Mass Evaluation of Lateral Flow Immunoassays for the Detection of SARS-CoV-2 Antibody Responses in Immunosuppressed People (The MELODY Study)
Conditions: Solid Organ Transplant Recipients, Autoimmune Diseases, Haematological Malignancies
Sponsor: Imperial College London

 
 
 
 
 
 
 
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Melody protocol v3.3 07.03.2022 
 
  
 
The MELODY Study 
 
 
 
 
 
Mass evaluation of lateral flow immunoassays for the detection of 
SARS-CoV-2 antibody responses in immunosuppressed people 
 
 
V3.3 07.03.2022 
 
 
MAIN SPONSOR: Imperial College London  
FUNDERS: Medical Research Council [MR/W029200/1] 
STUDY COORDINATION CENTRE: NHS Blood and Transplant Clinical Trials Unit 
 
IRAS Project ID: 306710 
REC reference: 21/HRA/4858 
 
 
 
Protocol authorised by: 
 
Name & Role 
Date 
Signature 
M.Willicombe (CI) 
07.03.2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
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Melody protocol v3.3 07.03.2022 
 
  
 
Study Management Group 
 
Chief Investigator
Dr Michelle Willicombe (Imperial College London)
Co-investigators
Dr Fiona Pearce (University of Nottingham) 
Professor Liz Lightstone (Imperial College London) 
Dr Peter Lanyon (Nottingham University Hospitals NHS Trust ) 
Dr Sean Lim (Southampton University) 
Professor Helen Ward (Imperial College London) 
Professor Graham Cooke (Imperial College London) 
Dr Stephen McAdoo (Imperial College London) 
Professor Gavin Pettigrew (Cambridge University) 
Statistician
Mrs Helen Thomas (NHS Blood and Transplant) 
Study Management
Claire Foley (NHS Blood and Transplant CTU) 
 
 
Study Coordination Centre  
For general queries, supply of study documentation, and collection of data, please 
contact: 
 
Study Coordinator:Gillian Powter  
 
 
 
 
E-mail:  MELODY@nhsbt.nhs.uk  
 
 
 
 
 
 
 
 
Clinical Queries 
Clinical queries should be directed to the study co-ordinator, who will direct the query 
to the appropriate person 
 
 
Sponsor 
 
Imperial College London is the research Sponsor for this study.  For further information 
regarding the sponsorship conditions, please contact the Head of Regulatory 
Compliance at: 
Research Governance and Integrity Team 
Imperial College London and Imperial College Healthcare NHS Trust 
Room 215, Level 2, Medical School Building 
Norfolk Place 
London, W2 1PG 
Tel: 0207 594 9459/ 0207 594 1862 
http://www3.imperial.ac.uk/clinicalresearchgovernanceoffice  
 

 
 
 
 
 
 
 
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Funder 
 
The study is funded by the Medical Research Council, UKRI infections and immunity 
research grant.  
 
This protocol describes the MELODY study and provides information about procedures 
for entering participants.  Every care was taken in its drafting, but corrections or 
amendments may be necessary. These will be circulated to investigators in the study.  
Problems relating to this study should be referred, in the first instance, to the Chief 
Investigator.  
 
This study will adhere to the principles outlined in the UK Policy Frame Work for Health 
and Social Care Research. It will be conducted in compliance with the protocol, the 
Data Protection Act and other regulatory requirements as appropriate.  
 
 
 

 
 
 
 
 
 
 
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TABLE OF CONTENTS                                                           
 
 
PAGE NO 
Table of Contents                                                              
4 
Glossary of Abbreviations 
                                        5 
Keywords 
                                        5 
Study Summary 
                                        6 
Reference diagram 
                                        8 
1. 
INTRODUCTION 
11 
1.1. 
BACKGROUND 
11 
1.2. 
RATIONALE FOR CURRENT STUDY 
12 
2. 
STUDY OBJECTIVES 
13 
3. 
STUDY DESIGN 
13 
3.1. 
STUDY OUTCOME MEASURES 
14 
4. 
RESEARCH PLAN 
14 
4.1. 
METHODS 
14 
4.2. 
ELIGIBILITY 
14 
4.3. 
14 
4.4. 
RECRUITMENT 
16 
4.5. 
WITHDRAWAL CRITERIA 
17 
5. 
ADVERSE EVENTS 
17 
5.1. 
DEFINITIONS 
17 
5.2. 
REPORTING PROCEDURES 
18 
6. 
ASSESSMENT AND FOLLOW-UP 
18 
7. 
STATISTICS AND DATA ANALYSIS 
19 
8. 
REGULATORY ISSUES 
21 
8.1. 
ETHICS APPROVAL 
21 
8.2. 
CONSENT 
21 
8.3. 
CONFIDENTIALITY AND DATA PROTECTION 
21 
8.4. 
INDEMNITY 
22 
8.5. 
SPONSOR 
22 
8.6. 
FUNDING 
22 
8.7. 
AUDITS 
22 
9. 
STUDY MANAGEMENT 
22 
10. 
PUBLICATION POLICY 
22 
11. 
REFERENCES 
22 
12. 
SUMMARY OF CHANGES 
23 
13. 
Appendix 1. REPRODUCIBILITY AND STATISTICAL DESIGN ANNEX 
25 
 
 
 
 

 
 
 
 
 
 
 
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GLOSSARY OF ABBREVIATIONS 
BAU 
Binding Antibody Units 
CPI
Confidential patient information 
DABA
Double binding antigen ELISA
HES
Hospital Episode Statistics
JCVI
Joint Committee of Vaccination and Immunisation
LFIAs
Lateral flow immunoassays
NCARDRS 
National Congenital Anomaly and Rare Disease Registration 
Service
NCRAS
National Cancer Registration and Analysis Service
NDRS
National Disease Registration Service
NHSBT
NHS Blood and Transplant
NIMS
National Immunisation Management Service
ONS
Office of National Statistics
PHE
Public Health England
REACT-2
REal-time Assessment of Community Transmission-2
RT-qPCR 
Real-Time Quantitative Reverse Transcription Polymerase Chain 
Reaction
SACT
Systemic anti-cancer therapy
SARS-CoV-2
Severe acute respiratory syndrome coronavirus 2
SAP
Statistical analysis plan
 
 
KEYWORDS 
Antibodies; Immunosuppressed; Lateral flow immunoassays; COVID-19 / SARS-
CoV-2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
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STUDY SUMMARY 
 
TITLE
Mass evaluation of lateral flow immunoassays for the detection of 
SARS-CoV-2 antibody responses in immunosuppressed people 
(The MELODY Study) 
DESIGN
Observational epidemiological study 
AIMS
 
To determine: 
1. The proportion of immunosuppressed people who have 
detectable SARS-CoV-2 antibodies following a primary 
vaccine course (at least 3 doses), and the demographic, 
disease, and treatment characteristics that influence 
antibody status.  
2. If the detection of antibodies inversely correlates with 
subsequent risk of severe acute respiratory syndrome 
coronavirus-2 infection and/or severity of disease in 
immunosuppressed people. 
 
OUTCOME 
MEASURES
1. The proportion of people with and without antibodies to 
SARS-CoV-2 at least 21 days post three vaccine doses will 
be presented. 
2. The proportion of people with and without antibodies to 
SARS-CoV-2 after a 4th vaccine dose will be presented.  
3. The incidence of participants having at least one RT-qPCR 
proven infection in the 6-month follow-up will be presented 
for those with and without antibodies to SARS-CoV-2 after 
3rd or 4th vaccine.  
4. The incidence of participants hospitalised due to COVID-19 
and deaths due to COVID-19 by 6 months will be presented 
for those with and without antibodies to SARS-CoV-2 
following 3rd or 4th vaccine, and compared as described 
above if there are sufficient events. 
5. Rates of those with and without antibodies to SARS-CoV-2 
after 3rd or 4th vaccine will be presented for different clinical 
characteristics and sociodemographic factors. 
 
POPULATION
Immunosuppressed people, with either: 
1. A solid organ transplant 
2. A rare autoimmune disease  
3. A haematological malignancy 
 

 
 
 
 
 
 
 
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ELIGIBILITY
Immunosuppressed adults (≥18 years old) or young persons (aged 
12 to 17 years) who have received at least 3 doses of a COVID-19 
vaccine. 
 
 
DURATION
6 months (from the point of registration) 
 
 

 
 
 
 
 
 
 
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REFERENCE DIAGRAM 
 
A. PARTICIPANT FLOW 
 

 
 
 
 
 
 
 
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B. PROCESS FLOW DIAGRAM 
 
 
 

 
 
 
 
 
 
 
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1. INTRODUCTION  
 
1.1. 
BACKGROUND 
 
Immunocompromised individuals make up 500,000 of the UK population and comprise 
diverse population groups including those with autoimmune disease, solid organ 
transplants, and cancer. These individuals, especially those receiving concomitant 
immunosuppression, have a poor prognosis to infection with SARS-CoV-2.  Whilst 
vaccination has conferred protection against severe infections for most individuals, the 
immunogenicity of SARS-CoV-2 vaccines have been shown to be suboptimal in 
immunocompromised individuals 1-4. Although immunogenicity does not necessarily 
correlate with clinical efficacy, the following data supports the continued risk of COVID-
19 to immunocompromised individuals: 256 COVID-19-related deaths were reported 
by The Office of National Statistics in doubly vaccinated individuals from January to 
July 2021, 13.1% of whom were immunocompromised5; 40% of 152 doubly vaccinated 
hospitalised COVID-19 patients were immunocompromised as a result of chronic 
corticosteroid use, solid organ transplantation or anti-cancer therapy6; early data from 
NHS Blood and Transplant (NHSBT) suggests the mortality rates from SARS-CoV-2 
infected vaccinated transplant patients is as high as 8%. With these emerging data, 
the Joint Committee of Vaccination and Immunisation (JCVI) approved 3rd vaccine 
doses for immunosuppressed people, in September 2021.  Existing data on 
serological responses to 3rd and 4th dose vaccines in immunocompromised individuals 
is limited, but reports suggest that up to 25% of immunosuppressed individuals will 
remain unprotected even after 3 doses of vaccine 7,8.  
 
Serological responses to 2 vaccine doses are known to be influenced by clinical 
factors, 
such 
as 
disease 
type, 
type 
of 
immunosuppression, 
timing 
of 
immunosuppression in relation to vaccine and type of vaccine1,9.  The reported real-
world clinical efficacy of SARS-CoV-2 vaccines in immunocompromised individuals in 
the UK is likely biased by patients’ behaviour, with many continuing to self-impose 
shielding because of anxiety over lack of vaccine efficacy.  Therefore, there is little 
data on true infection risk in this patient population, clinical factors contributing to lack 
of immunogenicity and clinical efficacy.  Such information could help provide stratified 
advice to specific patient groups, rather than classify all immunosuppressed patients 
at risk.  This information could also help estimate and/or identify the number of patients 
who may benefit from additional or alternative methods of prophylaxis, such as 
monoclonal antibody therapy10,11. 
 
The REal-time Assessment of Community Transmission-2 (REACT-2) study (IRAS ID 
210407), used lateral flow immunoassays (LFIAs) to track SARS-CoV-2 
seroprevalence in the UK, coinciding with the vaccine roll out12.  Evaluating the 
prevalence of SARS-CoV-2 antibodies, coupled with sociodemographic information, 
provided informative epidemiological data.  The LFIAs used within the REACT-2 study 

 
 
 
 
 
 
 
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have been validated in a cohort of kidney transplant patients, with a test performance 
adequate to be used for population screening13.  Using similar methodology to the 
REACT-2 study, we propose to investigate the proportion of immunosuppressed 
people with detectable SARS-CoV-2 antibodies following three and four vaccine 
doses, and correlate antibody status with subsequent infection risk and severity. 
 
With the emergence of the Omicron variant, the UK government announced an 
expedited approach to the COVID-19 vaccination programme on 29th November 
20211.  This involved people who are severely immunosuppressed being eligible for a 
4th vaccine dose 3 months after their 3rd primary dose.  Early data from France, 
suggest that up to 42% of transplant patients will develop de novo antibody responses 
following a 4th dose, if they have previously been non-responsive2,3.  Antibody 
concentrations remain generally low in this population, however, and are unlikely to 
be associated with significant protection. 
 
1.2. 
RATIONALE FOR CURRENT STUDY 
 
Effective protection and management strategies of SARS-CoV-2 infection in 
immunocompromised adults have wide-reaching implications.  For example, evidence 
has shown; a) they are more likely to have severe disease, which is associated with 
morbidity and mortality14-17; b) they are more likely to have prolonged infection, and 
viral shedding18; c) they are a risk for promoting viral evolution and escape mutations19.  
 
Immunocompromised young persons (aged 12-17 years old) are also considered to 
be at heightened risk from SARS-CoV-2 infection. Recent studies have suggested that 
this group mounts stronger responses following SARS-CoV-2 vaccination than are 
observed in vaccinated immunosuppressed adults, but numbers of participants were 
small, and the Joint Committee on Vaccination and Immunisation (JCVI) has 
recommended that the immunocompromised young also should receive a booster 
vaccine following three primary vaccines 4-6. Clarification of how this group respond to 
SARS-CoV-2 vaccination will inform future immunisation strategies.  
 
The aim of this proposal is to assess at a population level; 1) the proportion of 
immunosuppressed people who have detectable SARS-CoV-2 antibodies following at 
least 3 vaccine doses, and the sociodemographic, disease, and treatment 
characteristics that influence antibody status; 2) if the detection of antibodies inversely 
correlates with subsequent risk of SARS-CoV2 infection and/or severity of disease in 
immunosuppressed individuals. 
 
We aim to target patient groups least likely to mount an immune response to 
vaccination1,2,9: a) solid organ transplant recipients; b) patients with a rare autoimmune 
disease; c) patients with haematological malignancies, specifically lymphoid 
malignancies.  We will use comprehensive registries to identify and recruit patients 

 
 
 
 
 
 
 
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from these groups, and utilise the existing linkages these registries already have to 
obtain COVID-19 outcome information.  
 
We hypothesise that a sizeable proportion of immunosuppressed people will have no 
detectable SARS-CoV-2 antibodies following either their third or fourth vaccine dose, 
and that this cohort is particularly susceptible to SARS-CoV-2 infection and death. 
 
 
2. STUDY OBJECTIVES 
 
 
AIM: 
The aim of this proposal is to evaluate the detection of SARS-CoV-2 antibodies at a 
population level in immunosuppressed individuals, by determining: 
1. The proportion of immunosuppressed people who have detectable SARS-CoV-
2 antibodies following a primary vaccine course (at least 3 doses), and the 
demographic, disease, and treatment characteristics that influence antibody 
status.  
2. If the detection of antibodies inversely correlates with subsequent risk of SARS-
CoV-2 infection and/or severity of disease in immunosuppressed individuals. 
 
 
OBJECTIVES: 
1. To estimate the proportion of immunosuppressed people with detectable 
SARS-CoV-2 antibodies by LFIA testing following a primary vaccine course (3 
doses). 
2. To estimate the proportion of immunosuppressed people with detectable 
SARS-CoV-2 antibodies by LFIA testing following a 4th vaccine dose. 
3. To correlate antibody status with risk of subsequent RT-qPCR proven infection 
and disease severity in immunosuppressed people.  
4. To describe clinical characteristics, e.g. immunosuppression burden and 
sociodemographic factors, associated with lack of detectable SARS-CoV-2 
antibodies. 
 
 
 
3. STUDY DESIGN 
 
This is an observational epidemiology study, combining self-reported near patient 
testing results, questionnaire data and routinely collected electronic health records. 
 
Duration of study: 12 months 
 

 
 
 
 
 
 
 
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Number of participants: 36,000 immunosuppressed adults, and 750 
immunosuppressed young adults (12-17 years old). 
 
 
3.1. 
STUDY OUTCOME MEASURES 
 
1. The proportion of participants with and without antibodies to SARS-CoV-2 at 
least 21 days post 3rd vaccine will be presented.  
2. The proportion of people with and without antibodies to SARS-CoV-2 after a 4th 
vaccine dose will be presented.  
3. The incidence of participants having at least one RT-qPCR proven infection in 
the 6-month follow-up will be presented for those with and without antibodies to 
SARS-CoV-2 after 3rd or 4th vaccine.  
4. The incidence of participants hospitalised due to COVID-19 and deaths due to 
COVID-19 by 6 months will be presented for those with and without antibodies 
to SARS-CoV-2 following 3rd or 4th vaccine, and compared as described above 
if there are sufficient events. 
5. Rates of those with and without antibodies to SARS-CoV-2 after 3rd or 4th 
vaccine will be presented for different clinical characteristics and 
sociodemographic factors. 
 
 
 
4. RESEARCH PLAN 
 
4.1. 
METHODS 
We propose to undertake a survey of seroprevalence using self-administered lateral 
flow assays in 36,000 immunosuppressed adult participants and 750 young people, 
who are at least 21 days after completion of their primary COVID-19 vaccination 
course (3 vaccines of which may include a mix of vaccine types). People who have 
received a 4th booster dose, will also be included.  Infection data will be recorded over 
a 6-month period (from the date of registration).   
 
4.2. 
ELIGIBILITY  
 
Adults and young people ≥12 years of age, and are classified as being part of one of 
the following patient groups: 
1. A solid organ transplant recipient (n=12,000) 
2. Patients with a rare autoimmune disease (n=12,000) 
3. Patients with lymphoid malignancies (n=12,000) 
 
The above patients will all be registered within the following national patient 
registries: 
 
NHS BLOOD AND TRANSPLANT (NHSBT)  

 
 
 
 
 
 
 
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I. 
The UK Transplant Registry held by NHSBT records all people in the UK with 
a functioning solid organ transplant.  It already has successful linkage with 
Public Health England (PHE), National Immunisation Management Service 
(NIMS) and NHS Digital Tracing Service, providing data on vaccines, RT-qPCR 
COVID-19 infections and COVID-19 mortality data14,20. Linkage for COVID-19 
hospitalisations will be pursued for this project. 
II. 
Any adult or young person ≥12 years of age with a functioning transplant who 
has received 3 vaccines, will be eligible to participate.  
 
THE NATIONAL DISEASE REGISTRATION SERVICE (NDRS) 
NDRS, part of NHS Digital, comprises the National Congenital Anomaly and Rare 
Disease Registration Service (NCARDRS) and the National Cancer Registration and 
Analysis Service (NCRAS).   
 
NCARDRS Rare disease register (NCARDRS) 
I. 
The REgistration of COmplex Rare Diseases – Exemplars in Rheumatology 
(RECORDER) project within NCARDRS has already identified a cohort of 
170,000 people with rare autoimmune diseases, many of whom are on 
immunosuppressants. Early results from this project has shown this group have 
a COVID-19-related mortality 2.4 times higher than people of the same age and 
sex in the general population. This cohort have validated disease diagnoses, 
and the registry can further identify those treated with Rituximab who are most 
at risk of lack of seroconversion following COVID-19 vaccination.  
II. 
The RECORDER team already have access to National Immunisation 
Management Service (NIMS) data, RT-qPCR COVID-19 infection data, 
hospitalisation data (Hospital Episode Statistics) and Office for National 
Statistics mortality data for this cohort. 
III. 
Sampling will start with those people who have received treatment with 
Rituximab within the last 2 years, and a random sample of people with ANCA-
associated vasculitis (n=9,854) or Lupus (n=41,261), or myositis or 
scleroderma or Giant Cell Arteritis ≥18 years of age who have received 3 
vaccines will be eligible 
 
National Cancer Registration and Analysis Service (NCRAS) 
I. 
The National Cancer Registration and Analysis Service (NCRAS) is responsible 
for cancer registration in England to support cancer epidemiology, public health, 
service monitoring and research. 
II. 
NCRAS actively records from health care data all patients with a diagnosis of 
cancer, and captures those patients receiving systemic anti-cancer therapy 
(SACT). 
III. 
People ≥18 years of age with a haematological malignancy who have received 
3 vaccines will be eligible. 
 

 
 
 
 
 
 
 
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4.3. 
RECRUITMENT 
 
To ensure study start time is aligned with vaccine roll out, recruitment of transplant 
participants (via NHSBT) will differ from cancer and autoimmune participants.  This is 
a pragmatic decision based on differences in patient identifiable data recorded by the 
transplant and NDRS registries. The recruitment plans for the different cohorts are 
outlined below. Those wishing to participate in the study will register via a web portal 
developed by Ipsos MORI, as per REACT-2. They will consent electronically for 
participation in the study, subsequent data linkage on outcomes and willingness to be 
contacted about any future interventional research. 
 
Participate information sheets will be available for all potential recruits, both paper and 
electronic access, depending on method of recruitment. 
 
People with solid organ transplants:  Transplant recipients will be invited to join the 
study by self-registration via the study web portal. Eligibility for transplant recipients 
will be assessed by them providing their NHS/CHI number and year of most recent 
transplant at the time of opting-in to the study. This will be immediately verified against 
the data held by the UK Transplant Registry before the patient proceeds to register for 
the study. 
Participants will be invited to join (‘opt-in’ method) via communications via the British 
Transplant Society, NHSBT, transplant centres, patient charities and social media. 
Linkage to the NHSBT and NHS Digital registries will provide confirmation that the 
applicant is a solid organ transplant recipient and details of their vaccine status. Young 
transplant recipients (aged 12-15) will be additionally asked for their parent / guardian 
details upon registering on the study web portal.  
Simultaneous application for access to NHS Digital will be made. If the target number 
of 12,000 participants is not reached via the opt-in methods by the time approvals are 
obtained, invitations will be sent out to a random sample of patients on the transplant 
registry akin to the patients on the NDRS databases below.  Baseline participant 
characteristics of all participants on the transplant registry are available and will enable 
a description of the study participants in relation to the whole registry.  
As of 14th February 2022, 2,500 adult solid organ transplant recipients have self-
registered with the trial web portal and returned the results of the LFIA they received 
following their 3rd vaccine dose. Depending on availability of LFIA kits, participants 
who had an initial negative test will be further invited to perform an additional LFIA 
test, after receipt of their fourth or subsequent vaccine dose. These additional tests 
will provide data on the proportion of transplant recipients who seroconvert after their 
fourth or subsequent vaccine dose, despite being negative for anti-SARS-Cov-2 IgG 
antibodies after their third dose.  
 

 
 
 
 
 
 
 
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People with cancer and autoimmune disease:  A random selection of eligible 
patients with autoimmune disease or haematological malignancy registered on the 
NCARDRS and NCRAS databases respectively, will be invited to join the study via 
personalised letter. Up to 2 reminders will be sent to potential participants. Patients 
responding to the invitation will register via the Ipsos MORI web portal. Using the 
experience from REACT-2, we estimate a response rate of 70% at registration and 
85% at lateral flow test completion.   
 
Following registration on the web portal and completion of a short online questionnaire 
including information on immunosuppression and medical history, sociodemographic 
variables and COVID-19-related behaviours and COVID-19 infection history, 
participants will be sent a LFIA test, which is provided with a detailed instruction 
booklet and link to an instruction video. For consenting young persons aged 12-15, 
the LFIA kit will be addressed to the parent / guardian whose name they have provided. 
Participants will be asked to carry out the test and follow the instructions to read the 
result and to take a photograph of the completed test, if possible. They will then provide 
their results via the web portal. A telephone helpline will be established to deal with 
any queries that may arise.  Usability of the test has been assessed in REACT-2 study.   
 
 
4.4. 
WITHDRAWAL CRITERIA  
 
All participants are free to withdraw at any time without giving reasons and without 
prejudice. Data provided after registration will remain in the web portal as they cannot 
be deleted. Participants who have received a test kit but wish to withdraw can simply 
discard the test kit.  
 
 
5. ADVERSE EVENTS  
 
5.1. 
DEFINITIONS  
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study 
subject.   
 
Serious Adverse Event (SAE): any untoward medical occurrence or effect that: 
 
Results in death 
 
Is life-threatening – refers to an event in which the subject was at risk of 
death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it were more severe 
 
 
Requires hospitalisation, or prolongation of existing inpatients’ 
hospitalisation 
 
Results in persistent or significant disability or incapacity 
 
Is a congenital anomaly or birth defect 

 
 
 
 
 
 
 
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Medical judgement should be exercised in deciding whether an AE is serious in other 
situations.  Important AEs that are not immediately life-threatening or do not result in 
death or hospitalisation but may jeopardise the subject or may require intervention to 
prevent one of the other outcomes listed in the definition above, should also be 
considered serious. 
 
5.2. 
REPORTING PROCEDURES  
All adverse events should be reported.  Depending on the nature of the event the 
reporting procedures below should be followed.  Any questions concerning adverse 
event reporting should be directed to the Chief Investigator in the first instance.   
 
5.3.1 Non serious AEs 
All such events, whether expected or not, should be recorded- it should be specified if 
only some non-serious AEs will be recorded, any reporting should be consistent with 
the purpose of the trial end points.  
 
5.3.2 Serious AEs 
An SAE form should be completed and emailed to the Chief Investigator within 24 
hours.  However, infections and death due COVID-19, and hospitalisations for elective 
treatment of a pre-existing condition do not need reporting as SAEs. 
 
All SAEs should be reported to the London-Central REC where in the opinion of the 
Chief Investigator, the event was: 
 
‘related’, ie resulted from the administration of any of the research 
procedures; and 
 
‘unexpected’, ie an event that is not listed in the protocol as an expected 
occurrence 
 
Reports of related and unexpected SAEs should be submitted within 15 days of the 
Chief Investigator becoming aware of the event, using the NRES SAE form for non-
IMP studies.  The Chief Investigator must also notify the Sponsor of all related and 
unexpected SAEs. 
 
Contact details for reporting SAEs 
RGIT@imperial.ac.uk 
CI email (and contact details below) 
m.willicombe08@imperial.ac.uk 
 
 
6. ASSESSMENT AND FOLLOW-UP  
 

 
 
 
 
 
 
 
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Direct participant involvement will finish after the results of the LFIA test are uploaded.  
Follow up thereafter will be via the respective registries for 6 months.  The study will 
end at the time the last participant reaches 6 months from the point of their registration 
on the study web portal. 
 
Participants will have access to an information booklet which describes the meaning 
of the test results.  Participant’s GPs will not be informed of recruitment.  
 
7. STATISTICS AND DATA ANALYSIS 
 
Data collection will be undertaken in collaboration with implementation partner Ipsos 
MORI. The data from the participants is self-entered into an online questionnaire 
hosted on a secure web portal developed and managed by Ipsos MORI. 
 
Ipsos MORI will be responsible for setting up and managing the study web portal, 
receiving the questionnaire data, setting up and entering the questionnaire data into 
the study database and linking in the uploaded results of lateral flow assay. Once data 
is complete, a copy of the study database for each patient population will be sent 
securely from Ipsos MORI to NHSBT and NDRS where it will be linked to routinely 
collected healthcare data on COVID-19 infection, and severe outcome over the 
subsequent 6 months and analysed. 
 
Analysis for the a) solid organ transplant groups and b) rare autoimmune disease and 
cancer groups will be conducted using a common protocol and statistical analysis plan, 
but separately by NHSBT and NDRS respectively, due to the complexities of data 
sharing between the two registries and existing restrictions on the data sharing they 
have with NIMS and NHS Digital. The high-level analysis plan is as follows: 
 
1. The proportion of patients (and 95% CI) with and without antibodies to SARS-
CoV-2 at least 21days post 3rd vaccine and prior to 4th vaccine dose will be 
presented.  
2. The proportion of patients (and 95% CI) with and without antibodies to SARS-
CoV-2 following a 4th vaccine dose will be presented 
3. The cumulative incidence (and 95% CI) of participants having at least one RT-
qPCR proven infection in the 6-month follow-up will be presented for those with 
and without antibodies to SARS-CoV-2 at the time the study LFIA was 
undertaken. Risk-adjusted cumulative incidence rates will be compared using 
a Fine and Gray model. These methods allow for participants who may die 
during the follow-up period. 
4. The cumulative incidence (and 95% CI) of participants hospitalised due to 
COVID-19 and deaths due to COVID-19 by 6 months will be presented for those 
with and without antibodies to SARS-CoV-2 at the time the study LFIA was 
undertaken and compared as described above if there are sufficient events. 
5. Rates of those with and without antibodies to SARS-CoV-2 after 3rd or 4th 
vaccine (and 95% CI) will be presented for different clinical characteristics and 

 
 
 
 
 
 
 
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sociodemographic factors. Logistic regression models will be used to identify 
which of these factors are independently associated with developing antibodies 
following completion of the primary vaccine course. 
6. The proportion of re-sampled transplant recipients (and 95% CI) with and 
without antibodies to SARS-CoV-2 following 4th dose who did not have 
antibodies after 3rd dose.  
 
All outcomes will be adjusted for other known risk factors, and receipt of a subsequent 
4th vaccine dose, as and where appropriate. Multiple imputation based on full 
conditional specification will be used to impute values of potential risk adjustment 
factors. The set of variables that will feature in the multiple imputation models will be 
described in the SAP. Sensitivity analyses will be conducted with cases with missing 
primary outcome data excluded. 
A descriptive analysis aligned with above will additionally be performed for the young 
adult population. 
 
Sample size 
Each participant population will recruit 12,000 participants, and of these, we expect 
85% to return their LFIA result (n=10,200). Following a third vaccine, we estimate 
approximately a third of participants in each group will have no SARS-CoV-2 
antibodies, so this sample size will give a 95% confidence interval for the proportion 
of participants with no antibodies of ±0.91% for each participant group 7,8. Even if the 
proportion of participants with no antibodies is 50%, the confidence interval would be 
±0.97%, so the large study size gives very good precision. 
The study has been powered to detect a difference of 2% in the proportion of 
participants free from RT-qPCR proven infection in the 6-month follow-up period, with 
90% power and a 5% two-sided significance level. We have assumed a 2:1 ratio 
between those with and without SARS-CoV-2 antibodies, as described above. In the 
group with SARS-CoV-2 antibodies it has been estimated that 92.4% will be free from 
infection at 6 months and to detect a decrease to 90.4% in the group without SARS-
CoV-2 antibodies using survival analysis methods, we require 9,273 participants in 
total in each participant population (justification for the expected rate is provided in 
Appendix 1 (Reproducibility statistical and design annex). Our planned recruitment of 
10,200 LFIA results per group therefore allows for up to a 9% withdrawal rate, which 
we think is very achievable as patients will have opted-in to the study initially. Higher 
LFIA return rates, a lower withdrawal rate, or combining data across the patient 
populations would enable smaller differences to be detected.  We think that the timing 
of the study, and early evidence of strong engagement, would mean that only a 
relatively small number of patients would have received their 4th dose at the time of 
recruitment.  With the emergence of Omicron, we expect infection event rates 
observed in the study to now be higher than those used in the original sample size 
calculation, even if the 4th dose reduce rates of RT-qPCR positive infection. 
 

 
 
 
 
 
 
 
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Melody protocol v3.3 07.03.2022 
 
  
DIAGNOSTIC ACCURACY OF THE IMMUNOASSAYS 
 
Using the threshold value for positivity on serological testing of ≥7.10 BAU/ml (n=183) 
on Abbott Architect Assay and confirmatory DABA results for available discordant 
samples (n=3) as the reference standard, the overall performance of the test in these 
combined cohorts produce an estimate of sensitivity of 92.0% (115/125; 95% CI 85.8% 
to 96.1%) and specificity of 95.1% (58/61; 95% CI 86.3% to 99.0%).  
 
 
 
8. REGULATORY ISSUES  
 
8.1. 
ETHICS APPROVAL  
The Study has obtained approval from the London-Central Research Ethics 
Committee (REC) and Health Regulator Authority (HRA).  
 
8.2. 
CONSENT   
Participants interested in taking part will register their interest on the Ipsos MORI web 
portal and give separate consent to participate in the study, for subsequent data 
linkage on outcomes and willingness to be contacted about any future interventional 
research. At registration, young persons aged 12-15 years will be additionally asked 
to provide consent from their named parent / guardian, with the LFIA then posted to 
that parent / guardian.  
 
 
8.3. 
CONFIDENTIALITY AND DATA PROTECTION 
 
The Chief Investigator will hold the overall responsibility of preserving the 
confidentiality of participants taking part in the study from the Sponsor. 
 
Section 251 of the NHS Act 2006 will be used as the basis for sharing patient 
identifiable data (PID) without prior consent. Approval for Section 251 support was 
granted by the Confidentiality Advisory Group at the Health Research Authority. 
 
Ipsos MORI 
All personal data will be held securely in line with ISO27001 Information Security 
Standard and in accordance with data protection policies. 
 
NDRS 
All data will be held securely in accordance with NHS Digital’s data security 
standards and the National Congenital Anomaly and Rare Disease Registration 
Service’s (NCARDRS’) confidentiality policy. These are in line with ISO27001. 
 
NHSBT 

 
 
 
 
 
 
 
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Melody protocol v3.3 07.03.2022 
 
  
All data will be held securely in accordance with the NHSBT’s Information Security and 
Confidentiality and Data Protection policies. These follow the principles of ISO27001. 
NHSBT’s ICO Data protection registration Number is Z9210360. 
 
At the end of the study, Ipsos MORI will delete all personal data relating specifically to 
this study from their systems (i.e. exclusively obtained via study procedures). NHSBT 
and NDRS will hold data for up to 10 years 
 
8.4. 
INDEMNITY 
Imperial College London holds negligent harm and non-negligent harm insurance 
policies which apply to this study. 
 
8.5. 
SPONSOR  
Imperial College London will act as the Sponsor for this study.   
 
8.6. 
FUNDING  
The Medical Research Council are funding this study [MR/W029200/1], in 
collaboration with health charities (Kidney research UK, Blood Cancer UK, Vasculitis 
UK and the Cystic Fibrosis Trust).   
 
8.7. 
AUDITS   
The study may be subject to audit by Imperial College London under their remit as 
sponsor and other regulatory bodies to ensure adherence to GCP and the UK Policy 
Frame Work for Health and Social Care Research.  
 
 
9. STUDY MANAGEMENT  
 
The day-to-day management of the study will be co-ordinated through NHSBT CTU. 
A study management group will be convened to provide ongoing oversight of 
operational activity.   
 
10. PUBLICATION POLICY  
 
The study management group will be involved in reviewing drafts of the manuscripts, 
abstracts, press releases and any other publications arising from the study. Any 
publications arising will also be approved by funders.  Authors will acknowledge that 
the study funding as detailed in Section 8.6 above. Authorship will be determined in 
accordance with the ICMJE guidelines and other contributors will be acknowledged. 
 
11. REFERENCES  
 
1Prendecki, M., et al. Comparison of humoral and cellular responses in kidney transplant 
recipients receiving BNT162b2 and ChAdOx1 SARS-CoV-2 vaccines. medRxiv, (2021); 
2Prendecki, M., et al. Humoral and T-cell responses to SARS-CoV-2 vaccination in patients 
receiving immunosuppression. Ann Rheum Dis (2021);  

 
 
 
 
 
 
 
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Melody protocol v3.3 07.03.2022 
 
  
3Lopez Bernal, J., et al. Effectiveness of the Pfizer-BioNTech and Oxford-AstraZeneca 
vaccines on covid-19 related symptoms, hospital admissions, and mortality in older adults in 
England: test negative case-control study. BMJ 373, n1088-n1088 (2021);  
4Kearns, P. Examining the Immunological Effects of COVID-19 Vaccination in Patients with 
Conditions Potentially Leading to Diminished Immune Response Capacity – The OCTAVE 
Trial;  
5Deaths involving COVID-19 by vaccination status, England: deaths occurring between 2 
January and 2 July 2021; 
6Brosh-Nissimov, T., et al. BNT162b2 vaccine breakthrough: clinical characteristics of 152 fully 
vaccinated hospitalized COVID-19 patients in Israel. Clin Microbiol Infect (2021);  
7Kamar, N., et al. Three Doses of an mRNA Covid-19 Vaccine in Solid-Organ Transplant 
Recipients. New England Journal of Medicine (2021);  
8Benotmane, I., et al. Antibody Response After a Third Dose of the mRNA-1273 SARS-CoV-
2 Vaccine in Kidney Transplant Recipients With Minimal Serologic Response to 2 Doses. 
JAMA (2021);  
9 Lim, S.H., et al. Antibody responses after SARS-CoV-2 vaccination in patients with 
lymphoma. Lancet Haematol 8 (2021);  
10Casirivimab and imdevimab in patients admitted to hospital with COVID-19 (RECOVERY): 
a randomised, controlled, open-label, platform trial. medRxiv (2021);  
11O’Brien, M.P., et al. Subcutaneous REGEN-COV Antibody Combination to Prevent Covid-
19. New England Journal of Medicine (2021);  
12Ward, H., et al. REACT-2 Round 5: increasing prevalence of SARS-CoV-2 antibodies 
demonstrate impact of the second wave and of vaccine roll-out in England. medRxiv (2021); 
13Cann, A., et al. SARS-CoV-2 Antibody Lateral Flow Assay for antibody prevalence studies 
following vaccine roll out: a Diagnostic Accuracy Study. medRxiv (2021);   
14Ravanan, R., et al. Two Doses of SARS-CoV-2 Vaccines Reduce Risk of Death Due to 
COVID-19 in Solid Organ Transplant Recipients: Preliminary Outcomes From a UK Registry 
Linkage Analysis. Transplantation (2021);  
15Williamson, E.J., et al. Factors associated with COVID-19-related death using 
OpenSAFELY. Nature 584, 2020);  
16Peach, E., et al. Risk of death among people with rare autoimmune diseases compared with 
the general population in England during the 2020 COVID-19 pandemic. Rheumatology 60, 
(2021);  
17Rutter, M. COVID-19 Infection, Admission and Death Amongst People with Rare 
Autoimmune Rheumatic Disease in England. Results from the RECORDER Project;  
18Man, Z., Viral shedding prolongation in a kidney transplant patient with COVID-19 
pneumonia. Am J Transplant 20 (2020);  
19Corey, L., et al. SARS-CoV-2 Variants in Patients with Immunosuppression. New England 
Journal of Medicine 385, (2021);  
20Ravanan, R., et al. SARS-CoV-2 infection and early mortality of waitlisted and solid organ 
transplant recipients in England: A national cohort study. Am J Transplant 20, (2020) 
 
 
 
12. SUMMARY OF CHANGES 
 
Version  
Author 
 
Date 
Reason for revision 
2.1 
Michelle Willicombe 
Fiona Pearce 
07.12.2021 
To adjust endpoint to at least 21 days post 
3rd vaccine and take into account 
Government notification of 4th doses for 
clinically vulnerable people.

 
 
 
 
 
 
 
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Melody protocol v3.3 07.03.2022 
 
  
 
 
1. Outcome measures: Study 
Summary, page 6 and Eligibility p7 
2. 4th dose in Background Section 
1.1, p13 
3. New Study Objective listed in 
section 2, p 14 
4. New Outcome Measure re 4th dose 
added and outcome 1 updated to 
at least 90 days Section 3.1, p 15 
5.  Addition to Research Plan, 
Section 4.1, page 14 
6. Additions to the Statistics and Data 
Analysis listed in Section 7, pages 
20 and 21. 
7. Data Flow diagram on page 10 
updated to include Date of Birth 
8. Data Flow diagram on page 9 to 
include NHSBT data flow process 
akin to NDRS 
9. Trial coordinator details added to 
page 2
3.3 
Gillian Powter 
Gavin Pettigrew 
Michelle Willicombe 
15/02/2022 
 
Page 7- Eligibility -  Addition of 
Immunosuppressed young adults 
aged 12 to 17 years 
 
Page 14 – Amended to Adults 
and young people ≥12 years 
 
Page 14 NHSBT Cohort – 
amended to Any adult or young 
person ≥12 
 
Page 15 NDRS cohort – amended 
to adults or young people ≥12 
years 
 
Page 16 - To re-test adult 
transplant recipients who have 
completed MELODY study 
participation following their third 
vaccine dose, after receipt of their 
fourth dose. 
 
Page 21 -  young persons aged 
12-15 years will be additionally 
asked to provide consent from 
their named parent / guardian  
 
Page 26 – 3 additional references 
Nos 4 - 6 

 
 
 
 
 
 
 
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Melody protocol v3.3 07.03.2022 
 
  
13. APPENDIX 
1. REPRODUCIBILITY AND STATISTICAL DESIGN 
ANNEX  
 
This is an observational cohort study which will recruit 12,000 participants to each of 
3 patient populations - 36,000 in total. We anticipate a LFIA result return rate of 85%, 
giving data on 10,200 cases in each participant population. Participants will either be 
invited at random to participate (autoimmune and cancer), or sign-up themselves 
(transplant), minimising potential recruitment bias, although self-selection bias could 
occur for either approach. This can be assessed by comparing those recruited against 
wider data on the registries participating in the study, and risk adjustment for any 
important confounders associated with outcome will be conducted.  
 
LFIA diagnostic accuracy.  
The lateral flow device to be used is the Fortress COVID-19 Total Antibody Device for 
the detection of IgM and IgG SARS-CoV-2 against the spike protein. The device has 
been evaluated for use in detecting anti-S in transplant patients and found to have a 
sensitivity of 92% and specificity of 95%13. The sample size calculation is described 
previously. The event rate of freedom from infection at 6 months in the group with 
antibodies was estimated from data in the PHE report "COVID-19 vaccine surveillance 
report - week 37". Table 2 of this report presents case rates per 100,000 for those who 
have received 2 doses of vaccine, which we have taken as our best estimate for event 
rates in a group with SARS-CoV-2 antibodies. The age-adjusted mean event rate from 
this data shows a 0.79% infection rate over a 4 week period in August 2021. Multiplying 
this up for our 6-month (180 day) follow-up period would suggest an event rate of 
5.08%. However, this data relates to the UK population at large, not the higher risk 
immunosuppressed population that will be recruited to MELODY. Recent data on 
COVID-19 infection among those with rare autoimmune rheumatic disease showed 
that the age-standardised infection rate in this group was 1.54 times higher than in the 
general population17. We have therefore inflated our anticipated event rate by 50% to 
give the final event rate of 7.62% for this group (freedom from infection rate of 92.4%). 
Using the same process for the PHE data on unvaccinated individuals would suggest 
an infection rate 2.12% higher in the group with no SARS-CoV-2 antibodies. We have 
therefore powered the study to detect a 2% difference between those with and without 
antibodies. The statistical design and analysis was developed with co-applicants 
Helen Thomas, Fiona Pearce and Helen Ward.  
 
 
 
 
1. 
JCVI advice on the UK vaccine response to the Omicron variant. 
2. 
Kamar, N., et al. Assessment of 4 Doses of SARS-CoV-2 Messenger RNA-
Based Vaccine in Recipients of a Solid Organ Transplant. JAMA network 
open 4, e2136030 (2021). 

 
 
 
 
 
 
 
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3. 
Caillard, S., et al. Antibody response to a fourth mRNA Covid-19 vaccine 
boost in weak responder kidney transplant recipients. medRxiv, 
2021.2009.2003.21262691 (2021). 
4. 
Qin, C.X., et al. Antibody response to 2-dose SARS-CoV-2 mRNA vaccination 
in pediatric solid organ transplant recipients. American Journal of 
Transplantation 22, 669-672 (2022). 
5. 
Haskin, O., et al. Serological Response to the BNT162b2 COVID-19 mRNA 
Vaccine in Adolescent and Young Adult Kidney Transplant Recipients. 
Transplantation 105, e226-e233 (2021). 
6. 
 JCVI advice on COVID-19 vaccination of children and young people.