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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

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  • Zydus Therapeutics Inc

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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
Tauk 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
ndings 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 identied 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 inuenza-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, Pzer 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, Pzer, 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 rst 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-
rmed 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, 1855 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) (Identier: 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, conrmed by presence of serum-specic 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 conrmed 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 rst-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 ndings 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 contains5 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 rst 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 rst 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-
ed 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 dened 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 dened 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 participantsadverse reactions post vaccination and
compared safety proles across the dose groups. The antibodies
against SARS-CoV-2 were presented as geometric mean titers with
95% condence 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-specied statistical
analysis plan. The corresponding author had full access to all the data
in the study and had nal 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 rst 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 ve 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 rst vaccination; and one subject in arm 4 was withdrawn
due to asymptomatic positive COVID-19 rapid antigen detection test,
27 days after the rst 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 rst 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 signicant 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 signicant
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 ndings were reported till day 84.
3.3. Immune responses
Seroconversion was dened 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 quantication (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 specied category; NFIS = Needle Free Injection System; BMI = body mass index; SD
= standard deviation. Note: Percentages are based on the number of subjects in the specied 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 signicant (p
value = 0.0019) difference was found for day 70. Similarly for Arm 2
(1 mg: NFIS) vs Arm 4 (2 mg: NFIS), statistical signicant (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 signicant (pvalue = 0.0089) difference was found for day
84. Similarly for Arm 2 (1 mg: NFIS) vs Arm 4 (2 mg: NFIS), statistical
signicant (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
signicant (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 signicant (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 specied category; NFIS = Needle Free Injection System.
Seroconversion dened 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
signicant (pvalue = 0.0055) difference was found for day 84. Simi-
larly for Arm 2 (1 mg: NFIS) vs Arm 4 (2 mg: NFIS), statistical signi-
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
(dened 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 PBMCs and was maintained till Day 84 with median 45.5 SFC per
million PBMCs. A similar trend was observed with 1 mg NFIS arm
with Day 84 median 73 SFC per million PBMCs. 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 signicant 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 ndings 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 rst-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 rst
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 specied category; NFIS = Needle Free Injection System.
Seroconversion dened 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 prole 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 nd-
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 [17,1921].
The majority of solicited AEs reported in this trial were after the
rst 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 rst dose, an important aspect for the safety prole of SARS-
CoV-2 vaccines. One attractive feature of DNA vaccines, like ZyCoV-D,
is that the immunizations could be boosted without signicant
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 inefcient 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 signicant
SARS CoV-2 specic IgG, NAB titers and lower viral loads in animals
post challenge (Data on le). 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 specic antibod-
ies or T cells in building effective protection are not yet well-dened.
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 efcacy 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 specic
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 specic antibody amounts in subjects with
COVID-19 after recovery was also noted [23] suggesting that both
specic 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
denitive 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 rst-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 rst 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 signicantly 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 inuenza, 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 specic volume
with minimal vaccine wastage; auto disabling and eliminating possi-
bility of re-use; efcient vaccine delivery; and the workow 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-
le 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 prole 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 inuenza, 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 ndings suggested that those vac-
cines expressing full-length spike glycoprotein can induce good
immune responses and protective efcacy. 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 efcacy of vaccines which were already approved for emer-
gency use authorization. Currently ongoing clinical trials with
ZyCoV-D vaccine will provide important insights into efcacy 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 = condence interval; GMT = geometric mean titre; GMFR = geometric mean fold rise; N= number of subjects in respective treatment arm; n= number of
subjects in specied category; NFIS = Needle Free Injection System.
Seroconversion dened 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 specied category; NFIS = Needle Free Injection System.
Seroconversion dened 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 nal version of the manuscript for submission.
Data sharing statement
Deidentied 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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T. Momin et al. / EClinicalMedicine 38 (2021) 101020 13
... Another significant utility of ID injections is to administer vaccines, with multiple clinical studies demonstrating several advantages for the ID route, including strong immune responses, optimum dosing effects, patient tolerability, and cost-effectiveness (Lambert and Laurent, 2008;Zehrung et al., 2013;Tanizaki et al., 2015;Meunier et al., 2016;Roozen et al., 2022). To date, many vaccines implementing ID injection have been approved for clinical testing, including vaccines for the seasonal influenza, yellow fever, inactivated poliovirus (IPV), rabies, hepatitis B, and COVID-19 (Laurent et al., 2007;Lambert and Laurent, 2008;Zehrung et al., 2013;Schaumburg et al., 2019;Momin et al., 2021). A key consideration for intradermally administered biologic substances that act locally is that effectiveness is critically dependent on the molecular dispersion within the volume of cells surrounding the injection site. ...
... Utilizing four distinct size molecules (~1 nm FITC-dextran, 20 nm fluorescent particles,~100 nm Cy5-labeled DNA, and 3 µm fluorescent particles), we have characterized: 1) twodimensional area for the bleb and the puddle from a top view; 2) effect of injection volumes on bleb size; 3) puddle penetration depth; and 4) time-dependence for lateral dispersion of the puddle. These specific parameters are key factors for ID drug delivery, particularly for approaches that require a secondary delivery mechanism or when cellular uptake is required for function (Van Den Berg et al., 2009;Schultheis et al., 2018;Kim et al., 2019;Lallow et al., 2021;Momin et al., 2021;Benaouda et al., 2022;Jeong et al., 2022). ...
Article
Full-text available
Intradermal (ID) injection is a technique widely used in laboratorial and clinical applications. The boundary of the dome-like bleb formed during injection is assumed to represent the lateral extent of the injected material. This work systematically characterizes cargo molecule distribution (puddle) as a function of injection volume and molecular/particle size in rat skin post ID injection. In general, results indicate that the puddle forms a subdomain laterally contained within the bleb, with an area inversely correlating to the molecular size of the injected material. For 50 μL and 100 µL injections, the average area of the bleb was 40.97 ± 6.30 mm ² and 55.64 ± 8.20 mm ² , respectively, regardless of the molecular/particle size. On the other hand, the area of the puddle was dependent on the molecular size and ranged between 45.38 ± 8.29 mm ² and 6.14 ± 4.50 mm ² for 50 µL injections, and 66.64 ± 11.22 mm ² and 11.50 ± 9.67 mm ² for 100 µL injections. The lateral distribution appears to have no time-dependency up to 10 min post injection. The trend in the depth of cargo penetration is also similar, with smaller particles extending deeper into the dermis and subcutaneous fat layers. Because the area of puddle can be significantly less than that of the bleb, establishing base characterization is essential to understand cellular interactions with the injected biological substances.
... The study was conducted in a vaccine-naïve population during the early phase of the first peak Compared to other vaccine platforms, binding and neutralizing antibody responses induced by GLS-5310 administered with the GeneDerm suction device were similar to published results for those elicited by DNA vaccines using either electroporation (EP) or a needle-free injection Page | 16 system (NFIS) [14,15] but approximately 1-log lower than adenoviral vaccines and 2-log lower than mRNA vaccines. One of the DNA vaccines, ZyCoV-D, demonstrated 66.6% efficacy to protect against severe SARS-CoV-2 infection in a Phase 3 clinical trial conducted during the peak of the Delta wave [16] and has been approved in India. ...
... T cell responses as determined by ELISpot for GLS-5310 administered with GeneDerm were 15 to 30-fold greater than reported for other DNA vaccines administered with either EP or NFIS [14,15]. Based on reported data, T cell responses appear to be approximately 3 to 15-fold greater than those reported for viral vectored and mRNA vaccines [4,[18][19][20][21] and approximately 20-fold greater than heterologous vaccination with an adenoviral vaccine and an mRNA vaccine [19]. ...
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Objectives : The CoV2-001 Phase 1 randomized trial evaluated the safety and immunogenicity of the GLS-5310 bi-cistronic DNA vaccine through 48 weeks of follow-up. Design : Forty-five vaccine-naïve participants were recruited between 31 December 2020 and 30 March 2021. GLS-5310, encoding for the SARS-CoV-2 spike (S) and ORF3a proteins, was administered intradermally at 0.6 mg or 1.2 mg per dose, followed by application of the GeneDerm suction device as part of a two-dose regimen spaced either 8 or 12 weeks between vaccinations. Results : GLS-5310 was well tolerated with no SAE's reported. Antibody and T cell responses were dose-independent. Anti-S antibodies were induced in 95.5% of participants with average GMT of ∼480 four-weeks post-vaccination, and declined minimally through 48 weeks. Neutralizing antibodies were induced in 55.5% of participants with post-vaccination GMT of 28.4. T cell responses were induced in 97.8% of participants, averaged 716 SFU/10⁶ cells four-weeks post-vaccination, increasing to 1248 at week 24, and remained greater than 1000 through 48 weeks. Conclusion : GLS-5310 administered with the GeneDerm suction device was well tolerated and induced high levels of binding antibodies and T-cell responses. Antibody responses were similar to other DNA vaccines, whereas T cell responses were many-fold greater than DNA and non-DNA vaccines.
... In addition, the safety of the electric pulse-generating device itself needs to be guaranteed, as well as the safety of the therapeutic material. Pressurized microdroplet delivery of plasmid DNA using the PharmaJet needle-free injection system (NFIS) has been shown to yield immune responses similar to EP-mediated plasmid delivery (Jeong et al. 2022) and has regulatory approval in India for a COVID-19 vaccine (Khobragade et al. 2022;Momin et al. 2021). Technical difficulties with both EP and NFIS devices as well as adverse events associated with use urges the development of an alternative in vivo transfection platform for DNA vaccines (Lallow et al. 2021). ...
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pGO-1002, a non-viral DNA vaccine that expresses both spike and ORF3a antigens of SARS-CoV-2, is undergoing phase 1 and phase 2a clinical trials in Korea and the US. A preclinical repeated-dose toxicity study in New Zealand white rabbits in compliance with Good Laboratory Practice (GLP) was conducted to assess the potential toxicity, local tolerance, and immunogenicity of the vaccine and GeneDerm suction device. The dose rate was 1.2 mg/head pGO-1002, and this was administered intradermally to a group of animals (eight animals/sex/group) three times at 2-week intervals, followed by a 4-week recovery period. After each administration, suction was applied to the injection site using the GeneDerm device. Mortality, clinical signs, body weight, food consumption, skin irritation, ophthalmology, body temperature, urinalysis, and clinical pathology were also monitored. Gross observations and histopathological evaluation were performed. Overall, pGO-1002 administration-related changes were confined to minor damage or changes at the injection site, increased spleen weight and minimal increased cellularity in white pulp. All changes of injection site were considered local inflammatory changes or pharmacological actions due to the vaccine with the changes in spleen considered consistent with vaccine-induced immune activation. All findings showed reversibility during the 4-week recovery period. Animals vaccinated with pGO-1002, administered by intradermal injection and followed by application of suction with GeneDerm, developed humoral and cellular responses against the SARS-CoV-2 antigens consistent with prior studies in rats. Collectively, it was concluded that the pGO-1002 vaccine was safe and effective under these experimental conditions and these data supported future human study of the vaccine, now known as GLS-5310, for clinical trial use.
... We found that intradermal administration of the DNA vaccine via PJI effectively induced immune responses and viral protection in animal models 27 . Other groups have developed DNA vaccines with intradermal injectors [28][29][30][31][32] . ...
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Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global pandemic. New technologies have been utilized to develop several types of vaccines to prevent the spread of SARS-CoV-2 infection, including mRNA vaccines. Our group previously developed an effective DNA-based vaccine. However, emerging SARS-CoV-2 variants of concern (VOCs), such as the delta variant, have escaped mutations against vaccine-induced neutralizing antibodies. This suggests that modified vaccines accommodating VOCs need to be developed promptly. Here, we first modified the current DNA vaccine to enhance antigenicity. Compared with the parental DNA vaccine, the modified version (GP∆-DNA vaccine) induced rapid antibody production. Next, we updated the GP∆-DNA vaccine to spike glycoprotein of the delta variant (GP∆-delta DNA vaccine) and compared the efficacy of different injection routes, namely intramuscular injection using a needle and syringe and intradermal injection using a pyro-drive jet injector (PJI). We found that the levels of neutralizing antibodies induced by the intradermal PJI injection were higher than intramuscular injection. Furthermore, the PJI-injected GP∆-delta DNA vaccine effectively protected human angiotensin-converting enzyme 2 (hACE2) knock-in mice from delta-variant infection. These results indicate that the improved DNA vaccine was effective against emerging VOCs and was a potential DNA vaccine platform for future VOCs or global pandemics.
... The vaccine consists of a DNA plasmid constructed with the spike gene with a phosphate buffer and can be injected into individuals ≥ 12 years old. The vaccine is generally categorized as safe for use, with some side effects such as headache, muscle pain, fever, nausea, and diarrhea [74]. ...
Article
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) eruption has left not only illness and mortality in its wake, but also an overwhelming threat to health policy, human regality, food security, and struggle worldwide. The accessibility and potential distribution of a protective and successful vaccination to communities throughout the world are being considered now not just, as a potential of overcoming these hurdles, but also as an example of human perseverance in the face of catastrophe. A vaccine is the only tool that can efficaciously deal with the COVID-19 catastrophe. Currently, more than 47 vaccines are permitted for emergency use in distinct parts of the world. India will play a significant role in the development of the high-priced Moderna shots and Pfizer Inc, therefore assisting in the immunization of a large portion of the world. Moreover, many of the internationally researched and developed vaccine laboratories seek manufacturing in Indian firms and companies for efficient and low-cost production of vaccines intending to provide to the world, hence, making India, a major role player during these pandemic times. This review highlights the Indian contribution to the globe for COVID-19 management.
... Nous avons également montré que les CDD (CD1c + CD1a hi , CD1c + CD1a int , CD1c + CD1a -, et CD1c -CD1a -) et les macrophages dermiques CD163 + sont plus efficaces pour absorber OMV-DyLight 488 que les LC "en route" (CD1c + CD1a hi ). Dans la le Pharmajet Tropis (Momin et al., 2021). Il utilise une pression à l'aide d'un gaz pour percer les différentes couches de la peau et déverser le vaccin. ...
Thesis
Les stratégies vaccinales sont au cœur du développement d’un vaccin. Elles s’orientent autour de l’administration et de la formulation d’un antigène (Ag). Par exemple, l’environnement immunitaire de la peau est très riche en cellules présentatrices d’Ag (CPA) contrairement au muscle, ce qui la rend très prometteuse pour l’injection d’un vaccin. Cependant, la méthode conventionnelle Mantoux est difficile à utiliser, elle demande une injection très minutieuse, parallèle à l’épiderme. Les premiers travaux de cette thèse consistent à mettre en avant l’utilisation de nouvelles microaiguilles Bella-muTM, qui permet une application simple de la voie ID. Elle induit un ciblage des CPA de la peau avec l’activation des cellules de Langerhans et la prise en charge de l’Ag par les CPA du derme. Par ailleurs, la vaccination VIH est très complexe à cause de son extrême variabilité, ce qui bloque le développement d’un vaccin capable d’induire une réponse protectrice. Une formulation à base d’un peptide très conservé entre les souches formulées avec du Squalène montre une bonne qualité de réponse immunitaire adaptative avec la production d’anticorps neutralisant à large spectre contrairement à l’aluminium. Le deuxième projet de la thèse utilise la microscopie 3D pour étudier les différents éléments de la réponse immunitaire innée induits par ces deux adjuvants qui impactent la réponse humorale dans le ganglion. En conclusion, le choix des stratégies vaccinales tel que la voie d’administration et la formulation qui va déterminer les évènements de la réponse innée mise en place après l’injection qui impact la qualité de la réponse adaptative et donc l’efficacité du vaccin.
Article
The advent of coronavirus disease-2019 (COVID-19), in March 2020, has forced the entire world into continuously changing dimensions, the emergence of variants has put forward a challenge to scientists and doctors, indeed the entire world to cope with it. A lot of clinical research was done, starting with the use of Chloroquine, an anti-malarial drug to treat COVID-19, antivirals and indeed entire world experienced havoc and a death toll due to the coronavirus which led to the emergence of various vaccine platforms developed against changing variants of coronavirus and in this pandemic messenger Ribonucleic acid (mRNA) and nanotechnology added new dimensions to vaccine development. Vaccines may induce additional cellular or humoral immune regulations, including The cell (helper T-cell) responses and germinal center responses, and form relevant memory cells, which in turn enhance their efficiency. As the benefit outweighs the safety issues with vaccination so far, the World Health Organization (WHO), has approved a total of ten vaccines, to use in emergencies, including 3 Indian vaccines (Covovax™, Covaxin® and Sputnik V). The first part, of this mini-review highlights vaccine production, and the clinical trials landscape, and the second part discusses the emergence of global partnerships, and the equity of vaccines to all, taken together, combating pandemics.
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The COVID-19 pandemic and the need for additional safe, effective and affordable vaccines gave new impetus into development of vaccine genetic platforms. Here we report the findings from the phase 1, first-in-human, dose-escalation study of COVID-eVax, a DNA vaccine encoding the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein. Sixty-eight healthy adults received two doses of 0.5, 1 or 2mg 28 days apart, or a single 2mg dose, via intramuscular injection followed by electroporation, and were monitored for 6 months. All participants completed the primary safety and immunogenicity assessments after 8 weeks. COVID-eVax was well tolerated, with mainly mild to moderate solicited adverse events (tenderness, pain, bruising, headache and malaise/fatigue) less frequent after the second dose, and induced an immune response (binding antibodies and/or T cells) at all prime-boost doses tested in up to 90% of the volunteers at the highest dose. However, the vaccine did not induce neutralizing antibodies, while particularly relevant was the T cell-mediated immunity, with a robust Th1 response. This T cell-skewed immunological response adds significant information to the DNA vaccine platform and should be assessed in further studies for its protective capacity and potential usefulness also in other therapeutic areas, such as oncology.
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DNA vaccines, a type of nucleic acid vaccine, have emerged as one of the recent developments in immunology and recombinant DNA technology, offering great potential in terms of ease of manufacture, maintenance, and safety compared to conventional vaccines. Since their discovery, DNA vaccines have evolved immensely, resulting in the employment of new techniques such as gene guns, in vivo electroporation, and nanoparticle-based carriers to enhance the delivery of vaccines into the cells. Starting from the failures of the first-generation DNA vaccines to the near-success second-generation vaccines, several strategies including codon optimization, antigen design, and heterologous prime-boost have greatly helped in strengthening the vaccine's immunogenicity. The purpose of developing these third-generation vaccines is primarily to solve existing medical complications like cancer, along with therapeutic uses, to address health problems, and to aid the rapid eradication of sudden global outbreaks of infectious diseases including Ebola and COVID-19. In this review, we focus on the recent developments and strategies employed to improve the efficacy of DNA vaccines and discuss their mechanism of action, potential concerns, progress achieved, and a brief update on its clinical applications.
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Background Vaccines are needed to prevent coronavirus disease 2019 (Covid-19) and to protect persons who are at high risk for complications. The mRNA-1273 vaccine is a lipid nanoparticle–encapsulated mRNA-based vaccine that encodes the prefusion stabilized full-length spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes Covid-19. Methods This phase 3 randomized, observer-blinded, placebo-controlled trial was conducted at 99 centers across the United States. Persons at high risk for SARS-CoV-2 infection or its complications were randomly assigned in a 1:1 ratio to receive two intramuscular injections of mRNA-1273 (100 μg) or placebo 28 days apart. The primary end point was prevention of Covid-19 illness with onset at least 14 days after the second injection in participants who had not previously been infected with SARS-CoV-2. Results Download a PDF of the Research Summary. The trial enrolled 30,420 volunteers who were randomly assigned in a 1:1 ratio to receive either vaccine or placebo (15,210 participants in each group). More than 96% of participants received both injections, and 2.2% had evidence (serologic, virologic, or both) of SARS-CoV-2 infection at baseline. Symptomatic Covid-19 illness was confirmed in 185 participants in the placebo group (56.5 per 1000 person-years; 95% confidence interval [CI], 48.7 to 65.3) and in 11 participants in the mRNA-1273 group (3.3 per 1000 person-years; 95% CI, 1.7 to 6.0); vaccine efficacy was 94.1% (95% CI, 89.3 to 96.8%; P<0.001). Efficacy was similar across key secondary analyses, including assessment 14 days after the first dose, analyses that included participants who had evidence of SARS-CoV-2 infection at baseline, and analyses in participants 65 years of age or older. Severe Covid-19 occurred in 30 participants, with one fatality; all 30 were in the placebo group. Moderate, transient reactogenicity after vaccination occurred more frequently in the mRNA-1273 group. Serious adverse events were rare, and the incidence was similar in the two groups. Conclusions The mRNA-1273 vaccine showed 94.1% efficacy at preventing Covid-19 illness, including severe disease. Aside from transient local and systemic reactions, no safety concerns were identified. (Funded by the Biomedical Advanced Research and Development Authority and the National Institute of Allergy and Infectious Diseases; COVE ClinicalTrials.gov number, NCT04470427.)
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Background A vaccine against SARS-CoV-2 is of high urgency. Here the safety and immunogenicity induced by a DNA vaccine (INO-4800) targeting the full length spike antigen of SARS-CoV-2 are described. Methods INO-4800 was evaluated in two groups of 20 participants, receiving either 1.0 mg or 2.0 mg of vaccine intradermally followed by CELLECTRA® EP at 0 and 4 weeks. Thirty-nine subjects completed both doses; one subject in the 2.0 mg group discontinued trial participation prior to receiving the second dose. ClinicalTrials.gov identifier: NCT04336410. Findings The median age was 34.5, 55% (22/40) were men and 82.5% (33/40) white. Through week 8, only 6 related Grade 1 adverse events in 5 subjects were observed. None of these increased in frequency with the second administration. No serious adverse events were reported. All 38 subjects evaluable for immunogenicity had cellular and/or humoral immune responses following the second dose of INO-4800. By week 6, 95% (36/38) of the participants seroconverted based on their responses by generating binding (ELISA) and/or neutralizing antibodies (PRNT IC50), with responder geometric mean binding antibody titers of 655.5 [95% CI (255.6, 1681.0)] and 994.2 [95% CI (395.3, 2500.3)] in the 1.0 mg and 2.0 mg groups, respectively. For neutralizing antibody, 78% (14/18) and 84% (16/19) generated a response with corresponding geometric mean titers of 102.3 [95% CI (37.4, 280.3)] and 63.5 [95% CI (39.6, 101.8)], in the respective groups. By week 8, 74% (14/19) and 100% (19/19) of subjects generated T cell responses by IFN-ɣ ELISpot assay with the median SFU per 10⁶ PBMC of 46 [95% CI (21.1, 142.2)] and 71 [95% CI (32.2, 194.4)] in the 1.0 mg and 2.0 mg groups, respectively. Flow cytometry demonstrated a T cell response, dominated by CD8⁺ T cells co-producing IFN-ɣ and TNF-α, without increase in IL-4. Interpretation INO-4800 demonstrated excellent safety and tolerability and was immunogenic in 100% (38/38) of the vaccinated subjects by eliciting either or both humoral or cellular immune responses. Funding Coalition for Epidemic Preparedness Innovations (CEPI).
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Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (Covid-19) have afflicted tens of millions of people in a worldwide pandemic. Safe and effective vaccines are needed urgently. Methods Download a PDF of the Research Summary. In an ongoing multinational, placebo-controlled, observer-blinded, pivotal efficacy trial, we randomly assigned persons 16 years of age or older in a 1:1 ratio to receive two doses, 21 days apart, of either placebo or the BNT162b2 vaccine candidate (30 μg per dose). BNT162b2 is a lipid nanoparticle–formulated, nucleoside-modified RNA vaccine that encodes a prefusion stabilized, membrane-anchored SARS-CoV-2 full-length spike protein. The primary end points were efficacy of the vaccine against laboratory-confirmed Covid-19 and safety. Results A total of 43,548 participants underwent randomization, of whom 43,448 received injections: 21,720 with BNT162b2 and 21,728 with placebo. There were 8 cases of Covid-19 with onset at least 7 days after the second dose among participants assigned to receive BNT162b2 and 162 cases among those assigned to placebo; BNT162b2 was 95% effective in preventing Covid-19 (95% credible interval, 90.3 to 97.6). Similar vaccine efficacy (generally 90 to 100%) was observed across subgroups defined by age, sex, race, ethnicity, baseline body-mass index, and the presence of coexisting conditions. Among 10 cases of severe Covid-19 with onset after the first dose, 9 occurred in placebo recipients and 1 in a BNT162b2 recipient. The safety profile of BNT162b2 was characterized by short-term, mild-to-moderate pain at the injection site, fatigue, and headache. The incidence of serious adverse events was low and was similar in the vaccine and placebo groups. Conclusions A two-dose regimen of BNT162b2 conferred 95% protection against Covid-19 in persons 16 years of age or older. Safety over a median of 2 months was similar to that of other viral vaccines. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04368728.)
Article
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Background The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. Methods We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18–55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 10¹⁰ viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT50]; a microneutralisation assay [MNA50, MNA80, and MNA90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Findings Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p<0·05). There were no serious adverse events related to ChAdOx1 nCoV-19. In the ChAdOx1 nCoV-19 group, spike-specific T-cell responses peaked on day 14 (median 856 spot-forming cells per million peripheral blood mononuclear cells, IQR 493–1802; n=43). Anti-spike IgG responses rose by day 28 (median 157 ELISA units [EU], 96–317; n=127), and were boosted following a second dose (639 EU, 360–792; n=10). Neutralising antibody responses against SARS-CoV-2 were detected in 32 (91%) of 35 participants after a single dose when measured in MNA80 and in 35 (100%) participants when measured in PRNT50. After a booster dose, all participants had neutralising activity (nine of nine in MNA80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R²=0·67 by Marburg VN; p<0·001). Interpretation ChAdOx1 nCoV-19 showed an acceptable safety profile, and homologous boosting increased antibody responses. These results, together with the induction of both humoral and cellular immune responses, support large-scale evaluation of this candidate vaccine in an ongoing phase 3 programme. Funding UK Research and Innovation, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research (NIHR), NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and the German Center for Infection Research (DZIF), Partner site Gießen-Marburg-Langen.
Article
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We 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. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500µg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-γ levels.
Preprint
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We 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. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500μg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-γ levels.
Article
Background A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein 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 and allocated 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 registered with 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 low dose 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, and the 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 and neutralising 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.
Article
Background Since December, 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiological and clinical characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clinical course of illness, including viral shedding, have not been well described. Methods In this retrospective, multicentre cohort study, we included all adult inpatients (≥18 years old) with laboratory-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demographic, clinical, treatment, and laboratory data, including serial samples for viral RNA detection, were extracted from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors associated with in-hospital death. Findings 191 patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. 91 (48%) patients had a comorbidity, with hypertension being the most common (58 [30%] patients), followed by diabetes (36 [19%] patients) and coronary heart disease (15 [8%] patients). Multivariable regression showed increasing odds of in-hospital death associated with older age (odds ratio 1·10, 95% CI 1·03–1·17, per year increase; p=0·0043), higher Sequential Organ Failure Assessment (SOFA) score (5·65, 2·61–12·23; p<0·0001), and d-dimer greater than 1 μg/L (18·42, 2·64–128·55; p=0·0033) on admission. Median duration of viral shedding was 20·0 days (IQR 17·0–24·0) in survivors, but SARS-CoV-2 was detectable until death in non-survivors. The longest observed duration of viral shedding in survivors was 37 days. Interpretation The potential risk factors of older age, high SOFA score, and d-dimer greater than 1 μg/L could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future. Funding Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences; National Science Grant for Distinguished Young Scholars; National Key Research and Development Program of China; The Beijing Science and Technology Project; and Major Projects of National Science and Technology on New Drug Creation and Development.