Efficacy, safety, and immunogenicity of the DNA SARS-CoV-2 vaccine (ZyCoV-D): the interim efficacy results of a phase 3, randomised, double-blind, placebo-controlled study in India
Study design and participants
We investigated the efficacy, safety, and immunogenicity of ZyCoV-D at 2 mg dose administered intradermally via a needle-free injection system, compared with placebo, in a multicentre, double-blind, randomised, controlled trial at 49 hospitals in India. Healthy male and female individuals aged 12 years or older were eligible to participate in the study. Individuals with febrile illness, confirmed SARS-CoV-2, a history of SARS/MERS infection, history of contact with SARS-CoV-2-infected patients within the previous 14 days, confirmed immunosuppressive or immunodeficiency disorder, on any immunosuppressive or immunostimulant therapy, or other clinically significant systemic disorder were excluded from the study.
The study was initiated after approval from an ethics committee at each study site and local regulatory authorities. Written informed consent from all the participants was obtained before initiation of any study-related procedures. Additionally, assent was taken from participants aged 12–17 years and parents or guardians were asked for consent. The study was done in accordance with all national and local regulations as well as Indian Good Clinical Practice. The study was monitored by an independent data safety monitoring board.
Randomisation and masking
Eligible participants were randomly assigned (1:1) to ZyCoV-D vaccine or placebo using a randomisation schedule generated using SAS software (version 9.4 or higher) with the help of an interactive web response systems (IWRS). The IWRS was used for randomisation (blocks of four) of participants as well as to enrol individuals aged 60 years and older with or without comorbid conditions, and those aged 12–17 years. It was also used to identify 600 participants for immunogenicity (blocks of six).
Investigators at sites received random allocation information through the IWRS. The randomisation sequence was generated by an independent statistician using SAS and fed into the IWRS. Participants were enrolled by investigators with the help of the IWRS.
The population also included a subgroup of participants aged 12–17 years, participants aged older than 60 years, and participants with comorbidities (ie, hypertension, diabetes, obesity, chronic respiratory diseases, chronic kidney disease, or chronic heart disease).
The study medication was supplied in identical packages. The study medications were similar in colour and appearance, thereby maintaining double-blind conditions. The masking of individual allocation was maintained for all investigators and participants.
ZyCoV-D is comprised of a DNA plasmid vector pVAX1 carrying gene-expressing spikeS protein of SARS-CoV-2 and IgE signal peptide. The spike gene region was selected from submitted Wuhan Hu1 isolate (Genebank accession number MN908947.3).
All enrolled participants received three doses of ZyCoV-D vaccine (2 mg per dose) or placebo intradermally via a needle-free injection system 28 days apart (days 0, 28, and 56). Participants were kept under medical observation for 30 min after vaccine administration to assess adverse reactions.
After vaccine administration, a series of telephone follow-up visits were scheduled to detect suspected symptomatic COVID-19. Participants were then grouped in to one of three categories to identify asymptomatic and symptomatic individuals with SARS-CoV-2 infection as well as to assess the immunogenicity response.
Category 1 included participants who were symptomatic (pre-planned number of participants n=17 616). Telephone follow-up visits were done on days 70, 98, 112, 126, 140, 154, 168, 182, 196, 210, 224, 238, 252, 266, 280, 294, 308, 322, 336, and 350 (plus or minus 3 days) after the first vaccine dose to detect symptomatic COVID-19 cases following doses. If COVID-19 was suspected, nasopharyngeal and oropharyngeal swabs were collected for RT-PCR testing to confirm SARS-CoV-2 infection. The study used a case adjudication committee to review the source data of suspected cases, where RT-PCR results were equivocal, or the symptomology was suspected and not recorded correctly.
Category 2 included participants who were asymptomatic, and remained asymptomatic, or became symptomatic (pre-planned number of participants n=10 000). In addition to the procedures listed for category 1, in this group nasopharyngeal and oropharyngeal swabs to detect asymptomatic COVID-19 were collected every 4 weeks for up to 32 weeks after the third vaccine dose. During the telephone follow-up visits, if a suspected case of COVID-19 was identified, a nasopharyngeal and oropharyngeal sample was collected for RT-PCR testing to confirm SARS-CoV-2 infection. Telephone follow-up visits were scheduled for days 70, 98, 126, 154, 182, 210, 238, 252, 266, 280, 294, 308, 322, 336, and 350 (plus or minus 3 days) after the first vaccine dose.
Category 3 included participants who were symptomatic and had an immunogenicity assessment (pre-planned number of participants n=600). In addition to the procedures outlined in category 1, blood samples were collected for serum analysis for immunological assessment. Telephone follow-up visits were scheduled for days 70, 98, 126, 154, 182, 210, 224, 238, 266, 280, 294, 308, 322, 336, and 350 (plus or minus 3 days) after the first dose. During these follow-up visits, if COVID-19 was suspected, nasopharyngeal and oropharyngeal samples were collected for RT-PCR testing to confirm SARS-CoV-2 infection. Of the 600 participants, 100 participants were enrolled in the group aged 12–17 years and older than 60 years. Enrolment was ongoing at the time of interim analysis. The data cutoff date for the interim analysis was June 23, 2021, and interim analysis was done on June 26, 2021.
A diary card was issued to all participants for self-recording of solicited adverse events. The local solicited adverse events listed in the diary card were pain, redness, swelling, and itching. The systemic solicited adverse events listed in the diary card were fever, headache, tiredness, nausea, vomiting, diarrhoea, joint pain, chills, and muscle pain.
The primary efficacy endpoint was the number of participants with first occurrence of symptomatic RT-PCR-positive COVID-19, 28 days after the third dose of ZyCoV-D vaccine, until the targeted number of cases (n=158) had been achieved. The interim analysis of the primary endpoint was conducted once 50% (79 cases) of the target number of cases was met. Participants infected with SARS-CoV-2 were categorised as asymptomatic (RT-PCR-positive without any signs and symptoms), mild (RT-PCR-positive with signs and symptoms of COVID-19), moderate (RT-PCR-positive and pneumonia with no signs of severe disease), or severe (RT-PCR positive with severe pneumonia). For primary outcome assessment, the symptomatic mild, moderate, and severe cases were combined. Nasopharyngeal and oropharyngeal swabs were collected from the participants with any one symptom of COVID-19 lasting for at least 48 h.
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Seroconversion was defined as antibody-negative participants at baseline who became antibody positive after vaccination. The IgG concentration was analysed with a standard S1 ELISA using NIBSC standard with a concentration range of 45·23–1·4 EU. Additionally, a plaque reduction neutralisation test (PRNT50; ie, the concentration of serum to reduce plaques by 50% compared with the serum-free virus) was used for estimation of neutralising antibody titre in human serum samples against anti-SARS-CoV-2 on days 0, 56, and 84. In this assay, a reduction in the number of plaques formed by the virus correlates with the presence of neutralising antibody in the serum samples. The cellular response assessment included assessment of IFN-γ from PBMC samples. The methods for cellular assays has been previously described.
- Momin T
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Safety assessment included incidence and severity of solicited and unsolicited adverse events after each dose and incidence of serious adverse events throughout the study. Assessment of safety, tolerability, and baseline characteristics was based on the safety population, which consisted of all enrolled participants who were known to have received at least one dose of study vaccine or placebo.
Assuming vaccine efficacy of 60% after the third dose, approximately 158 confirmed COVID-19 cases would provide 90% power to conclude true vaccine efficacy of more than 30%, allowing early cessation for efficacy at interim analysis. A total of 11 286 evaluable participants per group (80% of 14 108 participants, accounting for 20% dropout or non-evaluable participants when randomly assigned [1:1] with placebo), means a total sample size of 28 216 was required. We assumed a 1% attack rate per year in the placebo group; thus, accrual of 158 COVID-19 cases within 6 months was required.
An interim analysis was done by independent statisticians after 50% of the target events (79 cases) were achieved. The interim analysis of a primary efficacy endpoint was based on the per-protocol population, which consisted of all participants with negative baseline SARS-CoV-2 status and who received three doses of vaccine or placebo. COVID-19 cases were adjudicated by a masked committee (comprising two independent members: one internal medicine physician and one pulmonologist).
Vaccine efficacy for the primary endpoint was analysed by calculating the infection rate ratio (IRR). The vaccine efficacy was compared using a standard statistical conditional exact test, based on the conditional binomial distribution of the number of infected cases in the vaccine group, given the total number of cases in both groups. Vaccine efficacy was defined as 100 × (1 – IRR), where IRR was calculated as the ratio of the first occurrence of symptomatic RT-PCR-positive COVID-19 cases, 28 days after the third dose of ZyCoV-D vaccination.
The secondary efficacy endpoints were evaluated using the same methods as the primary endpoint. Descriptive summary statistics for local reactions, systemic events, adverse events or serious adverse events, and laboratory parameters are presented.
The immunogenicity assessment including GMT, GMFR, and associated 95% CIs was presented for SARS-CoV-2 IgG on days 0, 56, and 84. Neutralisation titre (GMT, GMFR, and associated 95% CIs) was analysed in subgroups on days 0, 56, and 84. Cellular responses were analysed at days 0, 56, and 84.
This trial is registered with Clinical Trial Registry India, CTRI/2021/01/030416.
Role of the funding source
Cadila Healthcare designed and conducted the study and was involved in the study design, data collection, data analysis, data interpretation, and writing of the report.
Table 1Summary of demographics and baseline characteristics (safety population)
Data are mean (SD), median (range), or n (%). NA=not applicable.
12 350 participants in the ZyCoV-D group and 12 320 participants in the placebo group, who completed 28 days after the third dose, were included in the per-protocol population. 81 participants with COVID-19 were eligible for interim primary efficacy endpoint analysis per protocol. 20 cases occurred in the ZyCoV-D group and 61 in the placebo group. ZyCoV-D vaccine efficacy was 66·6% (95% CI 47·6–80·7).
During the study, one severe COVID-19 case occurred after the second dose. The event was considered a fatal adverse event. As per the ethics committee recommendation, the participant’s treatment code was unblinded, and the participant was found to have received placebo. Given no other severe COVID-19-related adverse events occurred in the vaccine group, ZyCoV-D was found to be 100% efficacious to prevent severe cases of COVID-19 after two doses. All three of the reported moderate COVID-19 cases were identified to have occurred in the placebo group. ZyCoV-D was therefore found to be 100% efficacious in moderate cases. 58 of 78 mild COVID-19 cases were identified in the placebo group and 20 cases in the ZyCoV-D group. ZyCoV-D therefore had an efficacy of 64·9% (95% CI 44·9–79·8) in mild cases.
Table 2Analysis of antibody titre (immunogenicity population)
GMT=geometric mean titre. GMFR=geometric mean fold rise.
Table 3Summary and comparison of seroconversion of neutralising antibodies (immunogenicity population)
Data are n (%). GMT=geometric mean titre. GMFR=geometric mean fold rise. Day-wise comparison of proportion of participants, with seroconversion rate based on IgG antibodies as compared with baseline, between ZyCov-D and placebo is evaluated using χ2 test. Significant p value (<0·05) indicates that there is a significant difference between ZyCov-D and placebo based on proportion of participants with seroconversion rate.
Table 4Analysis of neutralising antibodies (PRNT50; immunogenicity population)
GMT=geometric mean titre. GMFR=geometric mean fold rise.
At the data cutoff, 13 849 participants had received a first dose of ZyCoV-D vaccine and 13 852 had received a first dose of placebo; 13 153 had received a second dose of ZyCoV-D and 13 129 had received a second dose of placebo; and 12 773 had received a third dose of ZyCoV-D and 12 733 had received a third dose of placebo.
508 participants experienced 917 unsolicited adverse events during the study. The occurrence of unsolicited adverse events was also similar between the treatment groups (454 [3·27%] in the ZyCoV-D group vs 463 [3·34%] in the placebo group). In the ZyCoV-D group, 18 participants reported 43 events after the first dose, 26 participants reported 74 events after the second dose, and 198 participants reported 337 events after the third dose. In the placebo group, 20 participants reported 46 events after the first dose, 26 participants reported 64 events after the second dose, and 220 participants reported 353 events after the third dose.
Overall, there was no difference between the treatment groups in solicited or unsolicited adverse events. The most frequently reported solicited local adverse events in both the treatment groups (ZyCoV-D and placebo, respectively) were pain at injection site (92 [0·66%] and 82 [0·62%] after dose 1, 45 [0·34%] and 46 [0·35%] after dose 2, and 35 [0·27%] and 33 [0·26%] after dose 3), redness (43 [0·31%] and 39 [0·28%] after dose 1, 25 [0·19%] and 12 [0·09%] after dose 2, and 22 [0·17%] and 11 [0·09%] after dose 3), swelling (38 [0·27%] and 39 [0·28%] after dose 1, ten [0·08%] and eight [0·06%] after dose 2, and 12 [0·09%] and seven [0·05%] after dose 3), and itching (11 [0·08%] and 20 [0·14%] after dose 1, seven [0·05%] and nine [0·07%] after dose 2, and three [0·02%] and seven [0·05%] after dose 3). Most of the adverse events were mild or moderate in intensity. These events were similar between ZyCoV-D and placebo groups. The most commonly reported solicited systemic adverse events in both treatment groups (ZyCoV-D and placebo, respectively) were headache (34 [0·25%] and 30 [0·22%] after dose 1, 26 [0·20%] and 31 [0·24%] after dose 2, and 21 [0·16%] and 22 [0·17%] after dose 3), fever (28 [0·20%] and 19 [0·14%] after dose 1, 19 [0·14%] and 27 [0·21%] after dose 2, and 16 [0·13%] and 13 [0·10%] after dose 3), muscle pain (27 [0·19%] and 39 [0·28%] after dose 1, 15 [0·11%] and 24 [0·18%] after dose 2, and 14 [0·11%] and 12 [0·09%] after dose 3), and fatigue (26 [0·19%] both groups after dose 1, 18 [0·14%] and 21 [0·16%] after dose 2, and 11 [0·09%] and 17 [0·13%] after dose 3).
Most of the adverse events were mild or moderate in intensity. No different was observed with respect to successive dosing within each group or between the treatment groups.
Some of the commonly reported unsolicited adverse events were arthralgia, back pain, muscle spasms, myalgia, musculoskeletal pain, neck pain, vertigo, diarrhoea, gastritis, gastro-oesophageal reflux disease, nausea, vomiting, asthenia, chills, eye irritation, abdominal distension, abdominal pain, fatigue, pain, pyrexia, nasopharyngitis, pain in extremity, ageusia, anosmia, cerebral infarction, dizziness, headache, cough, dyspnoea, nasal dryness, oropharyngeal pain, rhinorrhoea, sneezing.
The safety profile in terms of solicited and unsolicited adverse events in adolescent participants (12–17 years), participants older than 60 years, and participants with comorbidities was similar to the general study population.
As of data cutoff, 15 serious adverse events were reported, including two fatal serious adverse events. These serious adverse events included cerebrovascular stroke (one event), cardiorespiratory arrest with septicaemia and alcoholic liver disease (one event), COVID-19 positive (seven events), cerebral infarct (one event), COVID-19 pneumonia (three events), hypoxaemia with COVID-19 (one event), and gram-negative enteritis with early shock (one event). All serious adverse events were mild to moderate in intensity except for the event of cerebrovascular stroke, COVID-19 pneumonia, and cardiorespiratory arrest, which were severe in intensity. All serious adverse events resolved, except for one event of COVID-19 pneumonia which remained unchanged at data cutoff as well as two deaths. None of the serious adverse events was considered causally related to vaccine or placebo.
The death (cardiorespiratory arrest with septicaemia and alcoholic liver disease) occurred after first dose and death event COVID-19 pneumonia occurred after second dose of study intervention. Both the events were considered unrelated to study intervention. For the death due to cardiorespiratory arrest, the treatment code was not unblinded, whereas for the death due to COVID-19 pneumonia, the treatment code was unblinded and the participant was found to be in the placebo group.
We report interim efficacy, safety, and immunogenicity findings from the phase 3 clinical study conducted in India for the DNA vaccine ZyCoV-D to prevent COVID-19. A three-dose regimen of ZyCoV-D, administered intradermally via a needle-free injection system, was found to be 66·6% effective against COVID-19. To our knowledge, this is the first time a DNA vaccine has been tested in a large population in India
Tracking of variants—April 26, 2021.
The number of delta variant cases detected in sample sequencing in April, May, and June, 2021, were 5356, 4947, and 3184, respectively. Cumulatively, India reported 19 766 (38%) delta sequences from Oct 15, 2020, to Sept 12, 2021.
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Therefore, we conclude that the ZyCoV-D vaccine is also effective against the delta variant. Considering that no severe or moderate COVID-19 cases were reported in the ZyCoV-D group, and based on the interim analysis results, the vaccine was found to be 100% effective against severe and moderate COVID-19 cases and 64·9% effective against mild COVID-19 cases. Therefore, it is possible that the severe and moderate cases that might result in fatalities and put enormous pressure on health-care systems could be prevented to a great extent with full vaccination using the ZyCoV-D vaccine.
The immunogenicity response of ZyCoV-D seen in phase 1/2 was maintained in the phase 3 study as well. The ZyCoV-D vaccine elicited a significantly high immunogenicity response at day 84 as evident from the seroconversion rate (93·33%) based on IgG against S1 antigen (by ELISA) and GMT (952·67 EU, 95% CI 707·9–1282·0) and GMFR (136·09, 95% CI 101·11–183·1) and neutralising antibody titre (GMT: 133·39 PRNT50, 95% CI 86·88–204·81; GMFR: 26·68, 95% CI 17·38–40·96). The vaccine also induced a significant cellular response as evident from higher IFN-γ levels in the ZyCoV-D group compared with the placebo group.
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Considerations for plasmid DNA vaccine for infectious disease indication. Guidance for industry.
The thermostability of the vaccine will aid transportation and storage of the vaccine and reduce any cold chain breakdown challenges, thereby preventing vaccine wastage. The plasmid DNA platform provides ease of manufacturing with minimal biosafety requirements. Being a plasmid DNA vaccine, ZyCoV-D does not share the problems often associated with vector-based immunity, such as poor immune response to target antigens following vaccination due to pre-existing antibody titres to vectors resulting from natural infections from vectors such as adenoviruses, measles, and influenza viruses. Other problems associated with vector-based immunity include a requirement for very high doses resulting in several side-effects, and a requirement for long periods between booster doses to minimise the effect of vector immunity interference. The plasmid DNA platform also allows for the rapid generation of new constructs to deal with mutations in the virus. The three-dose regimen that might be perceived as cumbersome is actually equivalent to a third booster dose, which is being considered by many countries in existing vaccine platforms.
The study has several limitations: it was conducted only in a predominantly male population in India; the sample size was not calculated on the basis of subgroup analysis; the efficacy analysis was not performed on the basis of age group; the efficacy analysis was not done after the first and second dose for mild, moderate, and severe cases; and the common laboratory investigations such as haematology, renal function tests, and liver function tests were not evaluated. The sample size was small and the duration of the study was short at the time of the interim analysis when the efficacy of ZyCoV-D in prevention of severe COVID-19 was assessed as 100%.
To our knowledge, no previous phase 3 studies have been published on the efficacy of a DNA plasmid vaccine on SARS-CoV-2. Our study therefore provides the first evidence that induction of immune responses against the spike protein using a plasmid construct carrying spike-S-gene provides protection against COVID-19 in humans. From the interim analysis of this phrase 3 trial, ZyCoV-D was found to be efficacious, safe, and immunogenic, and it could significantly contribute to the efforts to contain the COVID-19 outbreak in India and globally.
AK, SB, VR, SD, KG, HP, PS, IG, RR, RN, PK, Kalpesh Talati, Manish Hathila, Hari Shankar Gupta, Sharad Agarkhedkar, Sanjay Lalwani, Deepak Langade, Samir Gami, Rajendra Nerli, A G Srinivas Murthy, Vipul Khandelwal, Sandeep Jain, Dinesh Agarwal, Swapnav Borthakur, Tanmoy Mandal, Pankaj Bhardwaj, Parul Bhatt, Satyanarayan N Sharma, Animesh Choudhary, Shiva Narang, Vijay Kumar Shukla, Saurabh Agarwal, Sandeep Kumar Gupta, R Kulandaivel, Ravindra Mehta, A Vankateshwar Rao, K M Shivkumar, Raman Sharma, Dharmendra Gupta, Arepalli Sreedevi, N Srinivas Rao, V Rama Krishna CH, Veer Bahadur Singh, Manish Jain, Prashant Khandgave, Bhaskar Jedhe Deshmukh.
JS and KK were involved in conceptualisation and designing of clinical study. AD was involved in designing and developing vaccine candidate, and doing data analysis for ELISPOT and Luminex assay. TMCR was involved in immunogenicity analysis. Each author contributed important intellectual content during manuscript drafting 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.