An Omicron-specific, self-amplifying mRNA booster vaccine for COVID-19: a phase 2/3 randomized trial

Here we conducted a multicenter open-label, randomized phase 2 and 3 study to assess the safety and immunogenicity of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron-specific (BA.1/B.1.1.529), monovalent, thermostable, self-amplifying mRNA vaccine, GEMCOVAC-OM, when administered intradermally as a booster in healthy adults who had received two doses of BBV152 or ChAdOx1 nCoV-19. GEMCOVAC-OM was well tolerated with no related serious adverse events in both phase 2 and phase 3. In phase 2, the safety and immunogenicity of GEMCOVAC-OM was compared with our prototype mRNA vaccine GEMCOVAC-19 (D614G variant-specific) in 140 participants. At day 29 after vaccination, there was a significant rise in anti-spike (BA.1) IgG antibodies with GEMCOVAC-OM (P < 0.0001) and GEMCOVAC-19 (P < 0.0001). However, the IgG titers (primary endpoint) and seroconversion were higher with GEMCOVAC-OM (P < 0.0001). In phase 3, GEMCOVAC-OM was compared with ChAdOx1 nCoV-19 in 3,140 participants (safety cohort), which included an immunogenicity cohort of 420 participants. At day 29, neutralizing antibody titers against the BA.1 variant of SARS-CoV-2 were significantly higher than baseline in the GEMCOVAC-OM arm (P < 0.0001), but not in the ChAdOx1 nCoV-19 arm (P = 0.1490). GEMCOVAC-OM was noninferior (primary endpoint) and superior to ChAdOx1 nCoV-19 in terms of neutralizing antibody titers and seroconversion rate (lower bound 95% confidence interval of least square geometric mean ratio >1 and difference in seroconversion >0% for superiority). At day 29, anti-spike IgG antibodies and seroconversion (secondary endpoints) were significantly higher with GEMCOVAC-OM (P < 0.0001). These results demonstrate that GEMCOVAC-OM is safe and boosts immune responses against the B.1.1.529 variant. Clinical Trial Registry India identifier: CTRI/2022/10/046475.


INTRODUCTION
This Statistical Analysis Plan (SAP) describes a comprehensive and detailed description of the strategy and statistical technique to be used for the analysis of data for protocol GBL/GEMCOVAC-OM/2022/02, entitled 'A Prospective, Multi-centre, Open-labelled, Randomized, Phase II study seamlessly followed by a Phase III study to evaluate the Safety, Tolerability and Immunogenicity of GEMCOVAC-OM as a booster in Subjects 18 years of age and older'.
Phase II and III parallel-arm, multicentre clinical studies are being completed to assess the Safety, Tolerability and Immunogenicity of GEMCOVAC-OM as a booster in Subjects 18 years of age and older.
The reader of this SAP is encouraged to also read the clinical protocols for details on the conduct of this study and the operational aspects of clinical assessments and timing for completing a participant enrollment in this study.The purpose of this SAP is to outline the planned analyses to be completed to support the completion of the Clinical Study Report (CSR) for protocol GBL/GEMCOVAC-OM/2022/02.The planned analyses identified in this SAP will be included in regulatory submissions if applicable and/or future manuscripts.In addition, exploratory analyses not necessarily identified in this SAP may be performed to support the clinical development program.Any post-hoc or unplanned analyses which is not identified in this SAP and performed will be clearly identified in the respective CSR.

Protocol Title
A Prospective, Multi-centre, Open-labelled, Randomized, Phase II study seamlessly followed by a Phase III study to evaluate the Safety, Tolerability and Immunogenicity of GEMCOVAC-OM as a booster in Subjects 18 years of age and older.

Primary
Primary endpoints include: 1.To assess the safety of adult subjects who received GEMCOVAC-OM as a booster dose till Day 180 • Occurrence and severity of local and systemic reactogenicity adverse events (AEs) for 7 days following vaccination.

Study Design and Plan
This is a Phase II seamlessly followed by Phase III study to evaluate the immunogenicity and safety of a booster dose of the Omicron-specific mRNA vaccine -GEMCOVAC-OM.The study participants will be adult subjects who are fully vaccinated against COVID-19 with either COVAXIN™ or COVISHIELD™ and received last dose of primary vaccination at least 4 months prior to screening.This booster dose of GEMCOVAC-OM will be based on the sequence of Omicron variant BA.1 of SARS-CoV-2.The study will be conducted in two parts: Phase II part will be conducted in 140 subjects while Phase III part of the study will enrol 3140 subjects.

Phase II
Approximately 140 subjects will be randomized, and the enrolment will be competitive.
Hence, the subjects will be randomized in 1:1 ratio into two arms: Arm I: 70 Subjects who have received either COVAXIN™ or COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of GEMCOVAC-OM (intra-dermal).
Arm II: 70 Subjects who have received either COVAXIN™ or COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of GEMCOVAC-19 (Intramuscular) It will be ensured that at least 20% (n ~14) of the participants in each arm will have received COVAXIN™ and COVISHIELD™ as their primary vaccination.
All the subjects randomized in Arm I will receive 1 dose of GEMCOVAC-OM and those who are randomized in Arm II will receive 1 dose of GEMCOVAC-19 (Intramuscular) on Day 1. GEMCOVAC-OM will be administered intradermally by PharmaJet Tropis Needle-Free Injector.

Gennova Biopharmaceuticals
Ltd. Approximately 3140 subjects will be randomized, and the enrolment will be competitive.The subjects will be randomized into two arms: Arm I: 3000 Subjects who have received either COVAXIN™ or COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of GEMCOVAC-OM Arm II: 140 Subjects who have received COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of COVISHIELD™ All the subjects randomized in Arm I will receive 1 dose of GEMCOVAC-OM and subjects randomized in Arm II will receive 1 dose of COVISHIELD™.

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The immunogenicity subset will include 280 subjects from Arm I and all the subjects from Arm II (n=140).The dose of GEMCOVAC-OM will be administered by PharmaJet Tropis Needle-Free Injector.
In the immunogenicity cohort of Arm I, it will be ensured that a minimum of 42 participants (15%) will have receieved COVAXIN™ and COVISHIELD™ as their primary vaccination.
In the safety cohort of Arm I, a minimum of 300 participants (10%) will receive COVAXIN™ and COVISHIELD™.
Each subject must agree to participate in screening procedures by signing the most recent Ethics Committee approved Informed Consent Form (ICF) before any screening procedure is initiated.Each subject will be assigned a unique screening number on first cum first basis.
Subjects satisfying the inclusion and none of the exclusion criteria will be randomized in the study.
Subjects who qualify screening assessments will be randomized in the study and will receive single dose of booster vaccine on Visit 1 (Day 1).
Subjects will be assessed for infection risk category at screening.Physical examination/ Assessment of the main vital signs will be performed at all onsite visits./SAEs will be recorded throughout the study.Urine pregnancy test will be performed at screening for female of childbearing potential.

Gennova Biopharmaceuticals
Ltd.The subjects will have post vaccination telephonic follow-up on Day 7.

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A thermometer will be provided to all the randomized subjects, and they will be instructed to record their daily body temperature, for a period of 1 week after booster dose administration/randomization in e-diary/paper diary, wherever usage of e-diary is not feasible.Immunogenicity assessments will be performed in all the randomized subjects as per schedule of events.
All subjects receiving booster dose will participate in this clinical trial for approximately 6 months (Day 180+14 days) after the administration of the vaccine.The participants will have one screening/ vaccination visit and three on-site follow-up visits during the trial period.
The sites will collect the subject data using appropriate case report forms.Institutional Review Board/Institutional Ethics Committee approval will be taken prior to study initiation.Study procedures will be as per assessment schedule.

Study Flow Chart
In Phase II, participants who have received 2 doses of either COVAXIN™ or COVISHIELD™ will be enrolled in both the arms.It will be ensured that at least 20% (n ~14) of the participants in each arm will have received COVAXIN™ and COVISHIELD™ as their primary vaccination.
In Phase III, participants who received 2 doses of either COVAXIN™ or COVISHIELD™ will be enrolled in the GEMCOVAC-OM booster arm.Whereas, in the COVISHIELD™ booster arm, participants who have received 2 doses of COVISHIELD™ only will be enrolled.In the immunogenicity cohort of Arm I, a minimum of 42 participants (15%) will have received COVAXIN™ and COVISHIELD™.In the safety cohort of Arm I, a minimum of 300 participants (10%) will have received COVAXIN™ and COVISHIELD™ as their primary vaccination.

Gennova Biopharmaceuticals
Ltd.All subjects receiving GEMCOVAC-19 will participate in this clinical trial for approximately 6 months (Day 180+14 days) after the administration of the vaccine.The participants will be administered as a single dose on Day 1. Vaccinated subjects included in the study will be followed up to approximately 6 months (180+ 14 days) of study period thereafter.

Selection of Study Population
Subjects who meet all the inclusion criteria and none of the exclusion criteria, will be randomized in the study.The study participants will be adult subjects who are fully vaccinated against COVID-19 with either COVAXIN™ or COVISHIELD™ and have received last dose of primary vaccination at least 4 months prior to screening.Each subject will be evaluated from the day of signing of informed consent form till the End of Study.

Study Subject Group
Phase II Approximately 140 subjects will be randomized, and the enrolment will be competitive.
Hence, the subjects will be randomized in 1:1 ratio into two arms: Arm I: 70 Subjects who have received either COVAXIN™ or COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of GEMCOVAC-OM (intra-dermal).
Arm II: 70 Subjects who have received either COVAXIN™ or COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of GEMCOVAC-19 (Intramuscular) It will be ensured that at least 20% (n ~14) of the participants in each arm will have received COVAXIN™ and COVISHIELD™ as their primary vaccination.
All the subjects randomized in Arm I will receive 1 dose of GEMCOVAC-OM and those who are randomized in Arm II will receive 1 dose of GEMCOVAC-19 (Intramuscular) on Day 1. GEMCOVAC-OM will be administered intradermally by PharmaJet Tropis Needle-Free Injector.

Phase III
Approximately 3140 subjects will be randomized, and the enrolment will be competitive.The subjects will be randomized into two arms: Arm I: 3000 Subjects who have received either COVAXIN™ or COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of GEMCOVAC-OM Arm II: 140 Subjects who have received COVISHIELD™ as primary vaccination (both doses) will receive a booster dose of COVISHIELD™ All the subjects randomized in Arm I will receive 1 dose of GEMCOVAC-OM and subjects randomized in Arm II will receive 1 dose of COVISHIELD™.
The immunogenicity subset will include 280 subjects from Arm I and all the subjects from Arm II (n=140).The dose of GEMCOVAC-OM will be administered by PharmaJet Tropis Needle-Free Injector.
In the immunogenicity cohort of Arm I, it will be ensured that a minimum of 42 participants (15%) will have received COVAXIN™ and COVISHIELD™ as their primary vaccination.
In the safety cohort of Arm I, a minimum of 300 participants (10%) will have receieved COVAXIN™ and COVISHIELD™ as their primary vaccination.
Each subject must agree to participate in screening procedures by signing the most recent Ethics Committee approved Informed Consent Form (ICF) before any screening procedure is initiated.Each subject will be assigned a unique screening number on first cum first basis.
Subjects satisfying the inclusion and none of the exclusion criteria will be randomized in the study.
Subjects who qualify screening assessments will be randomized in the study and will receive single dose of booster vaccine on Visit 1 (Day 1).

Gennova Biopharmaceuticals
Ltd.A sample size of 420 (280 in GEMCOVAC-OM and 140 in COVISHIELD™ arm) in the immunogenicity cohort will provide adequate numbers for the statistical analysis considering a non-inferiority margin of 0.67, standard deviation of 1.82, alpha error of 5% and power of 90%.

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Case

Non-inferiority of difference in seroresponse rate
The immunogenicity cohort was calculated based on the WHO guideline of non-inferiority defines as lower bound of 95% CI in seroresponse rate difference (Seroconversion Rate GEMCOVAC-OM -Seroconversion Rate COVISHIELD™) >-10%.
The sample size is allocated in a 2:1 ratio between GEMCOVAC-OM and COVISHIELD™ arms and includes 20% dropout rate.
A sample size of 381 (254 in GEMCOVAC-OM and 127 in COVISHEILD™) in the immunogenicity cohort will provide adequate numbers for the statistical analysis considering a non-inferiority of -10%, alpha error of 5% and power of 90%.

Gennova Biopharmaceuticals
Ltd.The sample size of 420 (280 in GEMCOVAC-OM and 140 in COVISHIELD™ arm) is considered in this study to provide adequate numbers for the statistical analysis of both the primary endpoints.

Analysis Population
For the statistical analysis, following populations of the subjects will be considered:

Safety population
This population will include all the subjects with confirmation of receiving the study vaccine (Answer to 'Was IP administered:'=Yes').This population will serve as the primary population for the immunogenicity objectives for testing non-inferiority hypotheses.This population will also be used for the immunogenicity analysis related to secondary objectives along with FAS population.

Method of Treatment Assignment, Randomization and Blinding
Subjects who meet the inclusion/exclusion criteria and have successfully completed all screening procedures will be randomized in 1:1 ratio into two arms for phase II (70 Subjects each) and in two arms for phase III (Arm I: 3000 Subjects and Arm II: 140 Subjects), through block randomization.This is an open-labelled study i.e., a study where designated investigator site staff will remain unblinded to the study drug.The treatment is known to the subjects, the investigator and the independent biostatistician.Subjects who have received COVAXIN™/COVISHIELD™ as primary vaccination (both doses) will receive either Study Vaccine/Comparator Vaccine.
Unique randomization codes will be assigned to the subjects and will remain unchanged until the completion of the trial.The randomization codes will be generated through Proc Plan using SAS ® version 9.4 or higher (SAS Institute Inc, Cary, North Carolina) by an independent biostatistician.
The final randomization list will be kept strictly confidential, filed securely by the independent biostatistician, and accessible only to authorized persons per Sponsor (or designee) standard operating procedures until completion of the study.

Baseline
Baseline value will be defined /as the last non-missing assessment prior to the first study treatment dose.
In this study, the Baseline Visit is Visit 1/Day 1.

Change from Baseline
Value of change from baseline at any post baseline visit will be defined as the difference of the non-missing baseline value to the non-missing post baseline value i.e.
Change from baseline (∆) = post-baseline value at visit X -baseline value, where both values are non-missing.
Percent change from baseline will be calculated as: (Assessment value at post-baseline visit X -baseline value) / baseline value * 100.

End of Study
End of study value is defined as the last non-missing assessment at the end of study treatment visit.In this study, the end of visit is Visit 4/Day 180 + 14 in-person visit.
If the subject has not completed the study, then the last available non-missing value prior to the end of study treatment visit will be used as end of study value and that visit will be considered as early termination visit.

Treatment Start Day
The vaccine will be administered at Visit 1/Day 1, which will be the treatment start day.

Treatment End Day
As the vaccine will be administered only once, treatment end day is not applicable.

Gennova Biopharmaceuticals
Ltd.For early withdrawal of a subject from the study, all safety assessments related to end of study visit will be performed.If the subject notifies of his/her intention to withdraw the consent, stops visiting the study physician or there will be no communication with him/her during the study, such subject may be deemed as left the study.In such case, the analysis will be done till the last available visit.

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No imputation will be used for missing data, data will be presented as such.

Analysis Software
All analysis will be performed using SAS ® Software version 9.4 or later.

Demographics
Demographic characterstics including age, gender and race and anthropometric variables, height, weight and BMI will be summarized descriptively by treatment arms.Variables that are measured on a continuous scale, such as the age of the patient at the time of enrollment, the number of non-missing observations, mean, median, SD, minimum, and maximum will be tabulated by treatment assignment and overall, for the randomized population.Variables that are measured on a categorical scale will be summarized using frequencies and percentages by treatment assignment and overall, for the safety population.

Prior and Concomitant Medications
The prior and concomitant medications will be coded using World Health Organization Drug Dictionary (WHODD), 01 March 2021 or later.The frequency count and percentage of subjects will be summarized according to the coded terms of system organ class and preferred term across all the treatment groups.
Approximate duration of the study is 6 months (Day 180+14 days).
FAS population includes subjects in the safety population who received the planned vaccine dose and have pre-vaccination (Prior to Dose 1) and Day 29 post Dose 1 (Visit 2) immunogenicity measurement(s) available for analysis.FAS is based on ITT (intention to treat analysis) principle which includes all subjects randomized in the study.Per-protocol (PP) Population set:PP population includes all subjects in FAS population who received the vaccine dose of study vaccines as per the assigned treatment group and have immunogenicity measurement(s) at pre-vaccination (Prior to Dose 1) and Day 29 post Dose 1 (Visit 2) with no major protocol and Document Date: 1.0 & 02JAN2023 deviations that were determined to potentially interfere with immune response to the study vaccine.

Secondary Secondary endpoints include: Gennova Biopharmaceuticals Ltd. JSS Medical Research Asia Pacific Private Limited Data Management -BIS Annexure I Form Title: Statistical Analysis Plan-Module 1
(PRNT) assay with COVISHIELD™ at Day 29 by non-inferiority • Comparison of seroconversion rates as assessed by ≥ 2-fold rise in neutralizing

JSS Medical Research Asia Pacific Private Limited Data Management -BIS Annexure I Form Title: Statistical Analysis Plan-Module 1
• Occurrence and severity of local and systemic reactogenicity adverse events (AEs) for 7 days following vaccination • Occurrence of unsolicited adverse events up to day 29 post vaccination • Occurrence of related unsolicited adverse events throughout the duration of the study • Occurrence of serious adverse events (SAEs): throughout the duration of the study 2. To evaluate the immunogenicity as detected by IgG ELISA against the SARS-CoV-2 Spike protein with GEMCOVAC-OM in comparison with COVISHIELD™ at Day 29 • Comparion of anti-Spike (omicron variant) IgG ELISA against the SARS-CoV-2 Spike protein of GEMCOVAC-OM at Day 90 • GMT measured by IgG-ELISA against SARS-CoV-2 Spike (omicron variant) protein post booster administration at Day