TITLE: An experimental medicine study of seasonal influenza vaccination responses in Lymph nodE single-cell Genomics in AnCestrY (LEGACY01)
AUTHORS: Katrina M Pollock, Calliope Dendrou

[DESCRIPTION]
This is a collection of protocols of an experimental medicine study of seasonal influenza vaccination responses in Lymph nodE single-cell Genomics in AnCestrY (LEGACY01)

[GUIDELINES]
GENERAL INFORMATION

This protocol was constructed using the Imperial College Research Governance and Integrity Team template (Template Ref: RGIT_TEMP_027, Template V6.0 04Nov2021). The authors wish to credit the MRC CTU at UCL for use of their Protocol Template version 8.0 in drafting of this protocol, which describes the LEGACY01 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. 

COMPLIANCE

The study will be conducted in compliance with the approved protocol, the Declaration of Helsinki 1996, the principles of Good Clinical Practice (GCP) ICH topic E6 (R2), and revision E6 (R3) EWG, General Data Protection Regulation and the UK Data Protection Act 2018, and the UK Policy Framework for Health and Social Care Research.

PROTOCOL DEVELOPMENT TEAM

 
      Name  Address  Email and telephone    Mark Coles (MC)  Kennedy Institute of Rheumatology, Reuben College   University of Oxford Oxford, OX3 7FY  mark.coles2@kennedy.ox.ac.uk    (44) 1865   612675    Calli Dendrou (CD)  Wellcome Centre for Human Genetics, University of   Oxford, Oxford OX3 7BN  cdendrou@well.ox.ac.uk   calliope.dendrou@imm.ox.ac.uk    (44) 1865   287 657     Pontiano Kaleebu (PK)  Uganda Virus Research Institute, Plot 51-57   Nakiwogo Road, PO Box 49, Entebbe, Uganda  pontiano.kaleebu@mrcuganda.org    Tel:   Direct: +256 (0) 417 704103;     Tel: PA   +256 (0) 417 704145    Cell phone   +256-772 500 905    Hashem Koohy (HK)  MRC Human Immunology Unit, MRC Weatherall   Institute of Molecular Medicine University of Oxford Headley Way OX3 9DS   hashem.koohy@rdm.ox.ac.uk      Teresa Lambe (TL)  Old Road Campus Research Building, Roosevelt   Drive, Oxford OX3 7DQ  teresa.lambe@paediatrics.ox.ac.uk      Brian Marsden (BM)  Kennedy Institute of Rheumatology NDORMS   University of Oxford Old Road Campus Roosevelt Drive Headington OX3 7FY  brian.marsden@cmd.ox.ac.uk    (44) 1865   612658    Anita Milicic (AM)  The Jenner Institute  anita.milicic@ndm.ox.ac.uk    Old Road   Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ  (44) 1865 617613    Aime Palomeras (AP)  NIHR Imperial Clinical Research Facility,   Hammersmith Hospital Du Cane Rd, London W12 0HS  aime.boakye@nhs.net    (44) 20   3313 8070    Samantha Vanderslott (SV)  Oxford Vaccine Group University of Oxford,   Oxford, OX3 7LE   samantha.vanderslott@paediatrics.ox.ac.uk    (44) 1865   857420  
 Patient and public involvement and engagement (PPIE) representatives:

Two PPIE representatives have reviewed the study protocol prior to regulatory submission:

Dolapo Ogunleye, Patient Representative for NHS DigiTrials and PPIE representative for the LEGACY Network

Saira Tamboo PPIE representative for the LEGACY Network

Statisticians:

Calliope Dendrou, Sir Henry Dale Fellow, Nuffield Department of Medicine, University of Oxford

Hashem Koohy, Associate Professor of Systems Immunology, University of Oxford
 
STUDY SITE AND COORDINATION CENTRE 
NIHR Imperial Clinical Research Facility (Imperial CRF)
Hammersmith Hospital
Imperial College Healthcare NHS Trust
Du Cane Road
London W12 0HS

For general queries, supply of study documentation, and collection of data, please contact:
Study Coordinator: Aime Palomeras
Tel: 020 3313 8070 E-mail:aime.boakye@nhs.net
Web address: https://www.imperial.ac.uk/nihr-crf/

CLINICAL QUERIES

Clinical queries should be directed to Katrina Pollock who will direct the query to the appropriate person.

SPONSOR

Imperial College London is the main 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 1862
Imperial College - Research Governance and Integrity Team (RGIT) Website

STUDY REGISTRATION

The LEGACY study is registered ISRCTN13657999 https://doi.org/10.1186/ISRCTN13657999 

STUDY SUMMARY 
        TITLE  An   experimental medicine study of seasonal influenza vaccination responses in   Lymph nodE single-cell Genomics in AnCestrY    ACRONYM  LEGACY01    IRAS   ID  314444    SPONSOR  Imperial   College London    DESIGN  Experimental   medicine study; single arm, non-randomised, open label    SETTING  Secondary   care (NHS) and academic research facilities    AIM  To   investigate human immune responses in lymph node cells before and after   immunisation with a seasonal influenza vaccine    OBJECTIVES  Primary   objective: To generate a single cell atlas of lymph node cells before and   after immunisation with seasonal influenza vaccine.    Secondary   objective: To compare serum antibody responses before and after immunisation.    Exploratory:   To compare cellular immune responses in various immune compartments e.g.   blood and lymph nodes, against antigens including influenza before and after   immunisation with seasonal influenza vaccine.    Exploratory: To compare immune responses in various immune   compartments (e.g., blood and lymph nodes) against antigens including   influenza before and after immunisation to help inform vaccine development   and testing across different ethnicities.    Capacity   building and training: To build capacity with respect to staff expertise and   resource between the three partner institutions to support this project and   future similar research.    OUTCOME   MEASURES  Outcome   measures may include but are not limited to the following assays    1.       Single cell RNA sequencing analysis of LNC and matched paired   PBMC    2.       Binding ELISA specific for influenza/A antigens e.g.   haemagglutinin    3.       Intracellular cytokine secretion or activation induced marker   assay of PBMC and LNC    4.       Genotypic assays of areas of the genome of immunological   relevance may include tests such as HLA-testing.    POPULATION  Healthy   adults aged 18 – 55 years n=30    Cohort   1 in influenza season 2022 to 2023    Cohort   2 in influenza season 2023 to 2024    ELIGIBILITY  Individuals   with African or Asian ancestry    DURATION  Three   years  
 
FLOW DIAGRAM AND STUDY SCHEDULE
 
     
 
 
 Table 1. Schedule of investigations, treatments, and assessments
          Study visit  V1  V2  V2a  V3  V4  V5    Visit location  Site  Site  Remote (by   phone)  Site  Site  Site    Visit type  Screening  Enrolment:   FNA1  Follow up  Vaccination  Follow-up:   FNA2  Follow up    Study week  minus 24 to minus 1  0  0  1  2  5    Study day4  minus 168 to minus 1  0  5  7  12  35    Window (days)  NA  NA  minus 1 to plus 1  0 to plus 161  minus 2 to plus 2*  minus 2 to   plus 14    Informed consent  x                   Demographics  x                   Medical history  x                   Weight and height, calculate BMI  x                   Blood borne virus screen (approx. 6mL)1  x                   Laboratory safety tests (approx. 10mL)2  x                   Concomitant medication3  x  x  x  x  x  x    COVID-19 symptoms and trigger COVID-19 test3  x  x  -  x  x  x    Urinary pregnancy test3  x        x          Symptom directed physical examination3  x  x     x  x  x    Inspection of the FNA site3  x  x     x  x  x    Vital signs3  x  x     x  x  x    Ultrasound scan3     x        x       Lymph node fine needle aspiration     x        x       Vaccination           x          Blood for serum immunoassays (6mL)3     x     x  x  x    Blood for cellular and plasma immunoassays (42mL)3     x     x  x  x    Blood for RNA PAXgene tube (2.5mL)3           x  x       Blood for HLA testing (3-4 mL)3     x                Adverse events check3     x  x  x  x  x    Blood volume (approx.) (mL)  16  52  -  50.5  50.5  48  
   

INDEMNITY

Imperial College London holds negligent harm and non-negligent harm insurance policies which apply to this study. Imperial College Healthcare NHS Trust holds standard NHS Hospital Indemnity and insurance cover with NHS Resolution for NHS Trusts in England, which apply to this study.

SPONSOR

Imperial College London will act as the main Sponsor for this study.Delegated responsibilities will be assigned to Imperial College Healthcare NHS Trust.

FUNDING

The Chan Zuckerberg Initiative is funding this study. Participants will be paid for each visit they complete, for their inconvenience and travel, at the end of their participation in the study, as follows:
Screening (V1): £10
Visits which include an FNA (V2 and V4): £120
Vaccination visit (V3): £80
Follow-up visit V5: £60 

PATIENT AND PUBLIC INVOLVEMENT

Patient and Public Involvement and Engagement (PPIE) in research is research being carried out ‘with’ or ‘by’ members of the public rather than ‘to’, ‘about’ or ‘for’ them. The term “patient and public” includes patients, participants, carers and people who use health and social care services as well as people from specific communities and from organisations that represent people who use services.
The protocol has been reviewed and approved by the LEGACY01 PPIE committee.

PUBLICATION POLICY

The preparation of a manuscript for publication in a peer-reviewed professional journal or an abstract for presentation, oral or written, to a learned society or symposium will be discussed on the study calls and with the PPIE Advisory Group. Details of dissemination can be found in the study specific communication plan. 

Authorship will reflect work done by the investigators and other personnel involved in the analysis and interpretation of the data, in accordance with generally recognised principles of scientific collaboration.

PROTOCOL AMENDMENTS

The protocol v2.0 will be the first version approved for use.

REFERENCES

Huang C, Wang Y, Li X, et al. (2020) Clinical Features of Patients Infected With 2019 Novel Coronavirus in Wuhan, China. Lancet 395:497-506. 
Petersen LR, Jamieson DJ, Powers AM, Honein MA (2016) Zika virus. N Engl J Med 374:1552-1563. 
WHO Ebola Response Team (2014) Ebola virus disease in West Africa—the first 9 months of the epidemic and forward projections. N Engl J Med 371:1481-1495. 
WHO Disease Outbreak News, https://www.who.int/emergencies/disease-outbreak-news
Ball P (2021) The lightning-fast quest for COVID vaccines – and what it means for other diseases. Nature 589:16-18.  
Christy C, Pichichero ME, Reed GF, et al. (1995) Effect of gender, race, and parental education on immunogenicity and reported reactogenicity of acellular and whole-cell pertussis vaccines. Pediatrics 96:584-587. 
Kurupati R, Kossenkov A, Haut L, et al. (2016) Race-related differences in antibody responses to the inactivated influenza vaccine are linked to distinct pre-vaccination gene expression profiles in blood. Oncotarget 7:62898-628911.  
Haralambieva IH, Salk HM, Lambert ND, et al. (2014) Associations between race, sex and immune response variations to rubella vaccination in two independent cohorts. Vaccine 32:1946-1953. 
Sharma S, Hagbom M, Svensson L, Nordgren J (2020) The impact of human genetic polymorphisms on rotavirus susceptibility, epidemiology, and vaccine take. Viruses 12:324. 
D’Souza RS, Wolfe I (2021) COVID-19 vaccines in high-risk ethnic groups. Lancet 397:1348. 
Peng K, Safonova Y, Shugay M (2021) Diversity in immunogenomics: the value and the challenge. Nat Methods doi: 10.1038/s41592-021-01169-5. 
Razai MS, Osama T, McKechnie DGJ, Majeed A (2021) COVID-19 vaccine hesitancy among ethnic minority groups. BMJ 372:n513. 
Liang F, Lindgren G, Sandgren KJ et al. (2017) Vaccine priming is restricted to draining lymph nodes and controlled by adjuvant-mediated antigen uptake. Sci Transl Med 9:eaal2094. 
Cirelli KM, Carnathan DG, Nogal B et al. (2019) Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance. Cell 177:1153-1171. 
Havenar-Daughton C, Carnathan DG, Torrents de la Peña A et al. (2016) Direct probing of germinal center responses reveals immunological features and bottlenecks for neutralizing antibody responses to HIV Env trimer. Cell Rep 17:2195-2209. 
Havenar-Daughton C, Newton IG, Zare SY et al. (2020) Normal human lymph node T follicular helper cells and germinal center B cells accessed via fine needle aspirations. J. Immunol. Methods 479:112746. 
Turner JS, Zhou JQ, Han J (2020) Human germinal centres engage memory and naïve B cells after influenza vaccination. Nature 586:127-132. 
https://www.humancellatlas.org/
https://www.ema.europa.eu/en/documents/product-information/fluad-tetra-epar-product-information_en.pdf
https://www.fda.gov/media/135686/download
Fluad, Suspension for injection in pre-filled syringe - Summary of Product Characteristics (SmPC) - (emc) (medicines.org.uk)
Corridoni D, Antanaviciute A, Gupta T, et al. (2020) Single-cell atlas of colonic CD8+ T cells in ulcerative colitis. Nat Med 26: 1480-1490. 
Huang B, Chen Z, Geng L, et al. (2019) Mucosal profiling of pediatric-onset colitis and IBD reveals common pathogenics and therapeutic pathways. Cell 179: 1160-1176.   
COvid-19 Multi-omics Blood ATlas (COMBAT) Consortium. (2022) A blood atlas of COVID-19 defines hallmarks of disease severity and specificity. Cell 185: 916-938. 

APPENDIX 1. INFLUENZA VACCINE USE IN THE UK

Table S1 All influenza vaccines marketed in the UK for the 2022 to 2023 season (as of 14 Apr 2022)

 
          Supplier  Name of product  Vaccine type  Age indications  Ovalbumin content micrograms per dose  Contact details    AstraZeneca UK Ltd  Fluenz® Tetra  Quadrivalent LAIV (live attenuated influenza vaccine) supplied as nasal   spray suspension  From 24 months to less than 18 years of age  Less than 0.024 micrograms per 0.2 ml dose  0845 139 0000    MASTA  Quadrivalent influenza vaccine  QIVe (standard egg-grown quadrivalent influenza vaccine), split virion,   inactivated  From 6 months  Equal to or less than 0.05 micrograms per 0.5 ml dose  0113 238 7552    MASTA  Quadrivalent Influvac® sub-unit Tetra▾  QIVe (standard egg-grown quadrivalent influenza vaccine), surface   antigen, inactivated  From 6 months  Equal to or less than 0.1 micrograms per 0.5 ml dose  0113 238 7552    Sanofi Pasteur  Quadrivalent influenza vaccine  QIVe (standard egg-grown quadrivalent influenza vaccine), split virion,   inactivated  From 6 months  Equal to or less than 0.05 micrograms per 0.5 ml dose  0800 854 430    Viatris (formerly Mylan)  Quadrivalent Influvac® sub-unit Tetra▾  QIVe (standard egg-grown quadrivalent influenza vaccine), surface   antigen, inactivated  From 6 months  Equal to or less than 0.1 micrograms per 0.5 ml dose  0800 358 7468    Seqirus UK Ltd  Cell-based quadrivalent influenza vaccine Seqirus▾  QIVc (cell-grown quadrivalent influenza vaccine), surface antigen,   inactivated  From 2 years  Egg-free  08457 451 500    Sanofi Pasteur  Supemtek▾  QIVr (quadrivalent influenza vaccine (recombinant, prepared in cell   culture))  From 18 years  Egg-free  0800 854 430    Seqirus UK Ltd  Adjuvanted Quadrivalent Influenza Vaccine Seqirus▾  aQIV (adjuvanted egg-grown quadrivalent influenza vaccine) surface   antigen, inactivated, adjuvanted with MF59C.1  From 65 years  Equal to or less than 1 micrograms per 0.5 ml dose  08457 451 500  
 
Table S2 All influenza vaccines marketed in the UK for the 2021 to 2022 season (as of 23 Jun 2021)
 
          Supplier  Name of product  Vaccine   type  Age   indications  Ovalbumin   content   Contact   details            micrograms/dose    AstraZeneca UK Ltd  Fluenz® Tetra  Quadrivalent   LAIV (live attenuated influenza vaccine) supplied as nasal spray suspension  From 24   months to less than 18 years of age  Less than   0.024 micrograms per 0.2 ml dose  0845 139   0000    MASTA  Quadrivalent Influenza vaccine  QIVe   (standard egg-grown quadrivalent influenza vaccine), split virion,   inactivated  From 6   months  Equal to   or less than 0.05 micrograms per 0.5 ml dose  0113 238   7552    Sanofi Pasteur Vaccines  Quadrivalent Influenza vaccine  QIVe   (standard egg-grown quadrivalent Influenza vaccine), split virion,   inactivated  From 6   months  Equal to   or less than 0.05 micrograms per 0.5 ml dose  0800 854   430    Viatris (formerly Mylan)  Quadrivalent Influvac® sub-unit Tetra▾  QIVe   (standard egg-grown quadrivalent Influenza vaccine), surface antigen,   inactivated  From 6   months  Equal to   or less than 0.1 micrograms per 0.5 ml dose  0800 358   7468    Seqirus UK Ltd  Flucelvax® Tetra▾  QIVc   (cell-grown quadrivalent Influenza vaccine), surface antigen, inactivated  From 2   years  Egg-free  08457 451   500    Sanofi Pasteur Vaccines  Supemtek▾  QIVr   (quadrivalent Influenza vaccine (recombinant, prepared in cell culture))  From 18   years  Egg-free  0800 854   430    Seqirus UK Ltd  Fluad Tetra▾  aQIV   (egg-grown quadrivalent Influenza vaccine), surface antigen, inactivated,   adjuvanted with MF59C.1  From 65   years  Equal to   or less than 1 micrograms per 0.5 ml dose  08457 451   500  
 
COMPOSITION

One 0.5 mL dose of aQIV contains 15 µg of haemagglutinin from two A and two B strains of influenza propagated in hens’ eggs and adjuvanted with MF59C.1 which contains per 0.5 mL dose, squalene (9.75 mg), polysorbate 80 (1.175 mg), sorbitan trioleate (1.175 mg), sodium citrate (0.66 mg) and citric acid (0.04 mg).

By comparison, one 0.5 mL dose of Supemtek contains 45 µg of influenza virus haemagglutinin from two A strains and two B strains produced by recombinant DNA technology using a baculovirus expression system in an insect cell line derived from Spodoptera frugiperda.

Table S3 Comparison of Supemtek and aQIV: safety data
 
         Very   common  Common  Uncommon  Rare    (≥1/10)  (≥1/100 to   <1/10)  (≥1/1,000   to <1/100)      aQIV: Adverse reactions reported following vaccination in   elderly subjects 65 years and older in clinical trials  Headache,   injection site pain, fatigue  Nausea,   diarrhoea, myalgia, arthralgia, ecchymosis, chills, erythema, induration, ILI  Vomiting,   fever ≥38C       Supemtek   Adverse reactions reported following vaccination in adults 18 years and older   during clinical trials and post-marketing surveillance  Headache,   fatigue, myalgia, arthralgia, local tenderness,  Nausea,   firmness / swelling, redness, fever, shivering / chills,  Cough,  Dizziness,   urticaria    local pain  oropharyngeal   pain, diarrhoea, pruritus, dermatitis, rash flu-like symptoms, injection site   pruritus,    
 
Table S4 Comparison of Supemtek and aQIV: immunogenicity data in older adults*
 
          Lineage  A  A  B  B       A/H1N1  A/H3N2  B/Yamagata  B/Victoria    aQIV: 65 years and older GMT                   65  294.9  24.7  30.8       (57.8; 73.1)  (261.9;   332.1)  (22.7;   26.8)  (28.3;   33.5)      aQIV: 65   years and older seroconversion rate                35.2  39.3  16.4  13.4    (32.0; 38.5)  (36.1;   42.7)  (14.0;   19.0)  (11.2;   15.9)      Supemtek  A/California/7/2009 (H1N1)  A/Texas/50/2012 (H3N2)  B/Massachusetts/02/2012   (Yamagata lineage)  B/Brisbane/60/2008 (Victoria   lineage)    adults > 50 years    GMT                   190 (164;221)  522   (462;589)  55 (48;64)  29 (26;33)      Supemtek                adults > 50 years  44.9 (39.3; 50.6)  54.5   (48.8; 60.1)  38.9   (33.4; 44.5)  21.0   (16.6; 25.9)    seroconversion rate              
 
APPENDIX 2 FINE NEEDLE ASPIRATION OF THE LYMPH NODE

A medical practitioner will carry out the FNA using clinical facilities at Imperial College Healthcare NHS Trust, London, UK. 
Eligibility to undergo the procedure will be confirmed, paying attention to 

Blood thinning medication likely to induce bruising taken prior to aspiration

Signs of local infection

Pain or swelling at any sites of potential lymph node sampling

Allergy to local anaesthetic 


Any other medical reason, which the PI deems significant to warrant exclusion from the FNA 

Participants will have a set of observations performed including temperature, blood pressure and pulse rate.

The FNA will be conducted using standard aseptic technique under ultrasound guidance. During the procedure, the ipsilateral and contralateral lymph nodes in the axilla will be located by physical examination of the full lymphatic system, and then under US guidance. A sterile needle and syringe will be used to aspirate material from lymph nodes on each side using 3-5 passes. Where necessary local anaesthesia will be employed to numb the area prior to sampling, using a standard local anaesthetic e.g., 1% lidocaine. 

At each visit for FNA sampling a paired peripheral blood sample will be taken using standard non touch aseptic phlebotomy technique.  

Lymph node samples will be placed into pre-prepared and labelled specimen pots and placed with the blood tubes in an appropriate transportation container. They will be transferred to the receiving laboratory where they will be processed upon receipt.  The equipment necessary will all be made available on the day, including an US machine, and equipment for FNA (disinfectant, local anaesthetic, needles, 5ml syringes, air-tight specimen tubes prepared with R10 transport medium). 

Participants will be observed for a minimum of 30 min after the procedure. There will be an AE check and FNA site inspection at least 30 min post-FNA.

EXPECTED ADVERSE EVENTS AND GRADING 

Expected adverse events following lymph node aspiration include sample site pain or tenderness. Haematoma is a rare risk, and minimal bleeding may occur after the aspiration but should resolve spontaneously, and participants at increased risk due to blood-thinning medication will be excluded. Bruising may occur but is expected to fade after 2 weeks. Participants will be provided with information regarding expected adverse events in a participant information leaflet and adverse events will be monitored and reported as per standard AE reporting for the LEGACY01 study.

APPENDIX 3. POST MARKETING SURVEILLANCE FOR FLUAD

Adverse reactions reported in post marketing surveillance of the aTIV, FLUAD include thrombocytopenia including severe thrombocytopaenia in very rare cases, lymphadenopathy, asthenia, Influenza-Like Illness (ILI), swelling and redness of injected limb, allergic reactions including, rarely anaphylactic shock, anaphylaxis and angioedema, pain in the extremity, muscular weakness, encephalomyelitis, Guillain-Barré Syndrome, convulsions, neuritis, neuralgia, paraesthesia, syncope, presyncope, generalised skin reactions including erythema multiforme, urticaria, pruritus or non-specific rash, vasculitis which possibly associated with transient renal involvement.
https://www.medicines.org.uk/emc/product/9223/smpc

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