Immunogenicity to SARS-CoV-2 of Co-administering Bivalent mRNA BNT162b2 Booster and Quadrivalent Influenza Vaccine Among Healthy Volunteers: A Pilot Study
Phase: EARLY_PHASE1
Conditions: COVID - 19
Sponsor: Prince of Songkla University

NCT06831786 
Study Protocol 
Official Title: 
Co-administration of COVID-19 and Influenza Vaccines: A Pilot Randomized Controlled Trial 
in Healthy Adults 
NCT Number: NCT06831786 
Document Type: Study Protocol 
Document Date: 2 January 2024 
Sponsor/Institution: Faculty of Medicine, Prince of Songkla University, Hat Yai, Thailand 
 
 

NCT06831786 
Background 
Coronavirus disease 2019 (COVID-19) was declared a pandemic by the World Health 
Organization (WHO) in March 2020, owing to its rapid global spread and the resulting high 
rates of illness and death. In May 2023, the WHO indicated that the COVID-19 public health 
emergency of international concern had concluded, officially marking the end of the 
pandemic [1]. However, COVID-19 remains a persistent disease and is now transitioning 
toward an endemic status [1]. Control of COVID-19 transmission was achieved through herd 
immunity from vaccination or previous infection. The Centers for Disease Control and 
Prevention advises annual updates for both COVID-19 and flu vaccinations [2]. Recent 
studies suggest that the influenza vaccine may help reduce COVID-19 infection rates and 
related deaths [3–6]. Analyses at the national level in the United States have shown an 
inverse relationship between flu vaccine coverage in older adults and COVID-19 deaths [7]; 
similar results were observed in Italy [8]. The protective effect could be explained by cross-
reactive immunity, which is mediated by memory T-cells triggering both cell-mediated 
immune response (CMIR) and humoral immune response (HIR) [9]. A previous study 
identified cross-reactive antibodies between glycan epitopes of the COVID-19 virus spike 
protein and the influenza virus surface protein. Although non-neutralizing antibodies, they 
may facilitate antibody-dependent cellular cytotoxicity [10]. Another proposed mechanism 
involves the broad immunological effects of vaccines, which enhance trained immunity by 
modifying the gene expression patterns of innate immune cells [11]. A previous study 
reported markedly higher immunity against severe acute respiratory syndrome 
coronavirus 2 (SARS-CoV-2) neutralization titers among workers vaccinated with both flu 
and pneumococcal vaccines or with the flu vaccine alone [12]. Another study found higher 
levels of anti-spike receptor-binding domain (RBD) antibodies in individuals who received 
the flu vaccine, in contrast to those who were unvaccinated before receiving the BNT162b2 
vaccine [13]. 
Additionally, six randomized controlled trials (RCTs) examined the safety or 
immunogenicity of administering mRNA COVID-19 doses alongside influenza vaccines [14–
19]. All these studies have confirmed the safety of administering the vaccines together; 
however, the findings on immunogenicity were mixed. Out of the six RCTs, five evaluated 
HIR and observed reduced anti-SARS-CoV-2 antibody concentrations in those receiving 
both vaccines [15–19]. Only two studies met the non-inferiority margin for immunogenicity 
outcomes [15, 18]. However, none have yet evaluated CMIR.  
Owing to the lack of prior research evaluating CMIR, we conducted a pilot study to assess 
both HIR and CMIR for COVID-19 and influenza vaccines administered together compared 
with individually administering each vaccine. We hypothesized that the CMIR may be 
stronger in the co-administration group, which could help explain the reduced mortality 
from COVID-19 infections reported in previous studies for patients vaccinated with the 
influenza vaccine. If confirmed, a stronger CMIR may serve as a proof-of-concept, 
demonstrating the potential immunological benefits of vaccine co-administration. 
Additionally, our study may provide insightful findings on the co-administration policy, the 
implementation of which may increase vaccine coverage and reduce healthcare burden. 

NCT06831786 
Objectives 
Primary Objective: 
• 
To evaluate and compare post-vaccination cell-mediated immune responses (CMIR) and 
humoral immune responses (HIR) between participants receiving co-administration of 
COVID-19 and influenza vaccines versus COVID-19 vaccine alone. 
Secondary Objectives: 
• 
To assess the magnitude of immune responses within each study group. 
• 
To compare the magnitude of immune enhancement between groups. 
• 
To evaluate the frequency and severity of adverse events following vaccination. 
Study Design and Participants 
This was a pilot, single-blind, randomized controlled trial conducted at Songklanagarind 
Hospital, Faculty of Medicine, Prince of Songkla University, Hat Yai, Thailand. Eligible 
participants were healthy adult volunteers aged 18–60 years.  
Inclusion Criteria 
• 
Thai adults aged 18–60 years who had received COVID-19 and influenza vaccines more 
than 6 months prior. 
• 
Able and willing to comply with trial requirements, including 1-month follow-up. 
• 
In good health at enrollment as judged by medical history, physical exam, and 
investigator assessment. 
• 
Able to provide informed consent and sign the ICF. 
Exclusion Criteria 
• 
History of influenza or COVID-19 infection within 6 months. 
• 
History or family history of convulsions, epilepsy, encephalopathy, or psychosis. 
• 
Allergy to vaccine components or prior severe hypersensitivity reactions. 
• 
History of Guillain-Barré syndrome. 
• 
Positive pregnancy test, pregnant, breastfeeding, or pregnancy plan within 6 months. 
• 
Infectious diseases including HIV or suspected SARS-CoV-2 infection. 
• 
Prior SARS-CoV-2 infection within 6 months. 
• 
Severe uncontrolled chronic diseases (e.g., poorly controlled diabetes mellitus, 
uncontrolled hypertension). 
• 
History of urticaria within 1 year before vaccination. 
• 
Known coagulation disorders or thrombocytopenia contraindicating intramuscular 
injection. 
• 
Needle phobia. 
• 
Use of immunosuppressive therapy, cytotoxic therapy, or systemic corticosteroids. 
• 
Receipt of blood products within 4 months before vaccination. 

NCT06831786 
• 
Ongoing anti-tuberculosis treatment. 
• 
Inability to follow protocol or understand informed consent, as judged by investigator. 
Interventions 
Arm 1 – Active Comparator: Bivalent mRNA COVID-19 vaccine (BNT162b2) 
• 
Intervention: Biological/Vaccine – Bivalent mRNA SARS-CoV-2 vaccine 
• 
Description: Administering a booster dose of the bivalent mRNA vaccine BNT162b2 
(Pfizer, Bangkok, Thailand). 
Arm 2 – Experimental: Quadrivalent Influenza Vaccine 
• 
Intervention: Biological/Vaccine – Quadrivalent influenza vaccine 
• 
Description: Administering a VaxigripTetra™ (Sanofi-Aventis, Bangkok, Thailand). 
Arm 3 – Experimental: Co-administration 
• 
Interventions: 
o 
Biological/Vaccine – Bivalent mRNA SARS-CoV-2 vaccine (BNT162b2) 
o 
Biological/Vaccine – Quadrivalent influenza vaccine (VaxigripTetra™) 
• 
Description: Co-administering both vaccines intramuscularly, either in different 
arms or in the same arm at least 25.4 mm apart. 
Outcomes 
Primary Outcomes 
• 
Cell-mediated immune response (CMIR): Post-vaccination interferon-gamma 
enzyme-linked immunospot (IFN-γ ELISpot) assay results comparing the co-
administration group with the COVID-19 vaccine–only group. 
• 
Humoral immune response (HIR): Post-vaccination anti-receptor-binding domain 
immunoglobulin G (anti-RBD IgG) concentrations comparing the co-administration 
group with the COVID-19 vaccine–only group. 
• 
Non-inferiority margin: Defined as the lower bound of the 95% confidence 
interval for the geometric mean ratio (GMR) >0.67, consistent with WHO vaccine 
evaluation guidelines. 
Secondary Outcomes 
• 
Magnitude of immune response within each group: GMR comparing pre- and 
post-vaccination values (GMRpost/pre) for both CMIR and HIR. 
• 
Comparison of immune enhancement between groups: Ratio of GMRpost/pre 
values across groups. 
• 
Adverse events (AEs): Incidence, type, and severity of local and systemic solicited 
AEs at 1 and 4 weeks, as well as unsolicited and serious AEs. 

NCT06831786 
Study Procedures 
Eligible volunteers provided written informed consent before participation. Participants 
were randomized in a 1:1:1 ratio, using block randomization (block size of 6) generated 
with R Statistical Software, to receive: 
1. Bivalent mRNA COVID-19 vaccine (BNT162b2, Pfizer) alone, 
2. Quadrivalent influenza vaccine (VaxigripTetra™, Sanofi-Aventis) alone, or 
3. Both vaccines administered concurrently. 
Vaccines were administered intramuscularly, either in different arms or in the same arm 
with injection sites at least 25.4 mm apart. 
Blood samples were collected at baseline (Day 0, prior to vaccination) and at Day 28. 
Samples were used for measurement of: 
• 
Humoral immune response (HIR): anti-receptor binding domain (RBD) IgG levels. 
• 
Cell-mediated immune response (CMIR): IFN-γ enzyme-linked immunospot 
(ELISpot) assay. 
Participants were monitored for immediate adverse reactions for 30 minutes after 
vaccination. Active follow-up was conducted at 1- and 4-weeks post-vaccination to assess 
solicited local and systemic adverse events, and unsolicited or serious adverse events were 
documented throughout the study period. 
Sample Size 
A total of 36 participants was planned. The sample size followed Julious’ rule-of-thumb[20] for 
pilot studies (≥12 per group) to obtain stable variance estimates and inform the design of a 
subsequent adequately powered non-inferiority RCT. The study was not powered for 
confirmatory hypothesis testing; non-inferiority analyses are exploratory. Estimates from this 
pilot (means, variances, GMRs) will guide the definitive trial’s sample-size calculation. 
Statistical Methods 
Analysis Population 
• 
All randomized participants who received at least one dose of study vaccine were 
included in the immunogenicity and safety analyses (modified intention-to-treat). 
Data Handling and Transformation 
• 
Immune response outcomes (IFN-γ ELISpot and anti-RBD IgG antibody levels) were 
log-transformed prior to analysis to normalize distributions and stabilize variance. 
• 
Zero values were replaced with 1 to allow logarithmic transformation. 
Primary Analysis 

NCT06831786 
• 
Geometric mean ratios (GMRs) of post-vaccination responses were calculated to 
compare the co-administration group with the COVID-19 vaccine–only group. 
• 
Non-inferiority was concluded if the lower bound of the two-sided 95% confidence 
interval (CI) exceeded 0.67, consistent with WHO vaccine evaluation guidelines. 
Secondary Analyses 
1. Magnitude of immune response (within-group): 
o 
Calculated as GMRpost/pre for both CMIR and HIR. 
o 
Paired t-tests were used to assess the significance of pre- vs post-vaccination 
changes. 
2. Between-group comparisons (ratio of magnitude): 
o 
Ratios of GMRpost/pre between study groups were calculated to compare 
immune enhancement. 
3. Safety analysis: 
o 
Incidences of solicited local and systemic adverse events were summarized 
by frequency and percentage. 
o 
Group comparisons used Chi-square or Fisher’s exact tests, as appropriate. 
Software 
• 
All statistical analyses were conducted using R Statistical Software, version 4.3.2 
(R Foundation for Statistical Computing, Vienna, Austria). 
Laboratory Methods 
• 
Antibody analysis: Anti–SARS-CoV-2 RBD IgG (Wuhan-Hu-1 strain) will be 
measured by chemiluminescent microparticle immunoassay (Abbott, Abbott Park, 
IL, USA). Results in arbitrary units (AU/mL) will be converted to WHO binding 
antibody units (BAU/mL) using 1 BAU/mL = 0.142 AU/mL. 
• 
Ex vivo IFN-γ ELISpot assays: Peripheral blood mononuclear cells (PBMCs) will be 
stimulated with SARS-CoV-2 spike peptide pools (Wuhan strain) and assessed in 
triplicate wells. Spot-forming cells (SFCs) per 10⁶ PBMCs will be quantified using an 
automated ELISpot counter (AID Diagnostika, Konstanz, Germany), subtracting 
background from negative controls. 
Adverse Event Monitoring 
All participants were observed on-site for 30 minutes post-vaccination to detect any 
immediate reactions. 
Solicited Adverse Events (AEs): 
• 
Local AEs: pain, redness, swelling, and palpable mass at the injection site. 
• 
Systemic AEs: fever, myalgia, malaise, headache, nausea, and other constitutional 
symptoms. 

NCT06831786 
• 
These pre-specified solicited AEs were actively assessed at 1 week and 4 weeks 
after vaccination. 
Unsolicited and Serious Adverse Events (SAEs): 
• 
Any unsolicited AE reported during the study period was documented. 
• 
SAEs were recorded throughout the follow-up period, with severity grading and 
assessment of relationship to study vaccines. 
Ethics and Dissemination 
The study protocol was reviewed and approved by the Human Research Ethics Committee 
of Prince of Songkla University (REC. 66-462-14-1; approval date: 2 January 2024). The trial 
was conducted in accordance with the principles of the Declaration of Helsinki and 
applicable Good Clinical Practice (GCP) guidelines. 
All participants provided written informed consent before enrollment. Confidentiality of 
participant data was strictly maintained, and only de-identified data were used for analysis. 
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