Does Repeat Influenza Vaccination Constrain Influenza Immune Responses and Protection
Conditions: Influenza, Human, SARS-CoV-2 Infection
Sponsor: University of Melbourne

DOES REPEATED INFLUENZA 
VACCINATION CONSTRAIN INFLUENZA 
IMMUNE RESPONSES AND PROTECTION?
PROTOCOL ADDENDUM FOR FOLLOW-UP OF 
COVID-19 INFECTIONS AND VACCINATION
4 March 2021
Version 2
MH project number 2019.176
HREC reference: HREC/54245/MH-2019
Authors:
Sheena Sullivan, Annette Fox, Adam Kucharski, Vivian Leung
Sponsor: Melbourne Health
Funding: US National Institutes of Health (Administrative Supplement to 
Award:1R01AI141534)
CONFIDENTIAL
This document is confidential and the property of WHO Collaborating Centre for 
Reference and Research on Influenza. No part of it may be transmitted, reproduced, 
published, or used without prior written authorization from the institution.
Statement of Compliance
This study will be conducted in compliance with all stipulation of this protocol, the 
conditions of the ethics committee approval, the NHMRC National Statement on ethical 
Conduct in Human Research (2007) and the Note for Guidance on Good Clinical Practice 
(CPMP/ICH-135/95).

1
TABLE OF CONTENTS
1
Table of Contents..................................................................................................................................................... i
2
Glossary of Abbreviations and Terms...........................................................................................................iii
3
Introduction............................................................................................................................................................. 1
4
Significance of the Proposed Research..........................................................................................................1
5
Aims and Objectives.............................................................................................................................................. 2
5.1
Objective 1: To estimate risk factors and correlates of protection for SARS-CoV-
2 infection
............................................................................................................................................................... 2
5.1.1
Sub-objectives...................................................................................................................................... 2
5.1.2
Outcomes................................................................................................................................................ 2
5.2
Objective 2: To characterize viruses infecting HCW........................................................2
5.2.1
Sub-objectives:..................................................................................................................................... 2
5.2.2
Outcomes................................................................................................................................................ 3
5.3
Objective 3: To characterize immunological profiles following infection by 
SARS-CoV-2
............................................................................................................................................................... 3
5.3.1
Sub-objectives:..................................................................................................................................... 3
5.3.2
Outcomes................................................................................................................................................ 3
5.4
Objective 4: To characterize immunological profiles following vaccination for 
SARS-COV-2 ............................................................................................................................................................... 3
5.4.1
Sub-objectives:..................................................................................................................................... 3
5.4.2
Outcomes................................................................................................................................................ 3
6
Study Design............................................................................................................................................................. 4
6.1
Overview of Study Design...........................................................................................................4
6.2
Study Schedule................................................................................................................................ 5
7
Data Collection........................................................................................................................................................ 5
7.1
Overview of data collection........................................................................................................5
7.2
Blood sampling................................................................................................................................ 6
7.2.1
Convalescent blood collection after resumption of hospital duties................................6
7.2.2
Acute blood collection.......................................................................................................................6
7.2.3
SARS-CoV-2 vaccination blood collection.................................................................................7
7.2.4
Timing of blood collection...............................................................................................................7
7.2.5
Blood collection procedures...........................................................................................................7
7.2.6
Blood sample processing and storage........................................................................................7
7.3
Enhanced swabbing......................................................................................................................7
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8
Laboratory Procedures........................................................................................................................................ 8
8.1
Coronavirus testing and virus characterization.................................................................8
8.2
Sero-response to infection and identifying asymptomatic infections.......................8
8.2.1
Reporting of SARS-CoV-2 RT-PCR results.................................................................................8
8.2.2
Quantitative PCR................................................................................................................................. 8
8.3
B and T cell assays.........................................................................................................................8
8.4
Other immunological assays......................................................................................................8
8.5
Sero-response to vaccination....................................................................................................8
8.5.1
Reporting of SARS-CoV-2 vaccination results.........................................................................8
9
Data considerations............................................................................................................................................... 9
9.1
Sample Size....................................................................................................................................... 9
9.2
Data Analysis....................................................................................................................................9
9.2.1
Incidence of COVID-19 disease......................................................................................................9
9.2.2
Risk of severe illness..........................................................................................................................9
9.2.3
Correlates of protection....................................................................................................................9
9.2.4
Social contacts analysis..................................................................................................................10
9.3
Analyses relevant to Aim 2......................................................................................................10
9.3.1
Duration of viral shedding............................................................................................................10
9.3.2
Viral dynamics...................................................................................................................................10
9.3.3
Viral evolution................................................................................................................................... 10
9.4
Analyses relevant to Aim 3......................................................................................................10
9.4.1
Antibody kinetics..............................................................................................................................10
9.5
Analyses relevant to Aim 4......................................................................................................10
10
References......................................................................................................................................................... 11
11
Appendices........................................................................................................................................................ 12
Appendix H Rectal swab collection instructions......................................................................................12
Appendix I
Template of COVID antibody results for participants...................................................13
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2
GLOSSARY OF ABBREVIATIONS AND TERMS
Abbreviation
Description (using lay language)
ARI
Acute respiratory illness. Defined as one or more of the 
following symptoms include fever ≥37.8°C, headache, body 
aches, cough, sore throat, runny nose, sputum.
COVID-19
Coronavirus disease
SARS-CoV-2
Name of the virus causing coronavirus disease
HCW
Health care worker. Any personnel eligible for the free 
vaccination programme run at participating hospitals or 
health services.  Personnel may include staff, including 
administrative, research, clinical and support services, 
employed at the participating hospital. It may also include 
volunteers, students or honorary staff. 
GMT
Geometric mean titre. Arithmetic mean of the logarithms 
(base 2) of the last positive dilution of each serum.
MN assay
Microneutralization assay. A laboratory test which measures 
the ability of antibodies to neutralize virus infectivity.
PBMC
Peripheral blood mononuclear cell. A type of white blood cell 
that contains a single lobed nucleus.
RT-PCR
Real-time reverse transcriptase polymerase chain reaction. A 
laboratory test used to make many copies of a specific 
genetic sequence for analysis and can be used to diagnose 
disease.
Seropositive or seropositivity
Antibody titre of ≥40, as measured using a 
haemagglutination inhibition assay.
Sero-conversion
4-fold rise in antibody titre, as measured using a 
haemagglutination inhibition assay.
qPCR
Quantitative PCR
WHOCCRRI
World Health Organization Collaborating Centre for 
Reference and Research on Influenza.
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3
INTRODUCTION
Our existing protocol describes a study to recruit and follow a longitudinal cohort of healthcare workers 
(HCW) at 6 geographically dispersed Australian hospitals to understand why immunogenicity and 
effectiveness appear to attenuate with repeated administration of the influenza vaccine. We will recruit 
1500 HCW and follow them for up to 4 years.  We will collect sera at baseline (prior to influenza 
vaccination), 14-21 days post-vaccination and at the end of the influenza season (~7 months post). We 
will conduct weekly syndromic surveillance for acute respiratory illnesses (ARI), and request self-
collected swabs from HCWs when they experience ≥2 respiratory or ≥ 1 systemic + ≥1 respiratory 
symptoms. We will calculate influenza attack rates, assess correlates of protection, conduct detailed 
immunological assays, and use mathematical modelling to interpret dynamic antibody responses and 
assess the effectiveness of vaccination programs. 
Given the similarities between cases presenting with influenza and cases presenting with COVID-19, 
our funding agency has requested that we add on surveillance research activities to enhance our 
understanding of the COVID-19 pandemic.  Our cohort presents a number of opportunities for 
epidemiological, virological and immunological investigations of COVID-19. Other novel coronavirus 
outbreaks, including SARS-CoV and MERS-CoV, have been characterized by nosocomial transmission,1-3 
and while this does not appear to be the main driver of transmission for COVID-19,4 it is likely that our 
ARI surveillance will detect some COVID-19 cases in HCWs. 
With the availability of COVID vaccines (CoVax), studies comparing CoVax brands (Pfizer, AstraZeneca, 
other) are needed and likely to be numerous. Studies that investigate the cellular and molecular basis 
for any differences in antibody responses against the CoVax brands may be rarer. In addition, there may 
be limited analysis of responses to CoVax and influenza vaccination, whether the sequence of vaccines 
matters, and if so why.
We are already collecting sera around April and November, which will enable assessment of the 
asymptomatic infection rate. We intend to follow up HCW experiencing ARI to document the duration of 
symptoms and the illness outcome (e.g. GP visit, hospitalization, days absent from work). Importantly, 
we have already assembled a team ready to follow up HCW experiencing an influenza illness, and 
require little additional infrastructure to follow up HCW experiencing a COVID-19 illness or a HCW who 
has been vaccinated for COVID-19
This protocol describes additional surveillance activities directed at leveraging our existing cohort to 
improve our understanding of the epidemiology of the COVID-19 epidemic, the immunological 
responses to SARS-CoV-2 infection and vaccination, as well as further understand the virological 
characteristics. 
4
SIGNIFICANCE OF THE PROPOSED RESEARCH 
Completion of the proposed research will provide valuable insights into the virological and 
immunological dynamics of SARS-CoV-2 infection outcomes. We will estimate risk factors for infection 
and correlates of protection. We will identify drivers of transmission, identify transmission clusters and 
how they drive the COVID-19 epidemic, and explore virus evolution. We will conduct extensive 
exploratory immunological analyses to further generate hypotheses about immunological responses 
to SARS-CoV-2 infection and/or vaccination. An improved understanding of the risk factors and distinct 
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immunological profiles in people with asymptomatic, mild and severe disease will be useful for 
developing vaccines and therapeutics. 
5
AIMS AND OBJECTIVES 
Our overall aim is to increase our understanding of the epidemiological, virological and immunological 
characteristics of SARS-CoV-2 infections and/or vaccination in HCWs. 
5.1
Objective 1: To estimate risk factors and correlates of protection for SARS-CoV-2 
infection
Although there is limited understanding of the immune response to SARS-CoV-2, previous work from 
SARS suggests that protective immunity is induced following the production of protective antibodies.5 
We hypothesize that few HCWs will have antibodies to SARS-CoV-2 at baseline, but that exposure to 
COVID-19 patients will increase the risk of infection. Progression to severe disease (hospitalization) is 
expected to be associated with age, and pre-existing conditions, based on analyses from China. We 
additionally hypothesize that workplace risks, antibody titre to SARS-CoV-2 and other seasonal 
coronaviruses, the magnitude and kinetic of development of T and B cell responses, and viral load will 
also predict severity of infection.   
5.1.1
Sub-objectives
1.
To estimate the asymptomatic, mild and severe attack rates of COVID-19  
2.
To estimate risk factors for infection
3.
To identify differences in antibody titres between HCWs with asymptomatic, mild and severe 
disease
4.
To estimate the antibody titre associated with protection against SARS-CoV-2 infection 
(correlates of protection)
5.1.2
Outcomes
Completion of this aim will result in estimates of the symptomatic and asymptomatic attack rates, the 
hospitalisation risk estimates and identification of risk factors for severe disease. We 
1.
Estimated symptomatic COVID-19 attack rate based on PCR testing
2.
Estimated asymptomatic attack rate based on seroconversions
3.
Case-hospitalization risk 
4.
Risk factors for severe outcomes (death or intensive care or hospitalization versus 
asymptomatic or mild illness)
5.
Estimated protective antibody titres
6.
Identification of key behavioural drivers of transmission
5.2
Objective 2: To characterize viruses infecting HCW
We will estimate various aspects of within-host virus dynamics in a subset of HCW. We hypothesize that 
mildly ill HCW will experience a shorter duration of shedding, lower viral loads and less within-host 
virus evolution compared with severely ill HCWs.  
5.2.1
Sub-objectives:
1.
To characterize viral kinetics
2.
To estimate the degree of within-host virus evolution
3.
To identify the presence of transmission clusters
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5.2.2
Outcomes
1.
Estimated average duration of viral shedding and viral load over time and correlation with 
severity 
2.
Estimated speed of within-host virus evolution
3.
Within-site similarities in viruses recovered from HCWs
5.3
Objective 3: To characterize immunological profiles following infection by SARS-CoV-
2
We will conduct additional immunologic assays on subsets of our cohort to characterize the dynamics 
and types of innate and adaptive immune responses induced, and how these relate to viral load and 
severity. This will include analysis of gene expression, focusing on acute transcriptional profiles to 
characterize and compare innate anti-viral immunity; and quantitation of SARS-CoV-2 reactive B and T 
cells to determine the kinetics of adaptive immune responses, and to identify proteins and epitopes that 
are dominant targets of these responses. This will give us an opportunity to understand the 
contribution of innate and adaptive immune responses to severity.  We hypothesize that there will be a 
greater involvement of memory cells in acute responses to SARS-CoV-2 among HCW with mild versus 
severe infection. Additional hypotheses will be generated from these investigations as more is learned 
about this novel infection.
5.3.1
Sub-objectives:
1.
To characterise antibody kinetics during the course of infection
2.
To examine changes over time in frequency and effector function of the B and T cell populations 
induced by infection 
3.
To identify commonly recognised virus epitopes and whether they are targets of neutralizing 
antibodies
4.
To identify genes that are differentially expressed during the acute innate response compared 
to a post-recovery time-point. 
5.3.2
Outcomes
1.
Estimated post-infection serum antibody titres over time
2.
Enumeration of SARS-CoV-2-reactive B and T cells and identification of dominant epitopes
3.
Exploratory immunological findings to characterize the response to SARS-CoV-2 infection
5.4
Objective 4: To characterize immunological profiles following vaccination for SARS-
COV-2
We will conduct additional immunologic assays on subsets of our cohort to characterize the dynamics 
and types of innate and adaptive immune responses induced by vaccination. 
5.4.1
Sub-objectives:
1.
To measure and compare antibody responses to Adenovirus vector versus RNA formulations of 
COVID-19 vaccines
2.
To characterise and quantify innate immune responses induced by different COVID-19 vaccine 
formulations as well as influenza protein vaccine
3.
To characterize and quantify vaccine specific B and T cell responses induced by different COVID-
19 vaccine formulations as well as influenza protein vaccines 
5.4.2
Outcomes
1.
Estimated post-vaccination serum antibody titres over time for each vaccine formulation.
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2.
Enumeration of innate immune subsets.
3.
Identification of genes that are differentially expressed on d7 compared to d0 for each vaccine 
formulation, focusing on innate immune associated genes
4.
Enumeration of SARS-CoV-2-reactive B and T cells.
5.
Estimation of the relationship between antibody responses and innate, and B and T cell 
responses induced by each vaccine formulation
6.
Comparison of antibody (and B and T cell) responses induced against COVID-19 and influenza 
vaccines among participants who received COVID-19 versus influenza vaccine first or who were 
co-administered both vaccines. 
6
STUDY DESIGN
6.1
Overview of Study Design
The proposed study is a longitudinal cohort study (Figure 1). This study will collect data, blood and 
respiratory specimens from approximately 1500 HCWs to understand responses to influenza 
vaccination. 
Figure 1. Study flowchart for recruitment in the main study. 
We plan to recruit around 1500 HCWs, and expect that this will consist of at least 100 HCWs in each of the vaccination 
groups; the majority are likely to be frequent vaccinees. The expected proportion of each group is described in 9.1.
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Figure 2. Study flowchart for follow-up of COVID-19 infections among HCW. 
Up to 4 additional blood draws may be scheduled and additional daily swabs may be requested. These activities only 
pertain to HCWs identified with SARS-CoV-2 infections. 
6.2
Study Schedule
Follow-up of COVID-19 infections will be conducted for at least the first year of recruitment, and may 
continue into the second year of follow-up if infections continue to be identified. 
Follow up of COVID-19 vaccinations will be conducted in year two of the study or when vaccines 
become available, and may continue into subsequent years if vaccination is required periodically.
7
DATA COLLECTION
7.1
Overview of data collection  
The additional data that will be collected from participants with COVID-19 infection includes:
1.
Blood samples 
2.
Daily respiratory and rectal swab samples
For participants who are vaccinated for SARS-CoV-2, additional blood samples and vaccine details will 
be collected.
At each recruitment site, the same general procedures for obtaining informed consent and collection of 
specimens will be followed. A summary of the number of additional visits and procedures at each visit 
is below. 
Table 1. Additional study visits for participants testing positive for SARS-CoV-2 (does not show study 
visits described in the main protocol).
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Procedures
Assessment/ Procedure
Daily
d3
d7
d14
d30
End of 
season
Serum blood collection
x
x
x
x
X*
Heparin blood (Absolute 
counts, PBMCs, plasma)
x
x
x
x
x
PAXgene
x
x
Symptoms diary 
x
Respiratory swabs
x
Rectal swabs
x
*Serum will be collected anyway as part of the main study.
Table 3. Additional study visits for participants who receive SARS-CoV-2 vaccination and are in the 
main COVID-19 vaccination study (does not show study visits described in the main protocol).
Assessment/ Procedure
d0
d14-21 post dose 2 (booster)
Serum blood collection
x
x
Table 4. Additional study visits for participants who receive SARS-CoV-2 vaccination and are in the 
nested COVID-19 vaccination study (does not show study visits described in the main protocol).
Assessment/ Procedure
d0
D7*
D14*
Serum blood collection
x
x
x
Heparin blood (Absolute counts, PBMCs, plasma)
x
x
x
PAXgene
x
x
   * post dose 2 (booster)
7.2
Blood sampling
7.2.1
Convalescent blood collection after resumption of hospital duties
We expect that most HCW will be able to return to work within 14 days and that we should be able to 
safely collect convalescent blood draws. For the majority of SARS-CoV-2-infected HCWs, we will 
request:
1. 3 post-infection blood draws
a.
d14 
b.
d30 
c.
end of season
2. Collection of up to 54ml of blood at each time point
7.2.2
Acute blood collection
For a subset of HCWs who test positive for SARS-CoV-2, additional blood samples will be requested 
during the acute infection period.  
1. 2 additional post-infection
a.
d3 
b.
d7
2. Collection of up to 54ml of blood at each time point
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7.2.2.1
Home visits
In the likely event that a HCW has not returned to work by d3 or d7 these blood collections will be done 
in the home.  Home visits will be prearranged by a member of the study team.  PPE will be provided for 
team members going to a participant’s home for blood collection. Blood collection will proceed as 
normal. 
7.2.3
SARS-CoV-2 vaccination blood collection
For HCWs who are vaccinated for SARS-CoV-2, we will request:
1.
1 pre-vaccination blood draw (1 x 8 ml serum tube, +/- 2 x 9 ml Na Heparin tubes, 1 x 
PAXgene tube)
2.
1-2 post-vaccination blood draws (after the 2nd dose or otherwise completed course of 
SARS-CoV-2 vaccination)
a.
d7(1 x 8 ml serum tube, 2 x 9 ml Na Heparin tubes, 1 x PAXgene tube)
b.
d14-d21 (1 x 8 ml serum tube, +/- 2 x 9 ml Na Heparin tubes)
7.2.4
Timing of blood collection
For all groups, the exact timing of the post-vaccination bleed(s) and post-infection bleed(s) may change 
as more data about optimal timing become available. Where the number of days is specified, some 
leeway is permitted.  For example, blood samples taken 7 days post-infection may be taken 6-12 post; 
samples taken 14 days post-vaccination or infection may be taken 14-21 days post. 
7.2.5
Blood collection procedures
Blood will be collected in a combination of serum, sodium heparin and PAXgene tubes. 
As outlined in the main protocol, blood collection will be performed in line with each hospital’s 
phlebotomy policy. The use of a butterfly needle connected to a vacutainer tube is recommended as the 
best collection method to minimize haemolysis and to reduce the risk of needle stick injury. Blood 
collection will take place in a private room or behind a screen. 
7.2.6
Blood sample processing and storage
Blood samples will be processed and stored at each recruitment site, as per the main protocol. 
7.3
Enhanced swabbing
7.3.1.1
Respiratory specimen collection
Many questions remain unanswered about viral clearance, but longer duration of shedding appears to 
be associated with severity of symptoms.6 We will request HCWs to provide daily swabs while they 
remain symptomatic and for at least 2 days after symptoms have subsided. 
Swab kits will either be mailed in express post parcels or delivered by a member of the study team to 
the HCW’s door while s/he is in isolation. Swabs in universal transport medium can be stored in the 
home refrigerator until the HCW resumes duties. Swabs will be stored at the site and later forwarded to 
the laboratory in Melbourne for assessment.  
7.3.1.2
Rectal and other swabs
SARS-CoV-2 has been detected in the faecal samples of Chinese patients many days after respiratory 
swabs ceased to recover virus.7 Therefore, we may request rectal swabs from some of our participants 
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as these samples may later prove to be important for understanding the role of virus replication in the 
gut for transmission. Instructions will be provided (Appendix H).
8
LABORATORY PROCEDURES
8.1
Coronavirus testing and virus characterization
Swabs collected from HCWs reporting ARI symptoms will be tested using reverse transcription real-
time polymerase chain reaction (RT-PCR), according to standard operating procedures in each hospital 
laboratory. If the hospital cannot test, swabs will be forwarded to the Doherty (VIDRL) or another 
diagnostic laboratory for testing. 
All swabs will be forwarded to the Doherty for further virus characterization, including genetic 
sequencing.  
8.2
Sero-response to infection and identifying asymptomatic infections
Pre-vaccination (~April) and post-season serum samples will be tested for antibodies to SARS-CoV-2 
using commercial ELISA kits or serological assays developed in-house. Antibodies to other 
coronaviruses will also be tested. 
8.2.1
Reporting of SARS-CoV-2 RT-PCR results
If a HCW is identified as having SARS-CoV-2 through the study, the site manager will receive RT-PCR 
results from their relevant diagnostic laboratory and enter the results into the study database. 
Participants will be informed of their test results following the standard protocol at the hospital at the 
time. 
8.2.2
Quantitative PCR
Consecutive swabs taken over several days will be tested by quantitative PCR to assess the viral load. 
8.3
B and T cell assays
A combination of ELISPOT and flow cytometry will be used to enumerate SARS-CoV-2-reactive B and T 
cells, to characterize proportions that produce antibodies or key cytokines, and to identify proteins and 
epitopes that are dominant targets of these cells. T cells will be detected following brief (re)stimulation 
with (pseudo)virus or overlapping peptides whereas B cells will be detected using recombinant viral 
proteins, either directly ex-vivo or following in vitro differentiation into antibody secreting cells. 
8.4
Other immunological assays
Other immunological assays will be explored as more information becomes available about the SARS-
CoV-2 virus. 
8.5
Sero-response to vaccination
Pre- and post-vaccination serum samples will be tested for antibodies to SARS-CoV-2 spike protein 
using commercial ELISA kits and serological assays developed in-house. 
8.5.1
Reporting of SARS-CoV-2 vaccination results
Participants will receive results of their antibody response to vaccination (Appendix I).
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9
DATA CONSIDERATIONS
9.1
Sample Size
We have no prior information on which to predict how many HCWs will be infected with SARS-CoV-2. 
Some groups have estimated 20% infection rates among their staff, which could mean 300 infections in 
our cohort of 1500.  
9.2
Data Analysis
All statistical analyses will be performed in the statistical software package R.
9.2.1
Incidence of COVID-19 disease
9.2.1.1
Symptomatic attack rate
Symptomatic attack (incidence) rates will be calculated as the number of cases testing positive by RT-
PCR during the person-time at risk. 
9.2.1.2
Incidence proportion
Serological evidence of infection will be based on a 4-fold rise in antibody titres between baseline and 
any later time point. The incidence proportion will be calculated as the number of HCWs with evidence 
of sero-conversion among all HCWs followed during the same period.  
9.2.1.3
Asymptomatic incidence proportion
The asymptomatic incidence proportion will be calculated as the number of HCWs with evidence of 
sero-conversion and no ARI reported among all HCWs followed during the same period.
9.2.1.4
Hospitalization risk
The hospitalization risk (or incidence proportion) will be calculated as the number of HCWs 
hospitalized due to COVID-19 among all HCW with either asymptomatic or symptomatic evidence of 
infection during the same period.
9.2.2
Risk of severe illness
The predictors of severe infection will be estimated using a Cox proportional hazards regression model 
comparing the risk of COVID-19 illness (coded as 1 for hospitalised or 0 for infected but not 
hospitalised) among HCWs. If there are sufficient cases, various predictors of severity will be explored 
in either univariate or multivariate analysis. Predictors may include age, presence of comorbidities, and 
viral load.   
9.2.2.1
Duration of illness and predictors of illness duration
The number of days ill with COVID-19 (count) will be calculated and the risk factors for longer 
durations of illness will be explored. Because of the excess of 0 counts (people who never get infected), 
zero-inflated negative binomial regression will be used.
9.2.3
Correlates of protection
We will compare post-season geometric mean titres between those with asymptomatic and 
symptomatic infections. We will attempt to establish serological correlates of protection for SARS-CoV-
2, using a Bayesian implementation of logistic regression that we have used for influenza cohort 
studies.8 
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9.2.4
Social contacts analysis
Using social contacts data, we will attempt to infer the transmission dynamics for our HCW participants 
between each round of sample collection. We will use mathematical models social mixing data with 
infection risk to untangle specific behaviours/contact scaling that may be driving transmission.9,10 
These models may be extended to include genetic sequencing data, which has been previously used to 
reconstruct transmission clusters.11 
9.3
Analyses relevant to Aim 2 
9.3.1
Duration of viral shedding
For HCW identified as SARS-CoV-2-positive, we will request frequent self-collected swabs while in self-
isolation to estimate the duration of shedding up to day 15 post-infection. We will compare the 
estimated average duration of shedding among HCW with different risk profiles and illness outcomes, 
using regression methods.
9.3.2
Viral dynamics
We will use quantitative PCR (qPCR) to estimate the viral load over time to understand how long people 
remain infectious and corroborate these data with daily symptoms diaries to better understand 
whether viral load is positively associated with symptoms. 
Viral load will be included in analyses comparing asymptomatic, mild and severe infections. If possible 
we will explore the interactions of viral load with demographic (e.g. age) or medical (e.g. heart disease) 
characteristics.
9.3.3
Viral evolution
Using samples collected from individual HCWs over time, we will use deep sequencing and 
bioinformatics models to examine the within-host evolution of SARS-CoV-2 viruses. 
9.4
Analyses relevant to Aim 3
9.4.1
Antibody kinetics
Sera collected more frequently will be assessed for antibody titre and the titres compared over time. 
Geometric mean titres will be calculated and plotted to allow visual inspection of the antibody kinetics, 
overall and within groups (e.g. age groups, severity of infection). The mean rate of decay will be 
calculated using linear regression. Because little is known about the decay kinetics, various models will 
be explored to identify the model with best fit, based on visual inspection of the data and model fitting 
diagnostics. 
9.5
Analyses relevant to Aim 4
Mean antibody concentration will be calculated and compared for vaccine groups (CoVax vs influenza 
vaccine). Seroconversion post-vaccination will be calculated and compared between vaccine groups by 
logistic regression. Antibody levels will be correlated with fold changes in innate immune cells and in 
vaccine specific B and T cells detected at d14-21 versus d0.
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Chowell G, Abdirizak F, Lee S, et al. Transmission characteristics of MERS and SARS in the 
healthcare setting: a comparative study. BMC Med 2015; 13: 210.
2.
Lau EH, Hsiung CA, Cowling BJ, et al. A comparative epidemiologic analysis of SARS in Hong Kong, 
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11 APPENDICES
Appendix H Rectal swab collection instructions
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Appendix I
Template of COVID antibody results for participants
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