoV-D vaccine is found to be safe, well-tolerated and immunogenic in the Phase 1 trial. Our findings suggest that the DNA vaccine warrants further investigation.

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Research Paper
Safety and Immunogenicity of a DNA SARS-CoV-2 vaccine (ZyCoV-D):
Results of an open-label, non-randomized phase I part of phase I/II
clinical study by intradermal route in healthy subjects in India
Taufik Momin
a
, Kevinkumar Kansagra
a,
*, Hardik Patel
a
, Sunil Sharma
a
, Bhumika Sharma
a
,
Jatin Patel
a
, Ravindra Mittal
b
, Jayesh Sanmukhani
b
, Kapil Maithal
c
, Ayan Dey
c
,
Harish Chandra
c
, Chozhavel TM Rajanathan
c
, Hari PR Pericherla
c
, Pawan Kumar
c
,
Anjali Narkhede
d
, Deven Parmar
e
a
Zydus Research Center, Clinical R & D, Cadila Healthcare Limited, Sarkhej-Bavla N. H. No. 8 A, Moraiya, Ahmedabad, Gujarat 382213, India
b
Zydus Corporate Park, Ahmedabad, India
c
Vaccine Technology Center, Cadila Healthcare Ltd, Ahmedabad, India
d
Quality Assurance and Regulatory Affairs, Cadila Healthcare Limited, Ahmedabad, India
e
Zydus Discovery DMCC, Dubai, United Arab Emirates
ARTICLE INFO
Article History:
Received 13 April 2021
Revised 21 June 2021
Accepted 23 June 2021
Available online xxx
ABSTRACT
Background: ZyCoV-D is a DNA vaccine candidate, which comprises a plasmid DNA carrying spike-S gene of
SARS-CoV-2 virus along with gene coding for signal peptide. The spike(S) region includes the receptor-bind-
ing domain (RBD), which binds to the human angiotensin converting Enzyme (ACE)-2 receptor and mediates
the entry of virus inside the cell.
Methods: We conducted a single-center, open-label, non-randomized, Phase 1 trial in India between July
2020 and October 2020. Healthy adults aged between 18 and 55 years were sequentially enrolled and allo-
cated to one of four treatment arms in a dose escalation manner. Three doses of vaccine were administered
28 days apart and each subject was followed up for 28 days post third dose to evaluate safety and
immunogenicity.
Findings: Out of 126 individuals screened for eligibility. Forty-eight subjects (mean age 34¢9 years) were
enrolled and vaccinated in the Phase 1 study Overall, 12/48 (25%) subjects reported at least one AE (i.e. com-
bined solicited and unsolicited) during the study. There were no deaths or serious adverse events reported in
Phase 1 of the study. The proportion of subjects who seroconverted based on IgG titers on day 84 was 4/11
(36¢36%), 4/12 (33¢33%), 10/10 (100¢00%) and 8/10 (80¢00%) in the treatment Arm 1 (1 mg: Needle), Arm 2
(1 mg: NFIS), Arm 3 (2 mg: Needle) and Arm 4 (2 mg: NFIS), respectively.
Interpretation: ZyCoV-D vaccine is found to be safe, well-tolerated and immunogenic in the Phase 1 trial. Our
findings suggest that the DNA vaccine warrants further investigation.
© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Keywords:
DNA
SARS-CoV-2
Vaccine
Neutralizing antibody
COVID-19
1. Introduction
Severe Acute Respiratory Syndrome coronavirus 2019 (COVID-
19), emerged in December 2019 in Wuhan, China [1]. A novel corona-
virus was identified as the etiologic agent in January 2020. The
genetic sequence of the virus became available (MN908947.3) in Jan-
uary 2020. Within months of emergence, severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) infections and the resulting
disease, COVID-19, spread worldwide. On 11 March 2020, the World
Health Organization (WHO) declared the COVID-19 outbreak a pan-
demic [2].
COVID-19 disease is rapidly transmitted from human to human,
with influenza-like symptoms ranging from mild disease to severe
disease and multi-organ failure, eventually resulting in death, espe-
cially in aged patients with co-morbid conditions [3,4]. Vaccines are
considered to the most effective treatment to control the pandemic
and help to restore the global economy [5,6]. There are currently
more than 63 COVID-19 candidate vaccines undergoing clinical trials
and more than 172 COVID-19 candidate vaccines undergoing pre-
clinical development worldwide, including mRNA vaccines, replicat-
ing or non-replicating viral vectored vaccines, DNA vaccines,
* Corresponding author.
E-mail address: kevinkumarkansagra@zyduscadila.com (K. Kansagra).
https://doi.org/10.1016/j.eclinm.2021.101020
2589-5370/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
EClinicalMedicine 38 (2021) 101020
Contents lists available at ScienceDirect
EClinicalMedicine
journal homepage: https://www.journals.elsevier.com/eclinicalmedicine

autologous dendritic cell-based vaccine, and inactive virus vaccines
[7]. The results of the Phase 1 and 2 trials of several vaccines, such as
a chimpanzee adenovirus-vectored vaccine, recombinant adenovirus
type-5 (Ad5)vectored vaccine, inactivated vaccines and mRNA vac-
cines, have been published. The results of few phase 3 trials have also
been published now [8,9].
In December 2020, Pfizer Inc. and BioNTech SE received a tempo-
rary authorization for emergency use of COVID-19 mRNA vaccine
against COVID-19 from the USFDA. The Regulatory Agencies
approved this vaccine in the UK, Canada, Saudi Arabia and Bahrain as
well [10]. Moderna, Pfizer, Johnson & Johnson (USA and EU-approved
vaccines) as well as AstraZeneca and others (EU-approved vaccines)
also received a temporary authorization for emergency use. On 16th
January, 2021, the first COVID-19 vaccine received a temporary
authorization for emergency use by the Drug Controller General of
India (DCGI) in India and many more remain in development.
As of May 16, 2021, SARS-CoV-2 had infected more than 162 mil-
lion people and killed more than 3.3 million since the start of the
pandemic worldwide [11]. The number of reported SARS-CoV-2 cases
in India till May 19, 2021 is also on an increase with ~25 million con-
firmed cases and ~ 283,248 deaths [12]. The worldwide impact of this
pandemic on human society calls for the rapid development of safe
and effective therapeutics and vaccines.
Here, we report the safety and immunogenicity of DNA based
SARS-CoV-2 vaccine data from the Phase 1 clinical trial of an ongoing,
Phase 1/2 clinical study, which commenced in July 2020 to evaluate
the impact of ZyCoV-D vaccine in preventing Covid-19 in healthy
adult subjects, 1855 years of age. The data includes evaluation of
the 1 mg and 2 mg dose levels of ZyCoV-D vaccinated healthy adult
subjects. Collection of phase 2 data on vaccine immunogenicity and
the durability of the immune response following vaccination is ongo-
ing, and those data are not reported here.
We have developed a DNA vaccine candidate for prevention of
COVID-19. It is comprised of a plasmid DNA carrying spike-S gene of
SARS-CoV-2 virus along with gene coding for signal peptide. The
spike(S) region includes the receptor binding domain (RBD), which
binds to the human angiotensin converting Enzyme (ACE)-2 receptor
and mediates the entry of virus inside the cell. The DNA construct
was produced on large scale by transformation in E. coli [13]. The
immunogenicity potential of the plasmid DNA has been evaluated in
mice, guinea pig, and rabbit models by intradermal route at 25, 100
and 500 mg dose. Preliminary studies have demonstrated that the
DNA vaccine induces antibody response including neutralizing anti-
bodies (NAB) against SARS-CoV-2 and also provides Th-1 response as
evidenced by elevated IFN-glevels [13].
In fact, a similar approach has already been used in the past for
development of Middle East Respiratory Syndrome (MERS) and SARS
coronavirus vaccines [14,15]. The MERS DNA vaccine was found to be
well-tolerated in humans with a seroconversion rate of 94% in vacci-
nated volunteers, whereas, the SARS DNA vaccine induced antibody
response in 80% subjects. Based on the earlier published literature of
similar vaccines, the expected human dose of 2019-nCoV vaccine by
intradermal administration will be 1 mg or 2 mg of the 2019-nCoV
DNA vaccine candidate.
2. Methods
2.1. Study design and participants
We conducted a single-center Phase 1 trial of the DNA plasmid spike
protein COVID-19 vaccine candidate at a Clinical Unit of Zydus Research
center, Cadila Healthcare Limited in Ahmedabad, Gujarat, India.
This trial was initiated after obtaining the approvals of the Ethics
Committee (EC) and DCGI (dated 08 July 2020) and registering the
trial with the Clinical Trial Registry of India (CTRI) (Identifier: CTRI/
2020/07/026352). The study was performed in accordance with the
Declaration of Helsinki, Good Clinical Practice and applicable local
regulations. An independent data safety monitoring board was estab-
lished before the start of the study to provide oversight of the safety
data during the study. The authors had full access to all the data in
the study.
Eligible participants were healthy adults aged between 18 and 55
years; body weight >50 kg; body-mass index of between 18.5 and
29.9 kg/m
2
. For inclusion in the trial, participants needed to be able
to understand the content of informed consent and be willing to sign
the informed consent document; and be able and willing to complete
all the scheduled study visits. Exclusion criteria included SARS-CoV-2
infection, confirmed by presence of serum-specific antibody against
SARS-Co-V-2 detected by enzyme-linked immunosorbent assay
(ELISA) or chemiluminescence technology; positive results for
COVID-19 as detected by qualitative reverse transcription polymer-
ase chain reaction; history of SARS/ MERS infection; history of contact
with a confirmed active SARS-CoV-2 positive patient within 14 days;
participation in other clinical study of a SARS-CoV-2 candidate vac-
cine. Pregnant or breastfeeding women were also excluded. A com-
prehensive list of eligibility criteria is provided (Supplementary Table
S1). Written informed consent was obtained from each participant
before screening for eligibility.
2.2. Vaccine
The ZyCoV-D vaccine was developed by Cadila Healthcare Limited,
Ahmedabad, India. The DNA vaccine candidate against SARS-CoV-2 is
Research in context
Evidence before this study
We searched PubMed on March 23, 2021, using the search
terms “COVID-1900,“SARS-CoV-200 ,“vaccine”, and “clinical trial”.
Cadila Healthcare Limited, India developed a candidate vaccine
ZyCoV-D comprising of a DNA plasmid vector carrying the gene
encoding the spike protein (S) of the SARS-CoV-2 virus. Prelimi-
nary animal study demonstrates that the candidate DNA vac-
cine induces antibody response including neutralizing
antibodies against SARSCoV-2 and provided Th-1 response as
evidenced by elevated IFN-glevels.
Added value of this study
This first-in-human trial showed that the DNA vaccine was tol-
erable and immunogenic in healthy adults. The DNA vaccine
candidate induces antibody response against SARS-CoV-2 spike
(S) protein, following immunization with three doses adminis-
tered 28 days apart. Neutralizing antibody response was also
demonstrated against wild type SARS-CoV-2 strain, which may
play a substantial role in viral clearance and mitigation of
human clinical disease. This study has also evaluated safety and
immunogenicity of DNA vaccine administered by two different
methods of administration.
Implications of all the available evidence
Implications of all the available evidence Many vaccine candi-
dates are in rapid development, including recombinant-protein
based vaccines, replicating or non-replicating viral vector-
based vaccines, DNA vaccines, and mRNA vaccines (which
mostly have focused on the spike glycoprotein or receptor
binding domain), live attenuated vaccines, and inactivated virus
vaccines. Our findings indicate that DNA vaccine is safe and
immunogenic in healthy adults.
2T. Momin et al. / EClinicalMedicine 38 (2021) 101020

comprised of a DNA plasmid Vector pVAX1 carrying gene expressing
spike-S protein of SARS-CoV-2 and IgE signal peptide. The spike gene
region was selected from submitted Wuhan Hu-1 isolate (Genebank
Accession No. MN908947.3). For generation of the SARS-CoV-2 DNA
vaccine construct pVAX-1 plasmid vector was used. Chemically syn-
thesized Spike regions and signal peptide gene were inserted into
pVAX-1 plasmid DNA vaccine vector. Following the receipt of the
plasmid DNA constructs, transformations of the construct were car-
ried out in DH5-alphaTM chemically competent cells. The DH5-alpha
E coli carrying the plasmid DNA was further propagated for large
scale production in manufacturing suite approved by National Regu-
latory Authority under current Good Manufacturing Practice condi-
tions. Each 0.5 mL of ZyCoV-D vaccine contains5 mg of DNA
plasmid with spike protein gene region insert from SARS-CoV-2 Virus
suspended in phosphate buffer saline.
2.3. Procedures
Participants were recruited and followed up with according to
treatment arms. Participants were allocated to treatment arms
sequentially in a non-randomized, open label, dose escalation man-
ner (i.e. the first 12 subjects were allocated to treatment arm 1 [1 mg:
Needle]; the next 12 subjects were allocated to treatment arm 2
[1 mg; Needle-Free Injection System {NFIS}] once safety was estab-
lished in the previous treatment arm; same was followed for treat-
ment arm 3 [2 mg; Needle] and treatment arm 4 [2 mg; NFIS]).
Participants were immunized intradermally with three doses of vac-
cine (1 mg: Needle or NFIS; 2 mg: Needle or NFIS) four weeks apart
via syringe and needle or NFIS on days 0, 28 and 56. Follow-up visits
were scheduled after each vaccination until day 84 (End-of-study). In
this study, Pharmajet TropisÒdevice as NFIS was used for intradermal
administration of the vaccine. Participants were monitored in the
intensive observation unit for 24 h post the first dose of vaccine and
4 h post the second and third dose of vaccine for solicited adverse
reactions (injection site pain, redness, swelling and itching). Close
monitoring in terms of frequent vital signs and electrocardiogram
(ECG) assessments were done before and after each vaccine dose.
Subjects were also provided a diary card to record any solicited sys-
temic symptoms (fever, headache, fatigue, vomiting, diarrhea, nau-
sea, arthralgia, and muscle pain) and local adverse events (AEs) for
7 days post each vaccine dose and any other unsolicited AEs within
28 days post each dose. Serious AEs self-reported by participants
were documented throughout the study.
Adverse events were self-reported by the participants, but veri-
fied by investigators throughout the study after vaccination. Adverse
events were graded according to a standard toxicity grading scale
[16]. Laboratory safety tests including hematology, biochemistry, uri-
nalysis and serology were conducted as per the protocol (Appendix
1) to assess any toxic effects post-vaccination. Blood samples were
taken from participants as per the protocol for the immunogenicity
assessment. The follow-ups were scheduled at days 70 and 84 (end-
of-study) post vaccination for safety and immunogenicity assess-
ment.
2.4. Assessment of binding antibody (IgG and neutralizing antibody)
and cellular response
For Phase 1 part of the study, immunogenicity assessment for
serum IgG by ELISA, was done at baseline, day 28, day 42, day 56, day
70 and day 84. Neutralizing antibody titres and cellular response
were also assessed at baseline, day 28, day 56 and day 84.
An indirect ELISA was used to measure anti-S1 SARS CoV-2 IgG
antibodies present in the human sera samples post vaccination with
ZyCoV-D vaccine. We used antigen from Acro Biosystems. The anti-
gen from the same manufacturer was also used by Innovio in their
ELISA assay [17]. We used reference standard from National Institute
for Biological Standards and Control (NIBSC) which is a WHO refer-
ence laboratory. We obtained research reagent for anti-SARS-CoV-2
Ab NIBSC code 20/130 and against this we defined unitage as ELISA
Unit (EU). Plaque Reduction Neutralization Test (PRNT) was used for
estimation of NAB titer in human serum samples against anti SARS
CoV-2 virus. The SARS-CoV-2 virus (8004/IND/2020/PUNE), Accession
number MT416726 was used for PRNT assay.
Cell-mediated responses were assessed using IFN-gELISPOT assay
in separated peripheral blood mononuclear cells (PBMCs). Serum
samples from vaccinated subjects were also analyzed for the cyto-
kines levels (IFN-g, IL-2, IL-6, IL-4, IL-10, TNF alpha, Th-17A) using
MILLIPLEXÒMAP multiplex magnetic bead-based antibody detection
kits. Details regarding the methodology of these tests are provided in
supplementary material.
2.5. Outcomes
The primary endpoint was the overall incidence and severity of
adverse reactions within 7 days after each of the vaccination and AEs
within 28 days across the treatment groups were also analyzed as
safety endpoints.
The secondary endpoints included seroconversion rate based on
IgG antibodies against S1 antigen (by ELISA), NAB titers and IFN-g
cellular immune responses after 3 doses of vaccine. Seroconversion
was defined as antibody-negative subjects at baseline who become
antibody-positive after vaccination, and subjects having antibody
titre at baseline who have four fold rise in antibody titre after vacci-
nation.
2.6. Statistical analysis
The sample size was not determined on the basis of statistical
power calculations. Sample size was based on non-probability sam-
pling method. However, a minimum sample size of 48 participants
for this vaccine trial has been selected. We assessed the incidence
and severity of participants’adverse reactions post vaccination and
compared safety profiles across the dose groups. The antibodies
against SARS-CoV-2 were presented as geometric mean titers with
95% confidence intervals (CIs) and the cellular responses were shown
as a proportion of positive responders. We used the pearson chi-
square test to analyze categorical data, ANOVA to analyze the log
transformed antibody titers. Hypothesis testing was two-sided with
an avalue of 0.05. Statistical analyses were done by a statistician
using SAS (version 9.4).
Geometric mean titres (GMTs) was calculated as: anti-Ln(mean
[Ln Xi]) where Xi was the assay result for subject i. 95% CIs of GMTs
were calculated assuming log normal distribution.
Geometric Mean Fold Rise (GMFR) were calculated as:
GMFR = anti-Ln (mean [Ln Yi/ Bi]) where Yi was the post dose assay
result for subject i; and Bi was the baseline assay result for subject i.
Baselines were taken as Day 0 assay results.
2.7. Role of the funding source
The study sponsor, Cadila Healthcare Limited, designed the study
and oversaw its conduct and data analysis. The sponsor collected,
managed, and analyzed data according to a pre-specified statistical
analysis plan. The corresponding author had full access to all the data
in the study and had final responsibility for the decision to submit for
publication.
Development of ZyCoV-D was supported by a grant-in-aid from
COVID-19 Consortium under National Biopharma Mission, Depart-
ment of Biotechnology, Government of India, to Cadila Healthcare
Ltd. (Grant no. BT/COVID0003/01/20).
T. Momin et al. / EClinicalMedicine 38 (2021) 101020 3

3. Results
3.1. Study population demographics
Between July 2020 and October 2020, 126 participants were
screened, 51 subjects failed in screening, 27 subjects passed but not
enrolled in the study and a total of 48 subjects were enrolled into and
vaccinated in the Phase 1 study. A total of 43 (89¢0%) participants
completed the study and 5 subjects were discontinued from the
study (Fig. 1). All of the 48 participants (100%) were Asian male
healthy subjects. Baseline demographics (age, height, weight and
BMI) were well-balanced among the 4 treatment arms. Overall, the
mean (SD) age was 34¢9(7¢18) years and the mean BMI (SD) was
24¢28 §3¢0 kg/m
2
. The patient disposition, baseline and demographic
characteristics are provided in Table 1.
3.2. Vaccine safety and tolerability
A total of 48 subjects were vaccinated with the first dose of vac-
cine, 45 subjects were vaccinated with the second dose of vaccine
(except 1 subject in arm 1 and 2 subjects in arm 4 who received only
one dose) and 43 subjects were vaccinated with the third dose of vac-
cine (except 2 subjects in arm 3 who received only two doses). A total
of 43 subjects completed the study.
Of the five subjects who discontinued, two subjects were discon-
tinued because of withdrawal of consent (1 subject in each arm 1 and
3); one subject in arm 3 did not receive the third dose of vaccine due
to an ongoing unsolicited adverse event (typhoid fever) that occurred
14 days after the second vaccination which was considered not
related with vaccination; one subject in arm 4 was discontinued due
to an ongoing anti-rabies vaccination for dog bite reported 7 days
after the first vaccination; and one subject in arm 4 was withdrawn
due to asymptomatic positive COVID-19 rapid antigen detection test,
27 days after the first vaccination.
There were no deaths or SAEs reported in Phase 1 of the study.
Overall, 12/48 (25%) subjects reported at least one AE (i.e. combined
solicited and unsolicited) during the study. The number of subjects
with at least one solicited adverse event and at least one unsolicited
adverse event was 7 (14¢58%) subjects and 6 (12¢5%) subjects, respec-
tively (Figs. 2 and 3).
The number of subjects with solicited AEs across all four treat-
ment arms were similar [i.e. 2 subjects in each Arm 1 (1 mg: Needle),
Arm 2 (1 mg: NFIS) and Arm 4 (2 mg: NFIS); 1 subject in Arm 3
(2 mg: Needle)]. Overall, all solicited AEs reported were mild to mod-
erate in severity, related with study vaccination and resolved with or
without medication. The majority of solicited AEs were reported after
the first dose of vaccine (i.e. 6 subjects, 12¢5%) compared to the sec-
ond dose (0%) and the third dose of vaccination (1 subject, 2¢08%).
Most solicited AEs were mild in severity (6 subjects, 12¢5%) except 1
subject (2¢08%) who reported an adverse event of moderate severity.
No subject was discontinued from the study because of a solicited
adverse event. The reported solicited AEs were injection site pain (3
subjects), injection site pruritus (1 subject), pyrexia (1 subject),
arthralgia (1 subject) and diarrhea (1 subject).
There were no abnormal laboratory values that were deemed clin-
ically significant except proteinuria (1 subject on day 14), considered
possibly related to study drug and low WBC count (one subject on
day 56), considered not related to the study drug by the investigator
throughout the study period. There were no clinically significant
changes reported in vital signs and 12-lead ECG evaluated during the
monitoring period after vaccination of each dose as well as follow-up
visits till day 84. For all the physical examinations performed, no
major abnormal findings were reported till day 84.
3.3. Immune responses
Seroconversion was defined as antibody negative subjects at base-
line who become antibody positive after vaccination and subjects
Fig. 1. Subject allocation and participation in the completion of the study. Consort chart explaining subject participation (n= number of subject) in different treatment arms
NIFS = Needle-free injection system.
4T. Momin et al. / EClinicalMedicine 38 (2021) 101020

having antibody titre at baseline who have four-fold rise in antibody
titre after vaccination.
As mentioned in the method section, we used NIBSC reference
standard sera in our ELISA assay. Using this standard we established
the standard curve range from 1.41 EU as below limit of (BLQ) to
45.23 EU as upper limit of quantification (ULQ). We also tested panel
of negative pre-COVID-19 sera sample during assay validation and
samples were below the BLQ value of 1.41EU. NIBSC standard was
also used in ELISA assay performed by Oxford group for their
immunogenicity evaluation of ChADOx-1 SARS-CoV-2 vaccine candi-
date [18]. The proportion of subjects who seroconverted based on
IgG titers on day 84 (i.e. 28 days after third vaccine doses) was 4
(36¢36%), 4 (33¢33%), 10 (100¢00%) and 8 (80¢00%) in the treatment
Arm 1 (1 mg: Needle), Arm 2 (1 mg: NFIS), Arm 3 (2 mg: Needle) and
Arm 4 (2 mg: NFIS), respectively. This suggests a higher seroconver-
sion rate with the 2 mg vaccine dose, irrespective of method of deliv-
ery, compared to the 1 mg vaccine dose. When Arm 1 (1 mg: Needle),
and Arm 4 (2 mg: NFIS), seroconversion rates were compared for day
Table 1
Disposition, Baseline and Demographics Characteristics Safety Population.
Parameters/Statistic ZyCoV-D 1 mg (Needle)
(N= 12)
ZyCoV-D 1 mg (NFIS)
(N= 12)
ZyCoV-D 2 mg (Needle)
(N= 12)
ZyCoV-D 2 mg (NFIS)
(N= 12)
Overall (N= 48)
Disposition
All subjects, n(%) 12 (100) 12 (100) 12 (100) 12 (100) 48 (100)
Subjects who completed the
study, n(%)
11 (91.67) 12 (100) 10 (83.33) 10 (83.33) 43 (89.58)
Subjects discontinued from
the study, n(%)
1 (8.33) 0 (0) 2 (16.67) 2 (16.67) 5 (10.42)
Demographics
Age (Years)
Mean §SD 35.4 §6.56 31.8 §7.44 35.1 §6.95 37.2 §7.53 34.9 §7.18
Median (Range) 36.5 (27, 45) 31.5 (22, 48) 36.0 (20, 45) 36.5 (26, 48) 35.5 (20, 48)
Sex, n(%)
Male 12 (25%) 12 (25%) 12 (25%) 12 (25%) 48 (100%)
Height (cm)
Mean §SD 168.5 §4.08 166.9 §4.01 165.8 §4.53 169.3 §5.83 167.6 §4.72
Median (Range) 170.0 (161, 173) 167.0 (159, 173) 165.5 (159, 173) 170.0 (159, 181) 168.0 (159, 181)
Weight (kg)
Mean §SD 68.52 §7.878 65.32 §7.751 70.32 §8.562 67.97 §11.052 68.03 §8.810
Median (Range) 67.80 (57.6, 82.8) 66.65 (54.2, 76.2) 70.30 (56.8, 85.7) 66.60 (54.7, 87.5) 67.95 (54.2, 87.5)
BMI (kg/m
2
)
Mean §SD 24.178 §3.0745 23.416 §2.3624 25.593 §3.0485 23.720 §3.5076 24.227 §3.0470
Median (Range) 24.355 (19.98, 29.71) 23.860 (18.98, 26.40) 26.690 (18.98, 29.31) 23.805 (18.71, 29.58) 24.320 (18.71, 29.71)
Abbreviation(s): N = number of subjects in respective treatment arm; n = number of subjects in specified category; NFIS = Needle Free Injection System; BMI = body mass index; SD
= standard deviation. Note: Percentages are based on the number of subjects in the specified treatment arm.
Fig. 2. Solicited (local and systemic) adverse events reported within seven days after administration of each dose of vaccine. Adverse events were graded according to the common
terminology criteria for adverse events (CTCAE) scale.
T. Momin et al. / EClinicalMedicine 38 (2021) 101020 5

28, day 42, day 56, day 70 and day 84, a statistical significant (p
value = 0.0019) difference was found for day 70. Similarly for Arm 2
(1 mg: NFIS) vs Arm 4 (2 mg: NFIS), statistical significant (p
value = 0.0427) difference was found for day 70 and day 84 (Supple-
mentary Table S2).
The proportion of subjects who achieved seroconversion based
on IgG on day 28, day 42, day 56, day 70 and day 84 is men-
tioned in Table 2.WehavealsoincludedNABtitersofconva-
lescent sera sample from individuals recovered after SARS-CoV-2
infection (Fig. 4).
The proportion of subjects getting seroconverted based on NAB
titers on day 84 was 02 (18¢18%), 02 (16¢67%), 05 (50¢00%) and 08
(80¢00%) in the treatment Arm 1 (1 mg: Needle), Arm 2 (1 mg: NFIS),
Arm 3 (2 mg: Needle) and Arm 4 (2 mg: NIFS) respectively. The pro-
portion of subjects getting seroconverted based on NAB titers on day
56 was lower i.e. 00 (00¢00%), 02 (16¢67%), 02 (20¢00%) and 01
(10¢00%) in the treatment Arm 1 (1 mg: Needle), Arm 2 (1 mg: NFIS),
Arm 3 (2 mg: Needle) and Arm 4 (2 mg: NIFS) respectively. When
Arm 1 (1 mg: Needle), and Arm 4 (2 mg: NFIS), seroconversion rates
based on NAB titers were compared for day 56 and day 84, a
statistical significant (pvalue = 0.0089) difference was found for day
84. Similarly for Arm 2 (1 mg: NFIS) vs Arm 4 (2 mg: NFIS), statistical
significant (pvalue = 0.0083) difference was found for day 84 (Sup-
plementary Table S3).
Geometric mean titer of IgG (EU) on day 56 (28 days after two
doses of vaccine) was 34¢75 and 17¢46 in the treatment Arm 3 (2
mg: Needle) and Arm 4 (2 mg: NFIS), respectively, which
increased to 1019¢61 and 720¢25 in the treatment Arm 3 (2 mg:
Needle) and Arm 4 (2 mg: NFIS) respectively on day 70 (14 days
after three doses of vaccine) and remained stable at 748¢46 and
884¢04 in the treatment Arm 3 (2 mg: Needle) and Arm 4 (2 mg:
NFIS) respectively on day 84 (28 days after three doses of vac-
cine) See, Table 3 and Fig. 5.WhenArm1(1mg:Needle),and
Arm 4 (2 mg: NFIS), Geometric mean titer of IgG (EU) were com-
pared for day 28, day 42, day 56, day 70, and day 84 a statistical
significant (pvalue = 0.0006) difference was found for day 70 and
day 84 (pvalue = 0.0027). Similarly for Arm 2 (1 mg: NFIS) vs
Arm 4 (2 mg: NFIS), statistical significant (pvalue = 0.0376) dif-
ference was found for day 70 and p-values= 0.0259 for day 84
(Supplementary Table S4).
Fig. 3. Unsolicited (local and systemic) adverse events reported after seven days of each dose of vaccine. Adverse events were graded according to the Common Terminology Crite-
ria for Adverse Events (CTCAE) scale.
Table 2
Summary and Comparison of Seroconversion of IgG at day 28, 42, 56, 70 and 84.
Time point Seroconversion ZyCoV-D 1 mg (Needle) (N= 11) ZyCoV-D 1 mg (NFIS) (N= 12) ZyCoV-D 2 mg (Needle) (N= 10) ZyCoV-D 2 mg (NFIS) (N= 10)
Day-28, n(%) No 9 (81.82) 9 (75.00) 6 (60.00) 7 (70.00)
Yes 2 (18.18) 3 (25.00) 4 (40.00) 3 (30.00)
Day-42, n(%) No 10 (90.91) 9 (75.00) 7 (70.00) 7 (70.00)
Yes 1 (9.09) 3 (25.00) 3 (30.00) 3 (30.00)
Day-56, n(%) No 9 (81.82) 7 (58.33) 4 (40.00) 6 (60.00)
Yes 2 (18.18) 5 (41.67) 6 (60.00) 4 (40.00)
Day-70, n(%) No 10 (90.91) 8 (66.67) 0 (0.00) 2 (20.00)
Yes 1 (9.09) 4 (33.33) 10 (100.0) 8 (80.00)
Day-84, n(%) No 7 (63.64) 8 (66.67) 0 (0.00) 2 (20.00)
Yes 4 (36.36) 4 (33.33) 10 (100.0) 8 (80.00)
Abbreviation(s): N= number of subjects in respective treatment arm; n= number of subjects in specified category; NFIS = Needle Free Injection System.
Seroconversion defined as a positive antibody response as at least a four-fold increase in post-vaccination titer from baseline.
6T. Momin et al. / EClinicalMedicine 38 (2021) 101020

Geometric mean titer of NAB on day 84 (28 days after three doses
of vaccine) was 39.17 in the treatment Arm 4 (2 mg: NFIS). Geometric
mean titer and fold rise of NAB are presented in Table 4.
When Arm 1 (1 mg: Needle), and Arm 4 (2 mg: NFIS), Geometric
mean titer of NAB were compared for day 56, and day 84 a statistical
significant (pvalue = 0.0055) difference was found for day 84. Simi-
larly for Arm 2 (1 mg: NFIS) vs Arm 4 (2 mg: NFIS), statistical signifi-
cant (pvalue = 0.0251) difference was found for day 84
(Supplementary Table S5).
Seroconversion rates of IgG with 2 mg needle (Arm 3) and 2 mg
NFIS (Arm 4) on day 56 and day 84 were observed higher than 1 mg
needle and 1 mg NFIS (arm 1 and 2). Seroconversion rates of NAB
with 2 mg NFIS (Arm 4) on day 84 were observed higher than 1 mg
needle, 1 mg NFIS and 2 mg needle (Table 5). The seroconversion rate
(defined as subjects sero-negative at baseline becoming sero-positive
post vaccination and four fold rise in antibody titres post vaccination
in subjects sero-positive at baseline)) based on humoral responses
measured by ELISA were observed in 100% and 80% of the partici-
pants who received three doses of 2 mg vaccine either via needle and
syringe or NFIS device respectively. The seroconversion rate based on
NAB, measured by live virus neutralization assay, was seen in 50%
(05/10) and 80% (08/10) of participants who received three doses of
2 mg vaccine either via needle and syringe or NFIS device respec-
tively.
3.4. Cell-mediated responses
In our study, ZyCoV-D vaccine, when administered intradermally
via NFIS at 2 mg dose, showed peak cellular response in terms of IFN-
gELISPOT assay at Day 56 with 41.5 spot forming cells (SFC) per mil-
lion PBMC’s and was maintained till Day 84 with median 45.5 SFC per
million PBMC’s. A similar trend was observed with 1 mg NFIS arm
with Day 84 median 73 SFC per million PBMC’s. ZyCoV-D vaccine
when administered by conventional syringe and needle showed
some response in IFN-gELISPOT assay at Day 56 which declined on
Day 84 after reaching the peak on day 56 (Fig. 6).
In our study, there were no significant changes observed in cyto-
kine levels like. IFN-g, IL-2, IL-6, IL-4, IL-10, TNF alpha, Th-17A ana-
lysed by Luminex in all four-treatment arms throughout the study
compared to baseline.
4. Discussion
We report the findings from Phase 1 part of clinical trial on
the safety, tolerability and immunogenicity of ZyCoV-D, a SARS-
CoV-2 DNA vaccine encoding the spike protein. This first-in-
human Phase 1 study of ZyCoV-D DNA vaccine was carried out in
an intensive observational unit with frequent monitoring of vital
signs and ECGs for at least 24 h post administration of the first
Fig. 4. Neutralising antibody titres with error bars, representing geometric mean (95%CI) at baseline (Day 0) and Day 84 with comparative NAB of convalescent serum.
Table 3
GMTs of IgG at day 28, 42, 56, 70 and 84 post dose sero negative subjects only.
Time Point Statistics ZyCoV-D 1 mg (Needle) (N= 11) ZyCoV-D 1 mg (NFIS) (N= 12) ZyCoV-D 2 mg (Needle) (N= 10) ZyCoV-D 2 mg (NFIS) (N= 10)
Day-0 GMT(95% C.I) 15.82 (4.61, 54.25) 26.73 (7.45, 95.89) 7.00 (7.00, 7.00) 7.00 (7.00, 7.00)
Day-28 GMT(95% C.I) 26.92 (6.46, 112.12) 66.39 (14.11, 312.27) 11.42 (6.80, 19.17) 14.15 (4.79, 41.79)
GMFR(95% C.I) 1.70 (0.94, 3.07) 2.48 (0.93, 6.67) 1.63 (0.97, 2.74) 2.02 (0.68, 5.97)
Day-42 GMT(95% C.I) 20.41 (5.35, 77.84) 55.67 (12.48, 248.37) 11.01 (6.29, 19.26) 15.60 (4.57, 53.20)
GMFR(95% C.I) 1.29 (0.86, 1.93) 2.08 (0.83, 5.26) 1.57 (0.90, 2.75) 2.23 (0.65, 7.60)
Day-56 GMT(95% C.I) 23.37 (6.34, 86.22) 115.91 (23.41, 573.98) 34.75 (8.11, 148.89) 17.46 (4.81, 63.32)
GMFR(95% C.I) 1.48 (0.91, 2.40) 4.34 (1.14, 16.44) 4.96 (1.16, 21.27) 2.49 (0.69, 9.05)
Day-70 GMT(95% C.I) 20.10 (5.40, 74.77) 94.84 (18.49, 486.47) 1019.61 (326.04, 3188.54) 720.25 (123.28, 4207.96)
GMFR(95% C.I) 1.27 (0.85, 1.91) 3.55 (0.93, 13.56) 145.66 (46.58, 455.51) 102.89 (17.61, 601.14)
Day-84 GMT(95% C.I) 38.84 (9.80, 154.00) 96.49 (19.65, 473.89) 748.46 (237.75, 2356.25) 884.04 (138.69, 5635.19)
GMFR(95% C.I) 2.46 (0.92, 6.59) 3.61 (0.94, 13.93) 106.92 (33.96, 336.61) 126.29 (19.81, 805.03)
Abbreviation(s): N= number of subjects in respective treatment arm; n= number of subjects in specified category; NFIS = Needle Free Injection System.
Seroconversion defined as a positive antibody response as at least a four-fold increase in post-vaccination titer from baseline.
T. Momin et al. / EClinicalMedicine 38 (2021) 101020 7

dose of vaccine and for at least 4 h post administration of the
second and the third dose of vaccine. Each vaccination was fol-
lowed by frequent safety follow-up with subjects till 28 days of
the last dose of vaccine. ZyCoV-D vaccine was well-tolerated in
48 healthy adults in all four dose groups with no vaccine-related
severe or SAEs. The safety profile of ZyCoV-D vaccine supports
further development of ZyCoV-D in at-risk populations who are
at more serious risk of complications from SARS-CoV-2 infection,
including the elderly and subjects with comorbidities. Our find-
ings also correlate with previous clinical evaluation of other DNA
vaccine candidates which were reported to be safe and well-tol-
erated in healthy subjects [17,1921].
The majority of solicited AEs reported in this trial were after the
first dose of vaccine, while the second and third dose of vaccination
were found to be well-tolerated. The ZyCoV-D Phase 1 safety data
further suggest that the vaccine could be a safe booster as there was
no increase in frequency of side effects after the third dose compared
to the first dose, an important aspect for the safety profile of SARS-
CoV-2 vaccines. One attractive feature of DNA vaccines, like ZyCoV-D,
is that the immunizations could be boosted without significant
Fig. 5. (a) - 5 (f) IgG Antibody Titres with error bars, representing geometric mean titre (95%CI) at baseline (Day 0), Day 28, Day 42, Day 56, Day 70 and Day 84.
8T. Momin et al. / EClinicalMedicine 38 (2021) 101020

limitations such as dosing-incremented toxicities or anti-vector
responses and additional boosting with other DNA vaccines have
resulted in higher levels of cellular and humoral immune responses
without increased toxicity [22].
ZyCoV-D also generated balanced humoral and cellular immune
responses in participants displaying either or both antibody or Tcell
responses following three doses of vaccine. Humoral responses were
lower in subjects who received 1 mg vaccine irrespective of method
of administration. The exact reason for this is not known but it is
likely that when the vaccine is administered at the low dose of 1 mg
at single intradermal site, it may lead to inefficient transfection, in
the host cells and thus lower the expression of antigen. Our data cor-
roborates well with the Rhesus Macaques challenge study, where the
vaccination of 2 mg dose with Pharmajet NFIS elicited significant
SARS CoV-2 specific IgG, NAB titers and lower viral loads in animals
post challenge (Data on file). Further; a Phase II study in 1000 sub-
jects is currently ongoing which will provide better understanding of
immunogenicity of ZyCoV-D vaccine in a larger sample size.
In our study, three doses of 2 mg ZyCoV-D DNA vaccine adminis-
tered intradermally at two different sites via NFIS device 28 days
apart have shown good humoral and cellular immune response at
Day 70 onwards. Presently, correlation of protection for vaccine
against SARS-CoV-2 is unknown, and the roles of the specific antibod-
ies or T cells in building effective protection are not yet well-defined.
Therefore, we are only able to demonstrate immune response induc-
tion following vaccination and not protection to SARS-CoV-2 follow-
ing DNA vaccination on the basis of the vaccine-elicited immune
responses in this study. A double-blind, placebo controlled Phase III
Fig. 5. Continued.
T. Momin et al. / EClinicalMedicine 38 (2021) 101020 9

study in 28,216 subjects aged 12 years and above is also currently
ongoing which will help evaluate efficacy of the ZyCoV-D 2 mg dose
administered via NFIS device in protection against COVID-19 infec-
tion. The study is registered with CTRI/2021/01/030416.
Previous studies investigating SARS and Middle East Respiratory
Syndrome (MERS) found that there is a temporary rise in specific
antibodies which dropped rapidly in subjects after recovery, and the
CD4+ and CD8+ T-cell responses played a vital role in memory
response and protection against future exposure to virus [23]. A simi-
lar rapid decline of the specific antibody amounts in subjects with
COVID-19 after recovery was also noted [23] suggesting that both
specific cellular and humoral immunity are potentially important for
a successful COVID-19 vaccine. Here, we report immune response till
28 days after the last dose of vaccine.
ZyCoV-D vaccine also induced cellular response as measured by
IFN-gELISPOT which was maintained till Day 84 in subjects who
received vaccination 1 mg or 2 mg via NFIS device. This clearly indi-
cates that vaccination with ZyCoV-D induces cellular response with
fold rise. However, the sample size per arm is too small to reach a
definitive conclusion on the levels of IFN-gin different arms and the
results should be interpreted in the context of variability of the
immunological responses among individuals enrolled in the trial.
Fig. 5. Continued.
10 T. Momin et al. / EClinicalMedicine 38 (2021) 101020

Phase II data with a higher sample size will help to understand cellu-
lar response obtained with ZyCoV-D vaccine.
This first-in-human study of ZyCoV-D DNA vaccine has some limi-
tations. First, this open-label, non-randomized Phase 1 trial report is
based on a modest sample size (48) in all vaccine arms and, therefore
lacks a comparator group. Larger sample-sized randomized placebo
controlled blinded trials may be needed to show the true immunoge-
nicity difference between the dose groups. Second, this report only
involves healthy Indian male subjects aged between 18 and 55 years.
This is due to societal limitation, COVID 19 related lockdown and
completion of recruitment with male subjects at study center. The
results of this study are not generalizable to other ethnic groups and
female subjects. In this regard, female subjects were part of Phase
2 and 3 studies. SARS-CoV-2 infection has more severe and fatal
outcomes in older individuals. In this regard, the Phase 3 trial
will evaluate individuals of higher age group. Third, only data
within the first 84 days of vaccination is being reported, and this
report does not include data about the durability of the vaccine-
induced immunity. In previous clinical trials with similar DNA
vaccines, durable immune responses up to 1 year following vacci-
nation were reported [14,22]. Fourth, the study showed good
humoral and cellular immune response at Day 70 onwards after
administration of the third dose, while most other approved vac-
cinesshowedimmuneresponseafteradministrationofthesec-
ond dose.
In this study, two different vaccination strategies were used. One
is injection and needle, and the second is needle-free injection. i.e.
NFIS device. This technology has evolved significantly over the last
50 years and is now accepted in many routine immunization settings
as a safe and effective vaccine delivery method. Disposable syringe
jet injectors are now being used for the delivery of vaccines to eradi-
cate polio, measles, mumps, rubella and influenza, and are showing
promising results in vaccine clinical trials for the Zika virus and
human papillomavirus. Vaccine administration using NFIS device
offers some distinct advantages compared to the conventional
method of vaccination using needle and syringe, like improved com-
pliance and better coverage; no needle trash and needle stick inju-
ries; higher immunogenic response; calibrated for specific volume
with minimal vaccine wastage; auto disabling and eliminating possi-
bility of re-use; efficient vaccine delivery; and the workflow is 25%
faster than a conventional needle-syringe and is less painful [24].
Tebas et al. reported better immune response after administration of
two doses with intradermal DNA vaccine followed by electroporation
(EP) technique [17]. However, Pharmajet Tropis device has been used
in DNA vaccine clinical trials and has been reported as a better
administration technique in terms of ease of administration, reliabil-
ity, and precision. Use of Tropis is also reported to be cost-effective
and have better local tolerance compared with EP [25].
Our data suggests that ZyCoV-D demonstrates a good safety pro-
file and that vaccination induces both cellular and humoral
responses, supporting its further development to prevent infection
and death related to COVID-19 in the global population. The safety
and immunogenic profile are important parameters for vaccination
for high-risk populations, such as the elderly and those living with
co-morbid conditions.
Over the past decade, the vaccine industry and clinical research
centers have been asked to provide urgent responses to epidemics of
emerging infectious diseases, such as H1N1 influenza, Ebola virus,
Zika, MERS, and now SARS-CoV-2 [26]. The risk of COVID-19 caused
by SARS-CoV-2 is ongoing, making the need for effective vaccines
even more urgent [27]. Previous findings suggested that those vac-
cines expressing full-length spike glycoprotein can induce good
immune responses and protective efficacy. The full-length spike was
chosen in most of the viral vectored, mRNA, or DNA COVID-19 vac-
cines in development [23].
There have been recent reports of emergence of new SARS-CoV-2
viral strains like B.1.1.7 in UK, B.1.351 in South Africa, P.1 in Brazil
[28]. The emergence of new strains of virus has raised the doubts
about efficacy of vaccines which were already approved for emer-
gency use authorization. Currently ongoing clinical trials with
ZyCoV-D vaccine will provide important insights into efficacy and
safety of DNA vaccine platform. DNA vaccines are based on plug and
play platform, which allows rapid development of new constructs in
case mutant strains develop, and possibility of generating a new vac-
cine candidate in very short time, thus providing protection against
mutated viral strains.
Table 4
Summary results of neutralization titers at day 28, 56 and 84.
Time Point Statistics ZyCoV-D 1 mg (Needle) (N= 11) ZyCoV-D 1 mg (NFIS) (N= 12) ZyCoV-D 2 mg (Needle) (N= 10) ZyCoV-D 2 mg (NFIS) (N= 10)
Day-0 GMT(95% C.I) 5.98 (4.01, 8.92) 8.88 (4.52, 17.48) 5.00 (5.00, 5.00) 5.00 (5.00, 5.00)
Day-28 GMT(95% C.I) 6.79 (4.29, 10.73) 11.12 (4.91, 25.19) 5.00 (5.00, 5.00) 6.06 (3.93, 9.34)
GMFR(95% C.I) 1.13 (0.82, 1.57) 1.25 (0.62, 2.52) 1.00 (1.00, 1.00) 1.21 (0.79, 1.87)
Day-56 GMT(95% C.I) 6.48 (3.64, 11.52) 12.99 (5.60, 30.16) 8.72 (3.08, 24.72) 6.34 (3.70, 10.87)
GMFR(95% C.I) 1.08 (0.91, 1.29) 1.46 (0.63, 3.42) 1.74 (0.62, 4.94) 1.27 (0.74, 2.17)
Day-84 GMT(95% C.I) 8.52 (4.40, 16.49) 11.97 (5.72, 25.06) 14.13 (5.57, 35.82) 39.17 (15.43, 99.47)
GMFR(95% C.I) 1.42 (0.83, 2.45) 1.35 (0.68, 2.65) 2.83 (1.11, 7.16) 7.83 (3.09, 19.89)
Abbreviation(s): CI = confidence interval; GMT = geometric mean titre; GMFR = geometric mean fold rise; N= number of subjects in respective treatment arm; n= number of
subjects in specified category; NFIS = Needle Free Injection System.
Seroconversion defined as a positive antibody response as at least a four-fold increase in post-vaccination titer from baseline.
Table 5
Summary of Seroconversion for Neutralization Titers.
Time point Seroconversion ZyCoV-D 1 mg (Needle) (N= 11) ZyCoV-D 1 mg (NFIS) (N= 12) ZyCoV-D 2 mg (Needle) (N= 10) ZyCoV-D 2 mg (NFIS) (N= 10)
Day-28 n(%) No 10 (90.91) 10 (83.33) 10 (100.0) 9 (90.00)
Yes 1 (9.09) 2 (16.67) 0 (0.00) 1 (10.00)
Day-56 n(%) No 11 (100.0) 10 (83.33) 8 (80.00) 9 (90.00)
Yes 0 (0.00) 2 (16.67) 2 (20.00) 1 (10.00)
Day-84 n(%) No 9 (81.82) 10 (83.33) 5 (50.00) 2 (20.00)
Yes 2 (18.18) 2 (16.67) 5 (50.00) 8 (80.00)
Abbreviation(s): N= number of subjects in respective treatment arm; n= number of subjects in specified category; NFIS = Needle Free Injection System.
Seroconversion defined as a positive antibody response as at least a four-fold increase in post-vaccination titer from baseline.
T. Momin et al. / EClinicalMedicine 38 (2021) 101020 11

Declaration of Competing Interest
All authors declared no competing interests. TM, KK, HP, SS, BS, JP,
RM, JS, KM, AD, HC, CR, HPR, PK and AN are employee of Cadila
Healthcare Limited, Ahmedabad, India. DP is an employee of Zydus
Discovery DMCC, Dubai, United Arab Emirates.
Contributors
KK, JS, RM and DP were involved in conceptualization of the study.
TM and HP were the study investigators. TM and JP were involved in
data interpretation, manuscript writing, and manuscript review. SS
was involved in statistical analysis, designing, programming and gen-
eration of Tables, Listing, Figures and aided in interpretation of
results. BS was a pharmacist for this study. KM was involved in con-
ceptualizing, designing, developing the vaccine candidate and guid-
ing on data analysis, AD was involved in designing, developing
vaccine candidate, perform data analysis for ELISPOT and Luminex
assay. HC and CRTM were involved in development of analytical pro-
cedures for testing of the vaccine and data analysis for ELISA, neutral-
ization. HPRP was involved in developed process for vaccine
production and manufacture Phase-1 vaccine batches, AN was the
responsible for quality assurance and regulatory support. Each author
contributed important intellectual content during manuscript draft-
ing or revision and accepts accountability for the overall work by
ensuring that questions pertaining to the accuracy or integrity of any
portion of the work are appropriately investigated and resolved. All
authors approved the final version of the manuscript for submission.
Data sharing statement
Deidentified data are in the process of being deposited on the Data
Repository for the Cadila Healthcare Limited, and the corresponding
author can be contacted for data access.
Acknowledgment
The authors would like to acknowledge Hardik Patel, Vishal Nak-
rani, Jaydeep Kapdeeya for data review support and Chintan Shah
and Khushali Captain for data management support. The authors
would like to thank the PharmaJet, Inc. Golden, CO, USA for providing
us PharmaJet TropisÒNeedle-Free Injection System (NFIS) for vaccine
delivery. The authors would like to acknowledge all the participants
who participated in this trial. [ZRC communication number: 663]
Funding
Development of ZyCoV-D was supported by a grant-in-aid from
COVID-19 Consortium under National Biopharma Mission, Depart-
ment of Biotechnology, Government of India, to Cadila Healthcare
Ltd. (Grant no. BT/COVID0003/01/20).
Supplementary materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.eclinm.2021.101020.
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