Prospective Evaluation of COVID-19 Vaccine Induced Immunity
Conditions: Covid19
Sponsor: University of Manitoba

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Study Protocol 
 
 
Study Title:   Monitoring Diversity of COVID-19 Vaccine Induced Immunity 
 
 
Study Investigators: 
 
Public Health Agency of Canada/ National HIV & Retrovirology Laboratory: Dr. T. 
Blake Ball1, Dr. Catherine Card1, Dr. Paul McLaren1, Dr. Sandra Kiazyk1, Dr. Ruey-Chui 
Su1, Dr. Hezhao Ji1 
 
External/ University of Manitoba Collaborators: Dr. Yoav Keynan2, Dr. Lyle McKinnon2, 
Dr. Derek Stein2, Dr. Kelly MacDonald2, Dr. Joe Bueti3  
 
1National HIV and Retrovirology Laboratory, National Microbiology Laboratory, Public Health Agency of Canada 
2University of Manitoba, Department of Medical Microbiology and Infectious Disease 
3University of Manitoba, Section of Nephrology, Max Rady College of Medicine 
 
 
 
Background: 
 
The COVID-19 pandemic has led to a very rapid development of novel vaccines for the 
SARS-COV-2 virus.  The first two vaccines to show over 90% efficacy in phase III trials 
include novel mRNA vaccines produced by Pfizer /BioNtech1, and Moderna. Although 
these vaccines have, or likely will soon been approved for use in Canada (Pfizer 
approved on December 9, 2020, Moderna under review), the details of the immune 
response to the vaccine remain unclear. In particular, questions remain regarding the 
duration of protection, the correlates and mechanisms of protective immunity, and the 
durability of immunity among those with immune-compromising comorbidities.  
 
Vaccine efficacy and population-level immunity to infection requires both effective 
antibody responses (mediated by B-cells) and cell-mediated immunity (CMI; mediated 
through T-cells). The Pfizer vaccine, BNT162b1, a lipid nanoparticle-encapsulated 
mRNA vaccine targeting the receptor binding domain (RBD) of the SARS-CoV-2 spike 
protein, has been shown to produce robust neutralizing antibody responses in a wide 
range of individuals of diverse age and ethnicity 2, consistent with responses observed in 
recovered convalescent COVID-19 patients. Less is known about the T cell-mediated 
CMI response. A small study (60 participants) has shown T cell responsiveness to the 
vaccine in those under 55 3. These individuals were all healthy and had little ethnic 
diversity. Furthermore, the assessment of the T cell response was very basic and no 
information on the T cell subtypes, phenotypes or function (other than interferon (IFN)-
gamma production) was assessed.  

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Although the presence of antibodies is commonly used as an indicator of protective 
immunity following vaccination or infection, it may not be the only, nor the key, 
correlate of immune protection. Indeed, CMI responses are necessary for mounting 
proper antibody responses in addition to other immune functions. CMI responses to viral 
infection generally involve different classes of T cells including CD8+ cytotoxic T 
lymphocytes (CTLs), which kill infected cells, and CD4+ T-helper (Th) cells, which vary 
by subset in their ability to enhance CTL and antibody responses (Th1 and TFH), 
modulate immunity at mucosal surfaces (Th17) and regulate these responses and limit 
tissue damage (Tregs) 4. Each of these T cell subsets is critical for an effective and long-
lasting immune response; however, their function varies between individuals due to 
intrinsic factors, including genetic background, infection history, lifestyle and 
comorbidities. Variability in CMI responses has been observed in COVID-19 patients 
and has been associated with disease severity 5.  This variability can similarly be 
expected to impact the ultimate efficacy of a SARS-CoV2 vaccine.  
 
Much remains to be learned about the immune response to the new COVID-19 vaccines.  
This study will aim to assess the diversity and complexity of the CMI response to the 
COVID-19 vaccine. Importantly, we will monitor vaccine induced responses in both 
healthy groups, as well as those with co-morbid conditions including diabetes and kidney 
disease.  
 
 
Objectives: 
 
This study aims to address the following three objectives: 
 
1. 
Longitudinal evaluation of the development of CMI responses in response to 
SARS-CoV-2 Vaccine: T cells isolated from the blood of COVID-19 vaccine recipients 
will be evaluated for their functionality in response to vaccine antigens. The temporal and 
functional properties of CMI responses will be correlated with the humoral or antibody 
responsiveness. CMI responses will be measured in vaccine recipients prior to vaccination 
to determine whether the presence or functionality of pre-existing responses to common 
cold coronaviruses (CCCs) or previous SARS-CoV-2 infections affect the development of 
CMI responses to the COVID-19 vaccine. 
 
2. Identification of cellular and soluble factors that influence vaccine responsiveness:  
 
 While we know poor clinical outcomes in COVID-19 patients are strongly associated with 
markers of systemic inflammation, the influence these systemic markers will have on 
COVID-19 vaccine responsiveness is not clear. Using systems biology approaches, we will 
perform comprehensive profiling of cellular immune subsets, inflammatory signatures to 
identify determinants influencing the development of CMI responses to vaccine.  
 
3. 
Examine variability of immune and viral genes and their relationship to vaccine 
induced immune responses:  Human leukocyte antigen (HLA), T cell receptor (TCR) and 

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B cell receptor (BCR) proteins are highly genetically diverse and critical to development 
of protective immunity. We will perform HLA sequencing on whole blood-derived DNA 
samples and TCR and BCR sequencing on sorted, SARS-CoV2 vaccine antigen-specific T 
cells and B cells, respectively, to assess how different sequence combinations impact the 
CMI responses to vaccine. 
 
 
Methodology 
 
a) Study Populations 
 
Study populations will include individuals being administered one of the approved 
SARS-CoV-2/ COVID-19 vaccines. Several clinics within the Health Sciences Centre 
and microbiology laboratory staff in Winnipeg will be accessed to include both healthy 
participants as well as those with underlying health conditions known to increase risk for 
COVID-19 disease severity.  
 
Front line health care workers (hospital or laboratory)  n=100 
In partnership with Dr. Yoav Keynan who is currently running an occupational health 
screening program exposed health for COVID-19 care workers (HCW) at the Winnipeg 
Health Sciences Centre, we will enroll participants scheduled to be receiving the COVID-
19 vaccine. In addition we will work with Dr. Derek Stein at the Cadham Provincial 
Laboratory to facilitate enrollment of laboratory workers scheduled to receive the 
vaccine. 
We will also enroll study participants from the National Microbiology Lab, including 
staff members at the Canadian Science Centre for Human and Animal Health and the JC 
Wilt Infectious Diseases Laboratory in Winnipeg who are scheduled to receive the 
COVID-19 vaccine  
 
Dialysis Patients n=100 
In collaboration with Dr. Joe Bueti who heads the renal program at the Winnipeg Health 
Sciences Centre, we will enroll dialysis patients from the renal program scheduled to be 
receiving the COVID-19 vaccine.  
 
 
b) Recruitment: The study protocol, recruitment, consent to participate forms, and use 
of data are intended to comply with ethical principles set out in the 2nd edition of the Tri-
Council Policy Statement (TCPS 2) for the ethical conduct for research involving 
humans6.  
 
Participants at least 18 years of age or older attending the various clinics will be asked by 
a clinic nurse/physician if they are willing to participate in a study on vaccine induced 
immunity. If they agree to participate, a study nurse will approach the participant using the 
verbal script for recruitment. Participation in the study is entirely voluntary and participants 
can withdraw from the study at any time. Participants receiving any Health Canada 
approved COVID-19 vaccine are eligible to participate.   

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c) Informed Consent: The study nurse will describe the nature of the study, provide a 
brief description of the use of the samples, and discuss issues of confidentiality, and any 
risks or benefits associated with the provision of the samples. All participants must 
provide informed consent prior to the collection of samples. 
 
 
d) Questionnaire/Study Instruments  
 
Participant Enrollment Questionnaire: Together with the research nurse, participants will 
be asked to fill out a basic questionnaire at the time of enrollment. This form will collect 
basic demographic and clinical information critical for analysis of potential drivers or 
confounders of observed CMI responsiveness to vaccine:  
  
• Date of enrollment and first sample collection 
• Demographics: Age, sex, height, weight, ethnicity 
• Pre-existing co-morbidities 
• Current Medications 
• Whether participant received annual flu shot 
 
Participant Follow Up Questionnaire : On subsequent study visits, returning participants 
will be asked to answer some questions on whether they experienced any side effects 
following vaccination or if they have experienced any COVID-19 like symptoms or have 
had COVID-19 test since their last study visit.  
 
 
Data Extraction Form:  Participants who consent will have relevant clinical data extracted 
from their medical records. A research coordinator will extract information from patient 
charts pertaining to pre-existing comorbid conditions and the clinical measurements used 
to monitor the severity and treatment of that condition. These will then be used to 
determine if they are associated with CMI responses to vaccine in multivariate analysis. 
 
 
e) Sample Collection 
 
Participants will be approached prior to receiving their first dose of COVID-19 vaccine, 
and will consent to sampling pre and post vaccination. The specimens to be collected 
include:  
 
At each sampling timepoint, study personnel will collect: 
• 4 tubes of EDTA blood: taken from a vein in the arm. This will be used to isolate 
immune cells for evaluation of CMI responses (Objective 1), cellular biomarkers 

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(Objective 2) and analysis of immune genes (Objective 3), and blood plasma for 
analysis of soluble biomarkers (Objective 2). Collected at all time points  
 
• 1 NP swab in one nostril: a swab is inserted along the nasal septum to the 
nasopharynx and rotated for approximately 5 seconds to collect secretions then 
deposited into a tube containing transport medium. This will be used to evaluate 
cellular and soluble biomarkers (Objective 2). Note that an NP swab will ONLY 
be collected at time points 1, 3 and 4. 
 
• 1 buccal swab: a swab is inserted into the mouth and brushed against the inside of 
the cheek then deposited into a tube containing transport medium. This will be 
used to evaluate soluble biomarkers (Objective 2). Collected at all time point 
 
• 1 saliva sample: participant spits saliva into a collection tube. This will be used to 
evaluate soluble biomarkers (Objective 2).Collected at all time points 
 
 
 
f) Sampling Schedule  
 
1. Day 1: (Baseline sample) At the time of enrollment / prior to receiving the first dose 
of the COVID-19 vaccine 
2.  Day 3-4: After the initial first vaccine dose 
3. Day 10-14 post first vaccine dose  
4.  
5. Day 21:  Prior to receiving the second vaccine dose 4. Day10-14 post second vaccine 
dose :  Approximately 2 weeks post second vaccine dose  2 month post second 
vaccine dose 
6. 4 months post second vaccine dose 
7. 6 months post second dose:  Long term sampling timepoint at 6 months 
8.  1 year post second vaccine dose; :  Long term sampling timepoint at 1 year  
 
 
g) Logistics and Approvals  
Samples will be transported by study personnel to the JC Wilt Infectious Diseases 
Research Centre where researchers will process and store specimens for further analysis. 
 
Necessary study approvals and agreements will be in place prior to commencing 
participant enrollment. These include impact assessments for accessing patients and 
health records at the Health Sciences Centre, and approvals from the Assembly of 
Manitoba Chiefs, Health Information Research Governance Committee (HIRGC), as we 
are collecting ethnicity data.  
 
h) Testing and Analyses 
The different biological readouts for our study will vary by type of test and analysis, as 
outlined below.  

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Objective 1 – CMI responses: Cells isolated from the blood samples collected will be 
exposed to the COVID-19 vaccine antigens or other common viruses as a comparison 
(e.g. CCCs, influenza). The ability of the cells to produce immune mediators in response 
to viral antigens will be quantified by flow cytometry. Global responsiveness to vaccine 
antigen stimulation will also be assessed via transcriptomics (mRNA profiling). These 
responses will be compared to similar responses observed in natural infected individuals 
with disparate disease outcome (Separate PHAC study) as an indicator of vaccine 
response success. CMI responses will be correlated with the humoral or antibody 
responsiveness tested by a number of serological assays / platforms including:  two 
different POC rapid cassette / lateral flow antibody assays, the Roche NP and quantitative 
anti-RBD platforms and also by plaque reduction neutralization and /or surrogate 
neutralisation assays.   
 
Objective 2 – Inflammatory Signatures: Plasma samples, swabs and saliva will be analyzed 
for levels of inflammatory cytokines, chemokines, biomarkers of vascular dysfunction and 
profiles of regulatory RNA in serum exosomes. Inflammatory biomarkers will be evaluated 
as determinants of CMI responsiveness to vaccination. 
 
Objective 3 - HLA sequencing will be performed on whole blood-derived DNA samples 
from infected individuals. TCR and BCR sequencing will be performed on sorted, SARS-
CoV-2 vaccine-specific T- and B-cells using the 10X Genomics platform to assess how 
different combinations impact infection outcome. Genetic sequencing data cannot and will 
not be used to identify individuals.  
 
Analysis Plan:  
The data collected from these various objectives will be assembled to develop models for 
successful CMI responses to COVID-19. Demographic and clinical variables collected will 
be used to stratify study groups and will be incorporated into  multivariate analysis models 
in consultation with a biostatistician to identify confounders or true associations. In 
addition, should the study numbers allow, we will stratify data based on vaccine 
administered to identify  variations of immune responses to different vaccine formulations 
(ie: Pfiezer vs Moderna).   
As we do anticipate over-representation of indigenous ethnicity among participants 
enrolled in the dialysis clinic,  even though stratification based on ethnicity is not a primary 
objective of this study, should we observe any differences in vaccine responses due to 
ethnicity, this data may serve as preliminary data  for future research to be done in 
consultation with indigenous health research representation.  
 
i) Biological Sample Management 
Following collection, samples will be processed at JC Wilt Infectious Diseases Research 
Centre and stored for subsequent analysis. Biological samples may be stored for up to ten 
years, and in accordance with Canadian regulations.  
 
Ethical considerations 
 

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a) Participant confidentiality 
At the time of study enrollment, participants will be given a unique study ID code to keep 
their identity confidential. At no time will their personal identifiers be recorded on any 
specimen vials or tubes which will only be labeled with participant study number and date 
to protect confidentiality. Only the study coordinators will know participant names and 
corresponding study codes. The consent forms will be kept in a separate, secured file 
cabinet in paper copy only. All electronic research records will be computer password 
protected and accessible only to research personnel. At no time will the laboratory staff 
who ultimately will utilize the samples have access to the name or other identifying 
information of the individual who provided the sample.  Any information published or 
presented at public forums as a result of the research will not use personal identifiers, and 
results will be presented as a grouping of data. None of the information stored in study 
computers or notes will contain the participant’s name, and only study numbers will be 
included. Study records will be retained for up to 10 years following completion of research 
study, after which, records will be sent for proper disposition. 
Participants in the study must consent to and understand that they will not receive any of 
the results or data directly relating to the use of their collected samples. Participant identity 
will be anonymous to the researchers using their samples. 
 
b) Potential harms, discomforts, and inconveniences to study participants 
The collection of the nasopharyngeal swab involves insertion of a small swab into each 
nostril to the back of the throat for approximately five seconds. This may be 
uncomfortable but should not be painful. Blood collection involves insertion of a needle 
into a vein in the arm so that the blood can be drawn in to a sample tube. There may be 
momentary pain associated with the needle stick and occasionally people experience 
some bruising around the needle site. Although the needle site will be sterilized, blood 
collection carries a very minimal risk of infection at the site. The collection of saliva and 
buccal swabs are not anticipated to present any risk, discomfort or harm. 
 
c) Potential benefits to participants 
There is no direct benefit to the participant. Individual experimental results will not be 
communicated back with the participants 
 
d) Expected outcomes and proposed efforts for knowledge translation 
 
Results from this study will help to better characterize the cell mediated immune response 
to COVID-19 vaccination. Importantly, by monitoring vaccine response in both healthy 
participants and those with immune compromising underlying health conditions, we will 
be able to answer questions about the quality of the vaccine induced immune response in 
the patient groups most at risk for severe COVID-19 disease outcomes.  In addition, we 
hope to identify strong correlates of vaccine immunity, which can be used to evaluate the 
success of new vaccine candidates, and survey population level immunity. 
 
e) Potential biases  
 
We declare no conflict of interest associated with this study. 

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References 
 
1. Polack et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. 
NEJM 2020 
2. Mulligan, M. J. et al. Phase I/II study of COVID-19 RNA vaccine BNT162b1 in 
adults. Nature 586, 589–593 (2020) 
3. Sahin, U. et al. COVID-19 vaccine BNT162b1 elicits human antibody and T H 1 
T cell responses. Nature 586, 594–599 (2020). 
 
4. DuPage and Bluestone. Harnessing the plasticity of CD4(+) T cells to treat 
immune-mediated disease. Nat Rev Immunol 2016; 16(3): 149. 
5. Moderbaher et al. Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute 
COVID-19 and Associations with Age and Disease Severity. Cell 2020; 183:996. 
6. Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans – 
TCPS 2 (2018). https://ethics.gc.ca/eng/policy-politique_tcps2-eptc2_2018.html 
(accessed on 9 December 2020).