Kinetics of Mucosal and Systemic Immune Responses to Intranasal Live Attenuated Influenza Vaccine (LAIV)
Phase: NA
Conditions: Influenza, Vaccine Virus Shedding
Sponsor: Imperial College Healthcare NHS Trust

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 1 of 37 
 
Flu-M3 
(Influenza Mucosal Molecular Mechanisms) 
Nasal and systemic immune responses to nasal influenza vaccine 
Kinetics of Mucosal and Systemic Immune Responses to Intranasal Live Attenuated 
Influenza Vaccine (LAIV) 
V1.0 
07/03/2018 
Sponsor:  Imperial College London 
Funder: National Institute for Health Research (NIHR) Biomedical Research Cluster (BRC) at 
Imperial College London 
Study Coordination Centre: Imperial Clinical Respiratory Research Unit (ICRRU), Imperial 
College London 
IRAS Project ID: 243329 
REC Reference: 18/LO/0904 
ClinicalTrials.Gov Registration Number: xxxx 
 
Protocol Authorised by:  
 
 
 
 
 
Name & Role  
Date 
 
 
Signature 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 2 of 37 
Study Management Group 
Chief Investigator (CI): Professor Peter Openshaw 
Co-investigators: Prof Trevor Hansel, Prof Robin Shattock, Dr Ryan Thwaites 
Statisticians: Mrs Tanushree Tunstall, Dr Ryan Thwaites 
 
Study Coordination Centre 
For general queries, supply of trial documentation, and collection of data, please contact: 
Study Coordinator: Professor Peter Openshaw 
Address: Room 353, Department of Respiratory Medicine, National Heart and Lung Institute, 
Medical School Building, St Mary’s Hospital, Paddington, London, W2 1PG 
Email: p.openshaw@imperial.ac.uk 
Tel: 020 7594 3854 
Fax: 02033125751 
Clinical Queries 
Clinical queries should be direct to Peter Openshaw, who will direct the query to the 
appropriate member of the study team. 
 
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: 
Joint Research Compliance Office 
Imperial College London and Imperial College Healthcare NHS Trust 
Room 221, Level 2, Medical School Building 
Norfolk Place 
London, W2 1PG 
Tel: 020 7594 1862 
http://www3.imperial.ac.uk/clinicalresearchgovernanceoffice 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 3 of 37 
Funder 
The study is funded by the National Institute for Health Research Imperial Biomedical 
Research Cluster (NIHR-BRC). 
This protocol describes the Flu-M3 study and provides information about procedures for 
entering participants. The protocol should not be used as a guide for the treatment of other 
participants; every care was taken in its drafting, but corrections or amendments may be 
necessary. Any such amendments will be circulated to investigators in the study. 
Problems relating to this trial should be referred, in the first instance, to the study 
coordination centre. 
This trial will be conducted in compliance with the protocol, the Data Protection Act and 
other regulatory requirements as appropriate. 
 
Key Information 
Participant population: Young Adults (18-30 years; n=85 with n=40 receiving vaccination) 
Scientific Aims: Characterise mucosal and systemic influenza antibodies (IgG and IgA) 
induced by vaccination, viral shedding, innate and cellular immune responses 
Principal Investigator: Professor Peter Openshaw 
Sponsor: Imperial College London 
Site: Imperial Clinical Respiratory Research Unit, 1st Floor, Mint Wing, St Mary’s Hospital, 
Imperial College London 
 
 
 
 
 
 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 4 of 37 
Table of Contents 
Abstract ................................................................................................................................................... 6 
Introduction ............................................................................................................................................ 7 
Influenza .............................................................................................................................................. 7 
Influenza and Asthma ......................................................................................................................... 7 
IFN and Type 2 inflammation in Asthma............................................................................................. 7 
Influenza vaccines: LAIV versus inactivated vaccines ......................................................................... 8 
Viral Load ............................................................................................................................................ 8 
Innate Immunity ............................................................................................................................... 10 
Systems Biology ................................................................................................................................ 10 
Nasal Lavage...................................................................................................................................... 11 
Nasosorption ..................................................................................................................................... 11 
Project Impact ................................................................................................................................... 12 
Study Objectives ................................................................................................................................... 14 
Hypotheses ....................................................................................................................................... 14 
Aims: ................................................................................................................................................. 14 
Study Design ......................................................................................................................................... 16 
Participants and Recruitment ........................................................................................................... 16 
Study Outcome Measures ..................................................................................................................... 16 
Participant Entry ................................................................................................................................... 17 
Inclusion Criteria ............................................................................................................................... 17 
Exclusion Criteria............................................................................................................................... 17 
Withdrawal Criteria ........................................................................................................................... 18 
Randomisation and Enrolment Procedures .......................................................................................... 18 
Treatments ............................................................................................................................................ 18 
Premedication ................................................................................................................................... 19 
Interaction with other drugs ............................................................................................................. 19 
Dispensing and Accountability .......................................................................................................... 19 
Adverse events ...................................................................................................................................... 20 
Definitions ......................................................................................................................................... 20 
Reporting Procedures ....................................................................................................................... 20 
Assessments and Follow-ups ................................................................................................................ 21 
Trial Closure ...................................................................................................................................... 21 
Statistics and Data Analysis ............................................................................................................... 21 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 5 of 37 
Pilot Study: Nasal Sampling Methods and Measurement of Nasal Mucosal Mediators and Antibodies
 .............................................................................................................................................................. 22 
Main Study: Nasal Mucosal and Systemic Immune Responses to Intranasal LAIV in Healthy Adults of 
18-30 years ............................................................................................................................................ 23 
Nasal and Serum sample assays ........................................................................................................... 25 
Antibody Assays: Serum and Nasal samples ..................................................................................... 25 
Data Analysis: Bioinformatics and Systems Biology ............................................................................. 26 
Risks and Benefits to Study Participants ............................................................................................... 28 
Monitoring ............................................................................................................................................ 29 
Regulatory Issues .................................................................................................................................. 29 
Publication Policy .................................................................................................................................. 30 
APPENDIX 1: Main Study Sampling Schedule ....................................................................................... 31 
APPENDIX 2: Visit 2 (Vaccination day) sampling schedule ................................................................... 32 
Reference List ........................................................................................................................................ 33 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 6 of 37 
Abstract 
 
Intranasal live attenuated influenza vaccine (LAIV; trade name FluMist/Fluenz-Tetra, 
manufactured by AstraZeneca/Medimmune) is the standard influenza vaccine given to 
children aged 2-17 years of age in the UK. It is also licensed to be given to adults up to the 
age of 49 years in the USA. The systems biology of the human blood response to influenza 
vaccines has been studied in great detail, but there is a paramount need to study innate and 
specific, soluble and cellular immune responses at the nasal mucosal site of influenza 
infection.  In this way we hope to determine correlates of efficacy and vaccine take in serum 
and nasal mucosal lining fluid (MLF).  
 
We wish to take serial samples prior to vaccination and at intervals up to day 28 post-
vaccination to establish the kinetics of the nasal mucosal and blood systemic response to 
LAIV in young adults aged 18-30 years (n=40). In the nose we shall measure viral load, 
soluble mediators of inflammation and antibodies (humoral immunity) in mucosal lining 
fluid; while cellular immune responses and serology will be assessed in blood samples. 
Imperial College London (ICL) has been involved in the development of novel methods of 
non-invasive precision mucosal sampling, including absorption of MLF from the nose by 
nasosorption. ICL has also developed assays for influenza-specific IgA by ELISA, and we hope 
to compare against a repertoire of assays for serology in patients after LAIV administration.  
 
The study involves precisely assessing mucosal and systemic immune responses to the LAIV 
nasal vaccine. The primary endpoint will be based on nasal mucosal levels of IgA and IgG 
antibodies to the 4 constituent viral subtypes in LAIV: measured by ELISA and multiplex 
immunoassay (Mesoscale Diagnostics) and expressed as seroconversion rates, geometric 
mean titre (GMT) changes, and geometric mean fold rises (GMFR). The secondary endpoints 
will be: (1) haemagglutination inhibition (HAI) titres measured in serum and the nose, (2) 
influenza pseudotype neutralisation by antibodies in serum and the nose, (3) nasal cytokine 
and chemokine levels and (4) nasal viral load.  
 
It is thought that the immune response to LAIV in an individual is mediated by a combination 
of mucosal and systemic factors, involving innate and specific mechanisms that have 
different kinetics, and various cell types. By understanding the molecular and cellular basis of 
the nasal mucosal response to LAIV, we hope to be able to identify key molecular signatures 
and biomarkers associated with LAIV responses, and to assess protective pathways that 
could be stimulated by novel vaccines. The nasal vaccine challenge model could be used to 
test other new vaccines, and proceed to rational development of improved vaccines for 
influenza and other diseases. Furthermore nasal mucosal methods could be used in the clinic 
to identify subjects who have responded poorly to vaccines, or to assess vaccine efficacy in 
large populations.  
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 7 of 37 
Introduction 
Influenza  
Influenza viruses cause a continuous threat to global health, mutating and spreading in both 
human and animal populations. In the period from March to May 2009 a novel swine-origin 
influenza A (H1N1) virus emerged in Mexico and the United States (1, 2). Most cases were 
acute and self-limiting among children and young adults (2), although the global mortality 
over the first 12 months has been estimated at over 200,000 respiratory deaths (3). Those 
with severe illness typically presented with viral pneumonitis or pneumonia, sometimes 
complicated by multi-organ failure.  
 
By measuring cytokines and chemokines in the serum of patients with pH1N1 influenza, it 
has been found that  there  can be over exuberant  immune reactions to virus infection in 
severe flu,  with very high levels of cytokines such as interferons and even a “cytokine 
storm” (4-8).  Some studies have highlighted the importance of particular biomarkers: such 
as HMGB1 in H1N1 influenza-associated encephalopathy (9), and IL-6 in severe pH1N1 
influenza (10). In studies of fatal cases of H1N1 the presence of viral pneumonia and 
immunolocalisation of viral antigen with diffuse alveolar damage has been noted (11, 12). 
Influenza and Asthma 
The Influenza Clinical Information Network (FLU-CIN) recently reported the differences 
between asthmatics and non-asthmatics in 1520 patients hospitalised with influenza A 
infection across 75 UK hospitals  (13). This study noted that patients with asthma (present in 
25.3%) were less likely to have a severe outcome, despite more asthmatics requiring oxygen 
on admission. These investigators suggested that the association of asthma with less severe 
outcomes was due to corticosteroid use and earlier hospital admission. The better outcome 
of asthma patients when hospitalised with influenza has also been observed elsewhere (14, 
15). Despite this, a global pooled analysis of H1N1 found that asthma was the major 
comorbidity associated with severe outcomes (16), as noted in a study of hospitalized US 
patients from April-June 2009 (17). These studies reflect the complex association between 
asthma and influenza severity. 
IFN and Type 2 inflammation in Asthma 
Abnormal airway mucosal inflammatory responses are a feature of asthma (18), and an 
expanding number of asthma phenotypes are now recognised at a clinical and molecular 
level (19). Interferon (IFN) responses in asthmatics are controversial, since description of IFN 
deficiency was first described in cultured bronchial epithelial cells and bronchoalveolar 
lavage (20) cells stimulated with rhinovirus in vitro (21). Recent studies have shown normal 
or elevated interferon responses in mild asthma (22), in children with asthma (23) and in 
unchallenged severe asthma subjects (24). Type 2 inflammation is a major feature of 
asthma, with involvement of mast cells and eosinophils, and an expanding pathway of 
epithelial and lymphoid cytokines and chemokines. This has led to the development of 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 8 of 37 
biologic therapies for asthma including those targeting IL-5 and IL-13 (25, 26), since these 
cytokines are prominent in the eosinophilic phenotype of asthma that has type-2 
inflammation (27, 28). 
Influenza vaccines: LAIV versus inactivated vaccines 
There is a major ongoing effort to improve the efficacy of influenza vaccines (2). Indeed, 
there is a concerted effort to develop ‘universal’ influenza vaccines, which would provide 
broad protection against many influenza strains (29). However, antigenic drift and the high 
number of potentially circulating strains means that current vaccines are a ‘best guess’ of 
which strains are likely to cause disease in humans, and this must be modified each year (2, 
3).  
 
Intranasal vaccination against influenza virus is a favoured site for induction of mucosal and 
systemic responses (3). This is because the nose is the natural site of influenza infection, 
involving the respiratory pseudostratified epithelium, and immune responses in the local 
lymphoid tissue of Waldeyer’s ring: the adenoids and tonsils, with cervical lymph nodes (4). 
LAIV has been developed for use in infants and young children (5, 6) and is also licensed for 
us in adults up to 49 years of age in the USA (7). However, in adults the lower efficacy of LAIV 
relative to intramuscular inactivated influenza vaccine has resulted in LAIV only being 
licensed in the UK between 2-17 years of age (7, 30). Despite this, LAIV is frequently given to 
adults in the USA with an excellent safety profile (31, 32). This study will vaccinate healthy 
young adults (18-30 years) with LAIV. 
Viral Load 
Following LAIV infection shedding of the vaccine virus has been detected in nasopharyngeal 
swabs in children (8, 9). However, shedding of the vaccine virus has not been characterised 
in detail, and the contribution of viral load (the extent to which the virus replicates in the 
airway) to vaccine efficacy is unknown. The immune response to influenza is based on both 
respiratory mucosal and systemic factors: in an interaction between the innate immune 
response of the respiratory epithelium mucosa, coupled with mucosal and systemic specific 
antibody (B cell) and T cell responses (10, 11). After the 2009 H1N1 influenza epidemic it was 
found that CD8+ T cells specific to conserved viral epitopes gave cross-protection against 
symptomatic influenza (12).  
 
Quantitative PCR (qPCR) against the 4 influenza viral sub-types is analysed using a fast 
throughput multiplex assay to measure influenza viral load. This has been developed by 
Medimmune (Gaithersburg, ML) with Virosciences (Rotterdam, NL). Selection of influenza 
sub-type is based on WHO recommendations for influenza vaccination (2017/18) - LAIV 
Quadrivalent 
2017/18 
contained 
H1N1 
A/Slovenia/2903/2015, 
H3N2 
A/New 
Caledonia/71/2014, B/Phuket/3073/2013 (B/Yamagata-lineage) and B/Brisbane/60/2008 
(B/Victoria-lineage) (33).  These component vaccine strains are changed each year, in 
response to predictions of circulating influenza strains. 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 9 of 37 
 
For a protective immune response to be generated to LAIV, the vaccine viruses must infect 
the cells of the upper airway (called ‘vaccine take’). This is believed to be lower in adults 
than children due to pre-existing immunity to other influenza strains, which can take the 
form of antibodies in both the blood and the airway mucosal fluid. However, immune 
correlates of vaccine take are poorly defined for LAIV. 
 
Antibodies against Influenza 
A key protective mechanism of vaccination is the induction of antibodies, which participate 
in the prevention of future infection with pathogenic influenza. Antibody titre and function 
against influenza can be determined in a number of ways: 
 
• ELISA for antibodies 
Influenza subtype-specific IgA and IgG anti-HA antibodies can be measured by ELISA. 
Nasosorption using SAM strips enables higher detectable levels of total and H1-specific IgA 
when compared to flocked swabs or nasal wash samples (34). Following nasal LAIV this 
method allows the establishment of the magnitude and kinetics of nasal HA IgA antibody 
production. 
 
• Fast throughput multiplex immunoassay, based on MSD (developed by Medimmune, 
Gaithersburg) 
MSD immunoassays have been created to allow highly sensitive measurement of the 
concentration of antibody versus HA and NA recombinant antigens from the 4 vaccine virus 
subtypes.  This is determined as the concentration of influenza viral subtype-specific IgG and 
IgA in blood and nasal samples. In comparison to traditional ELISA, MSD assays require less 
sample volume and provide faster results with higher sensitivity and specificity. 
 
• Haemagglutination inhibition assay (HAI) 
Protective serum humoral immunity after natural viral infection can be assessed by HAI, as 
routinely performed by Public Health England (Colindale, UK) (13-15), although serum levels 
are not used to select subjects for LAIV vaccination. Seroconversion measuring influenza HA 
antibodies in the HAI assay is commonly assessed after vaccination. In an integrated review 
of LAIV experience in over 10,000 adults and children, it was found that serum HAI 
geometric mean fold rises (GMFR) for HAI antibodies were higher in children and higher in 
baseline seronegatives (35). However, HAI responses were modest overall, and post-
vaccination HAI titres were below those associated with protection for inactivated influenza 
vaccines.  
 
• Pseudotype virus neutralisation assays 
Pseudotypes and chimeric viruses utilised in assays are shown to be highly efficient for the 
study of cross-protective responses against multiple influenza subtypes (36). In combination 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 10 of 37 
with HAI, the late nasal mucosal and serum antibody response can be investigated. 
Pseudotype-based antibody neutralisation assays are preferred to microneutralisation test 
because they are cheaper, more standardised and their greater sensitivity suits them to 
measurements of mucosal samples.  
 
• Antibody dependent cellular cytotoxicity (ADCC)/Antibody dependent effector cell 
activation (ADCA) 
The ability of an antibody to drive cytotoxicity of infected cells, through the activation of 
immune cells including Natural Killer (NK) cells and neutrophils, is a key function of 
antibodies against influenza (37).  Techniques exist to measure this ability of antibodies from 
the serum (38, 39) and such techniques may be used to determine mucosal antibody 
function. 
• Antibody dependent phagocytosis 
Antibodies can also induce the phagocytosis of targets through antibody dependent 
phagocytosis (39). This property may also play a role in viral clearance and prevention of re-
infection with influenza. 
However, little is known about the role of local nasal mucosal immune responses to nasal 
vaccines and natural influenza infection, because virus load and the IgA immune response is 
generally measured in nasal lavage samples that are variably diluted (16-18). Hence, for LAIV 
nasosorption mucosal antibody measurements may be more relevant than serum HAI, and 
mucosal antibody measurements will be enabled by sampling of neat secretions by 
nasosorption.  
Innate Immunity 
Innate immune responses occur in the first few hours after intranasal LAIV, cytokines of this 
immune response, particularly interferons (IFN), may cause systemic adverse events that 
characterise a viral illness: fever, headache and malaise (6). In older adults, proinflammatory 
responses may be decreased by IL-10, causing decreased immune response to vaccines and 
lower efficacy (19). 
 
Study of nasal and bronchial mucosal inflammatory responses during rhinovirus-induced-
asthma exacerbation has shown asthmatics presented robust anti-viral responses: including 
elevated nasal IFN-γ, IFN-λ/IL-29, CXCL11/ITAC, CXCL10/IP10 and IL-15 (40). In addition there 
is elevation of nasal IFN-γ, IL-1β, CCL5/RANTES and IL-10 in RSV bronchiolitis of infancy (41).  
 
Systems Biology 
In recent years systems biology approaches have been used to investigate seasonal influenza 
infection in humans: involving integrated analysis of genetics, gene expression and proteins 
(21). This concept has been extended to studying blood systemic vaccine responses, 
regarding the vaccination as an “experimental challenge model” (22). However, these 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 11 of 37 
authors are clear on the future priority: “We emphasise here the paramount need for future 
studies deciphering the role of innate immune response to influenza virus at the site of 
infection” (23, 24). In our proposed study on LAIV vaccination, we propose to study nasal 
mucosal antibodies, the IFN response and viral load. 
Nasal Lavage 
Nasal lavage has been extensively used in the study of patients with allergic rhinitis (42), and 
also to measure nasal mucosal responses to intranasal LAIV (16, 17). However, nasal lavage 
with 3.0 to 5.0ml saline has the problems of dilution of secretions, variable recovery, and 
not being able to repeat for 24h for repeatable results (43). Nasal lavage shortly following 
vaccination also risks washing the vaccine virus out of the nose, preventing infection. This 
technique is therefore poorly suited to investigating the innate immune response to 
influenza. Levels of mediators in nasal lavage are uniformly lower than after nasosorption 
(34, 44, 45); we are therefore interested to study levels of antibodies and inflammatory 
mediators in alternative respiratory samples. 
Nasosorption 
The nose is much more accessible than the airways to obtain respiratory samples (46), and 
nasal and bronchial epithelial mucosal lining fluid (MLF) is a potentially important sample to 
study inflammatory mediators in a variety of respiratory diseases: ranging from a variety of 
infections, to chronic conditions including asthma, chronic obstructive pulmonary disease 
(COPD), interstitial lung diseases (47) and lung cancer.  
 
Strips of synthetic absorptive matrices (48) or synthetic sponge can be used to sample MLF, 
before eluting the fluid and measuring levels of mediators that cause and are a feature of 
inflammation. Both natural and synthetic sponges have been used to absorb MLF from mucosal 
surfaces. Ophthalmic Weck-Cel sponges composed of natural cellulose have been used to 
sample saliva, cervical and vaginal secretions (47, 49).  Different absorptive materials have been 
compared for sampling oral fluid prior to measuring antibodies (50), while polyurethane 
minisponges have been used to collect human tears (51). 
 
Filter paper or natural cellulose from the cotton plant has been widely used to absorb nasal 
secretions (52-56).  Nasal lavage has been used to detect IL-5 and IL-13 in nasal secretions 
after nasal allergen challenge (17, 57, 58). However, nasosorption with filter paper detected 
IL-5 and IL-13 at higher levels (59). Following this it was found that different batches of filter 
paper vary in their degree of protein binding, some failing to release cytokines (internal 
data), and thus we decided to identify a suitable synthetic absorptive matrix (SAM). 
 
SAMs are now generally used to obtain nasal MLF by nasosorption (44, 48). These absorbent 
materials are comfortable to use and can obtain MLF even from inflamed noses at frequent 
intervals over extended periods of time. The eluate from nasosorption contains cytokines 
and chemokines at high detectable levels by immunoassay. SAMs have been utilised for 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 12 of 37 
nasosorption in children with allergic rhinitis (60), infants with a family history of atopy (61), 
and in atopic adults after nasal allergen challenge (17) when employing therapeutic anti-IL-
13 monoclonal antibodies (48). A validation study has used a variety of SAMs after NAC to 
show tryptase, eosinophil cationic protein (ECP) and IL-5 responses (62). In contrast, a study 
with nasal LPS challenge showed IL-1β and IL-6 responses (63). In relation to experimental 
human rhinovirus (HRV) challenge in asthma, nasosorption was performed to measure IL-15 
(64), IL-25 (65), IL-33 (66) and IL-18 (67).  
 
Nasosorption devices are now manufactured by Hunt Developments, a specialist medical 
device company from Midhurst in West Sussex. They have clean rooms that ensure 
nasosorption medical devices can be prepared free from lipopolysaccharide (LPS), dust and 
microbial contamination. Nasosorption devices are CE (Conformité Européenne)-marked as 
Class I medical devices. Good Manufacturing Practice (GMP) is followed in a specialised 
medical device facility; with medical grade materials throughout, ensuring devices are 
sterile, LPS and dust free. 
 
This study proposes the use of precision mucosal sampling through nasosorption, in which a 
synthetic absorptive matrix (48) is used to absorb nasal mucosal lining fluid (MLF). In 
previous studies in asthma and hay fever we found that nasosorption sampling permits 
higher levels of mediators to be detected than can be found in nasal lavage. Nasosorption 
can also be repeated with high frequency, up to every few minutes (62), permitting detailed 
investigation of the kinetics of the immune response. Studies have indicated that 
nasosorption can be used to perform viral RNA analysis by reverse transcriptase-polymerase 
chain reaction (RT-PCR) to determine viral load (41).  
 
Project Impact 
 
 
 
• Mucosal sampling and assays 
There is a paramount need to study local nasal mucosal immune responses to nasal LAIV, 
and this will depend on the development of nasal sampling methods and sensitive assays for 
assessing the virus and the immune response. 
 
• Biomarkers and correlates of influenza LAIV Responses and Protection 
It is thought that the immune response to LAIV is mediated by a combination of mucosal and 
systemic factors, involving innate and specific mechanisms that have different kinetics and 
various cell types. Through understanding the molecular and cellular basis of the nasal 
mucosal response to LAIV, we hope to be able to identify key molecular signatures and 
biomarkers that correlate with LAIV responses, and to assess protective pathways that could 
be stimulated by novel vaccines.  
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 13 of 37 
• The nasal vaccine challenge model 
The nasal vaccine challenge model could be developed to test new vaccines, and proceed to 
rational development of improved vaccines for influenza and other diseases. Advanced 
mucosal sampling, assays and biomarkers could be used to accelerate clinical assessment of 
vaccine development. 
 
• Clinical applications in vaccinology 
Establishing correlates of protection and vaccine take may identify subjects who have 
responded poorly to vaccines, and allow assessment of vaccine efficacy in large populations 
including patients that are immunocompromised and vulnerable to influenza. Establishing 
correlates of protection may enable vaccines to be targeted to patients who require 
protection. Use of standardised agents (such as vaccine-grade attenuated vaccine viruses) 
and standardised techniques (such as nasosorption) the feasibility of large, multi-centre 
studies is greatly enhanced. These features could permit studies of vaccine efficacy across 
countries, ethnicities and other population demographics. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 14 of 37 
Study Objectives 
In our proposed study of intranasal LAIV we are especially interested in a number of aspects 
in relation to vaccine take and correlates of efficacy:  
 
1. Characterise nasal MLF IgA/G responses to specific viral sub-types in terms of the 
kinetics and magnitude of the immune response to LAIV specific influenza sub-type 
antibodies: measured using ELISA and by MSD multiplex immunoassays 
2. Quantify serum IgG antibody to 4 influenza sub-types: measured using ELISA and by 
MSD multiplex immunoassay 
3. Determine serum and nasal (if feasible) HAI to all 4 influenza vaccine viruses 
4. Determine serum and nasal (if feasible) antibody neutralisation of HA coated 
pseudotype viruses (representing the 4 influenza vaccine viruses) 
5. Quantify antibody dependent phagocytosis (ADP) of HA bearing particles using serum 
and MLF antibodies 
6. Develop antibody dependent cellular cytotoxicity (ADCC) and antibody dependent 
cellular activation (ADCA) assays using serum and MLF antibodies 
7. Quantify viral load (all 4 strains in the vaccine, by qPCR) 
8. Innate IFNs and IFN-related proteins 
9. Determine the relationship between local and systemic immune responses 
10. Determine the relationship between innate and adaptive immune responses 
11. Explore predictors of the response (responders): Mediators & cellular parameters 
that predict infection and/or antibody induction 
Hypotheses 
1. Influenza sub-type MLF IgA antibodies from the nose can be quantified by ELISA and 
MSD multiplex immunoassays and functional characteristics determined (e.g. 
neutralisation, HAI, ADP, ADCC/ADCA) 
2. Nasosorption can be used to follow the kinetics of LAIV influenza sub-type load (by 
qPCR) 
3. Baseline and early innate-immune induced cytokines (interferons) and chemokines 
(especially relating to IFN responses) will affect the specific immune responses to 
LAIV 
4. Pre-existing antibody immunity (serum or mucosal IgG or IgA) against the vaccine 
influenza sub-types prevent infection and/or decrease viral shedding load/duration 
Aims: 
1. Development of a clinical testing model to assess LAIV correlates of efficacy: 
a. Validation of novel sampling methods for the quantification of viral load and 
antibody measurements 
b. Determination of biomarkers of the innate and adaptive immune response to 
LAIV 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 15 of 37 
c. Determine the influence of pre-existing immunity and the early innate 
response on viral take and the ensuing specific immune response 
 
2. To provide a proof-of-concept human clinical testing model for novel vaccine 
approaches: 
a. To improve tools and provide biomarkers for the rational design and 
improvement of respiratory mucosal viral vaccines  
b. For testing different influenza vaccine strains (selected for the upcoming 
season) and attenuated vaccines with different formulations (e.g. 
nanomaterials) and adjuvants.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 16 of 37 
Study Design 
Participants and Recruitment  
 
Participants will be recruited from Imperial College London through local advertisement for 
participants aged 18-30. ICRRU also holds a database of participants who have previously 
given their contact information and consented to be contacted about future ethically 
approved studies. This database will also be used to contact potential participants with 
advertisement materials. For the pilot study and the main vaccination study the following 
groups will be recruited: 
• Non-asthmatic participants with allergy (atopics); n=15 (pilot study only) 
• Asthmatics; n=15 (pilot study only) 
• Healthy volunteers without allergies or asthma; n=55 (including n=15 for pilot study*) 
 
*Healthy volunteers who participate in the pilot study will be eligible to participate in the main 
vaccination study. 
Study Outcome Measures 
 
Primary Outcome Measure 
Induction of nasal mucosal antibodies to the vaccine influenza strains, defined by a four-fold 
or greater rise in antibody titre. 
 
Secondary Outcome Measures 
1. 
Serum levels of antibodies against the 4 constituent viral subtypes in LAIV: 
measured by ELISA and multiplex immunoassay (Mesoscale Diagnostics) 
2. 
Haemagglutination inhibition (HAI) titres against the 4 viral subtypes from day 0 
to day 28 measured in serum and the nose.  
3. 
Neutralising antibodies (measured by pseudotype assays) measured in the serum 
and nose against the 4 vaccine virus subtypes from day 0 to day 28 
4. 
Elevation in antibody effector function (serum and nasal antibodies): Including 
ADCC/ADCA and ADP 
5. 
Mucosal innate immune response: nasal MLF cytokines and chemokines 
generated within the first 8-hours post-vaccination. Including: IFN-α, IFN-β, IFN-γ, 
IFN-λ (IL-29), IP-10 and ITAC 
6. 
Mucosal LAIV viral load in the first week after vaccination 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 17 of 37 
7. 
Association of transcriptional signatures (nasal and blood) with induction of 
antibodies. 
Participant Entry 
 
Pre-Enrolment Evaluations 
At each participants screening visit their medical history, details of any drugs/medications 
used and a physical assessment will be performed. These investigations will seek to rule out 
the presence or history of any respiratory or other health conditions which meet the 
exclusion criteria.  An ECG will additionally be performed to exclude the possibility of any 
underlying cardiovascular condition. A urine pregnancy test will be performed on all female 
participants, as pregnancy may influence the study results. 
Inclusion Criteria 
 
• Capacity to provide informed consent. 
• Aged 18-30 years - selected to increase likelihood of 'vaccine take' and minimise risk. 
• Fluent English speaker, to ensure comprehensive understanding of study aims, 
methodologies and outputs. 
 
Exclusion Criteria 
 
• Current involvement in another study unless observational or in follow-up phase 
(non-interventional). 
• Received any influenza vaccine over the last 2 years. 
• Egg allergy (as per influenza vaccines patient leaflet). 
• Previous significant adverse reaction to any previous vaccination/immunisation. 
• Current regular (daily) smoker. 
• Pregnant - as this may influence the study results. 
• Taking medication that may affect the immune system in any way e.g. steroids, 
steroid nasal spray. 
• Regularly taking acetylsalicylic acid (aspirin) - as per LAIV guidance to reduce the risk 
of Reye’s syndrome. 
• Unable to give fully informed consent. 
• Current acute severe febrile illness - to avoid vaccination and inoculation in 
participants that may have current infection. 
• Taking long term antibiotics e.g. following splenectomy or sickle cell disease. 
• Clinically diagnosed with flu in the last 2 years. 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 18 of 37 
• A long-term health problem with heart disease, lung disease (including asthma), 
kidney disease, neurologic disease, liver disease, metabolic disease (e.g., diabetes), 
or anaemia or another blood disorder (LAIV contraindications list). 
• Use of drugs for the treatment of rheumatoid arthritis, Crohn’s disease, or psoriasis 
or anticancer drugs; or radiation treatments (LAIV contraindications list). 
• History of Guillain-Barré syndrome 
• Live with or expect to have close contact with a person whose immune system is 
severely compromised and who must be in protective isolation (e.g., an isolation 
room of a bone marrow transplant unit) 
• Received any other vaccinations in the past 4 weeks. 
 
Withdrawal Criteria 
 
In the event of a serious adverse event (SAE), vaccination visits will all be postponed. The 
cause of the SAE will then be investigated. If the SAE is considered to result from the study 
protocol then the study will be terminated. Participants will be vaccinated in groups of 
maximum 3 on any single day. At most 6 will be vaccinated in any single week. 
Participants will be free to withdraw from the study at any point. Any samples collected up 
to the point of their withdrawal will be retained for analysis in anonymised form. 
Randomisation and Enrolment Procedures 
 
As this is an investigational study assessing the mechanism underlying immune protection 
conferred by vaccination, and not a trial of efficacy, no randomisation of participants will be 
performed. Additionally, the study is not placebo controlled or blinded and will be conducted 
as an ‘Open Label’ study. All participants enrolled to the main vaccination study will 
therefore receive LAIV. 
Treatments 
 
All participants in the main vaccination study (not the pilot study) will receive the same LAIV. 
The formulation of this vaccine is altered each year to reflect the circulating strains of 
influenza in that year. This information is not released until May or June each year, so the 
exact formulation of LAIV for 2018/19 is not presently known. However, the vaccine typically 
contains 1x107 fluorescent focus units of each of the four vaccine virus strains. The vaccine 
will be administered intra-nasally in 100μl volumes to each nostril. Participants breathe 
normally during the vaccine administration, which takes less than one minute. 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 19 of 37 
LAIV for this study will be sourced from OTC direct clinical trials services ltd who are the 
registered distributor of LAIV for clinical trials in the UK. This will be received in its normal 
packaging for use in the UK market, which will not be altered prior to vaccination. The 
investigators will pay OTC for LAIV. Considerable safety data is available from young adults 
immunised with LAIV in countries, such as the USA, where LAIV is licensed for this 
population. As such, no dose modification will be conducted. 
Premedication 
No drugs will be prescribed before or during the trial. 
 
Interaction with other drugs 
 
Fluenz Tetra/FluMist should not be given in conjunction with salicylate (aspirin) therapy due 
to the reported associations between use of salicylates and wild type influenza with Reye’s 
syndrome. 
Antivirals such as Oseltamivir should not be used during the trial due to suppression of 
vaccine replication, though no adverse safety events have been reported. 
Efficacy of other live attenuated vaccines (e.g. Yellow Fever Vaccine) could be influenced by 
Fluenz Tetra/FluMist, though no clinically meaningful changes have been observed. As such 
we ask participants to discuss any planned live attenuated vaccinations with us during their 
involvement in the study. 
 
Dispensing and Accountability 
 
LAIV will be kept in a monitored fridge on ICRRU throughout the study duration and will be 
accounted for and administered by the study physician. 
 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 20 of 37 
Adverse events 
 
Definitions   
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study subject.   
 
Serious Adverse Event (SAE): any untoward and unexpected 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 
 
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. 
 
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.   
 
Non serious AEs 
All such events, whether expected or not, should be recorded.   
 
Serious AEs 
An SAE form should be completed and faxed to the Chief Investigator within 24 hours.  
However hospitalisations for elective treatment of a pre-existing condition do not need 
reporting as SAEs. 
 
All SAEs should be reported to the London Camberwell St Giles REC where in the opinion of 
the Chief Investigator, the event was: 
• 
‘related’, i.e. resulted from the administration of any of the research procedures; 
and 
• 
‘unexpected’, i.e. 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 SAEs. 
 
Local investigators should report any SAEs as required by their Local Research Ethics 
Committee, Sponsor and/or Research & Development Office. 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 21 of 37 
 
 
 
 
 
 
Assessments and Follow-ups 
 
The study comprises two phases, detailed below: 
1) A single visit pilot study, which does not involve LAIV administration and no follow 
up. 
2) The main 6 visit study, with administration of LAIV. If these participants are lost to 
follow up their loss will not be flagged. The existing excellent safety profile of LAIV 
makes any such loss to follow up unlikely to result from the vaccine. 
Trial Closure 
 
The trial will be closed at the end of the study period (29/05/2020). All study documents will 
be retained for a period of no-less than 10 years after trial closure.  
 
Statistics and Data Analysis 
 
As a study of the mechanism of action of LAIV, rather than a trial of efficacy, the endpoints 
and planned statistical assessments are exploratory. The sample size calculation is based on 
an estimated sero-conversion rate of 50%, with a documentable infection with vaccine 
virus(es) expected in 30% of participants (though this data is incomplete).  
 
 
 
 
Contact details for reporting SAEs and SUSARs: 
r.nicholson@imperial.ac.uk 
CI email: p.openshaw@imperial.ac.uk 
Fax: 02033125751, attention Peter Openshaw 
Please send SAE forms to: Peter Openshaw by any of the 
above methods 
Tel: 0207 594 3854 (Mon to Fri 09.00 – 17.00) 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 22 of 37 
Pilot Study: Nasal Sampling Methods and Measurement of Nasal Mucosal 
Mediators and Antibodies 
 
This Preliminary Pilot Study is carried out with the aim of selecting and validating the optimal 
methods of sampling and analysis for the main study. The pilot does not involve LAIV 
vaccination or physical examination. 
 
Subjects: We shall undertake a Preliminary Pilot Study in volunteers (n=45), including atopics 
(n=15), asthmatics (n=15) and healthy volunteers (n=15). 
 
Patient Information and Consent 
We shall have a separate Patient Information Sheet and Consent Form for this Preliminary Pilot 
Study. Live cells will be retained, along with samples for transcriptional studies (RNA), but not DNA.  
 
Samples: 
1. Blood sampling 
We shall take a single blood sample of up to 50ml blood: for optimisation of cellular immune 
and antibody assays. 
2. Gum crevicular fluid 
This will be collected using a specially designed Oracol swab. 
3. Nasal Sampling Methods: 
• Nasosorption: We shall compare different methods of nasosorption of nasal MLF 
involving different medical grade materials, including poly-urethane and Leukosorb. 
A sampling schedule will be performed as follows, in order to establish repeatability and 
reproducibility of measurements: 
- 
Left nostril: 0min, 30min 
- 
Right nostril: 0min, 30min 
Sample elution methods: 
We shall compare methods for elution of nasosorption samples with a variety of elution 
buffers and manual or centrifugation methods.  
• Nasal wash 
• Nasal curettage: We shall compare different methods of nasal microcurettage: 
Rhinoprobe versus CellSkim. We shall take up to 4 microcurettage specimens per 
volunteer. 
 
Assays: 
We shall measure markers of inflammation (e.g. cytokines and chemokines), mucosal antibodies, 
microRNA, mRNA, the microbiome and viral RNA. In particular we shall optimise techniques for the 
measurement of influenza IgG and IgA antibodies (nasal and serum) and their function. 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 23 of 37 
Main Study: Nasal Mucosal and Systemic Immune Responses to Intranasal 
LAIV in Healthy Adults of 18-30 years 
 
Subjects: The main study will recruit healthy volunteers (n=40) through local advertisement 
within Imperial College London. 
 
Patient Information and Consent 
We shall have a separate Patient Information Sheet and Consent Form for this Main Study. Live cells 
will be retained, along with samples for transcriptional studies (RNA), but not DNA. 
 
Primary End-Point 
Induction of nasal mucosal antibodies to the 4 constituent viral subtypes in LAIV: quantified 
by ELISA and multiplex immunoassay (Mesoscale Diagnostics) and expressed as 
seroconversion, geometric mean titre (GMT) and geometric mean fold rises (GMFR).  
Secondary End-Points 
1. Serum levels of antibodies against the 4 constituent viral subtypes in LAIV: measured 
by ELISA and multiplex immunoassay (Mesoscale Diagnostics) 
2. Haemagglutination inhibition (HAI) titres against the 4 viral subtypes from day 0 to 
day 28 measured in serum and the nose  
3. Neutralising antibodies (measured by pseudotype assays) measured in the serum and 
nose against the 4 viral subtypes from day 0 to day 28 
4. Antibody effector function (serum and nasal antibodies): Including ADCC/ADCA and 
ADP 
5. Mucosal innate immune response: nasal MLF cytokines and chemokines generated 
within the first 8-hours post-vaccination. Including: IFN-α, IFN-β, IFN-γ, IFN-λ (IL-29), 
IP-10 and ITAC 
6. Mucosal LAIV viral load in the first week after vaccination 
7. Association of transcriptional signatures (nasal and blood) with induction of 
antibodies. 
 
Schedules for Sampling 
For reference, also see appendix 1 of this document.  
Sampling at screening visit 
The screening visit will firstly assess candidates for eligibility, based on the study exclusion 
criteria and contraindications for LAIV vaccination, including completion of checklist for LAIV 
contraindications. This screening visit should take place 1-6 weeks prior to the vaccination 
visit. Once this screening has been completed, the following samples will be collected from 
volunteers: 
• Nasosorption sampling 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 24 of 37 
• Nasal wash  
• Nasal curettage 
• Blood samples (less than 50ml) 
• Gum swab using Oracol swabs 
 
If no contraindications for LAIV administration are found then participants will be invited to 
proceed to the main vaccination study. 
 
Sampling schedule in relation to LAIV administration on Day 0 (0d)  
For reference to the study schedule on the vaccination day (0d), see appendix 2: 
• Nasosorption sampling on vaccination day (0d): pre-vaccine, 3h, 4h, 5h, 6h, 7h, 8h 
• Nasosorption sampling on days after 0d: 1d, 3d, 7d and 28d 
• Nasal curettage: 3d 
• Nasal wash: 28d 
• Blood samples will be collected at  1d, 3d, 7d and 28d 
 
Clinical Assessments at study visits 
Physical assessment of symptoms and Nasal Inspiratory flow will be determined once on 
each of the following visits: Screening, 1d, 3d, 7d and 28d. 
On the vaccination visit 2 (0d), physical assessment of symptoms and nasal inspiratory flow 
will be determined immediately following nasosorption sampling at times: pre-vaccine, 4h, 
8h. 
Physical examination will involve a general examination of participant's nose, heart and 
lungs. This will include taking a history, measuring blood pressure, heart rate, breathing rate 
and temperature. 
 
 
 
 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 25 of 37 
 
Nasal and Serum sample assays 
 
Samples may be shipped directly to international collaborating study partners or contract 
research organisations for analysis, the patient information sheets and consent forms for 
this study highlight this point. 
 
Samples arising from this study will be used for laboratory experiments, including: 
 
• Viral Sub-Type qPCR: 4 Influenza strains in Flumist (Nasal only) 
Fast throughput qPCR assay developed through Medimmune (Gaithersburg, USA) 
and Virosciences (Rotterdam, NL).  
qPCR primers available for the 4 sub-types designated by the WHO for 2018/19 
 
• Cytokines/chemokines: 
IFN-α, IFN-β, IFN-γ, IFN-λ (IL-29), IP10, ITAC 
IL-1β, IL-6 
IL-5, IL-13, IL-33 
 
Antibody Assays: Serum and Nasal samples 
 
1. 
Viral sub-type specific IgG and IgA versus HA 
High sensitivity immunoassay for the quantification of antibodies against each vaccine 
strain; developed by Medimmune. To be performed on serum and nasal respiratory 
samples. 
 
2. 
HAI serum IgG haemagglutination inhibition antibodies    
In collaboration with Katja Hoschler, Public Health England 
HAI functional assays for the 4 individual vaccine virus subtypes.  
• 
Seronegative titre <8,  seropositive titre >8  
• 
Serum response defined as >4 fold rise 
• 
Geometric mean fold rises (GMFRs) 
Performance of respiratory samples (nasosorption or nasal wash) unknown: detergent in 
the samples could interfere with the assay, development is therefore needed. 
 
3. 
Pseudotype neutralisation antibodies        
In collaboration with Nigel Temperton, University of Kent             

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 26 of 37 
• Replication-defective HIV core, integrated with HA subtype on virion surface, 
expresses luciferase upon infection: high sensitivity and large dynamic range 
compared to classical neutralisation assays. 
• Available viral subtypes are close to the Flumist: H1N1 (California) and H3N2. New 
pseudotypes can be rapidly generated. 
• Requirement for whole HA RNA sequences, which are available online (33): ordered 
from Genscript HK (or Invitrogen) for cloning. 
• Determines neutralising antibody concentrations. 
Performance of respiratory samples (nasosorption or nasal wash) unknown: detergent 
in the samples could interfere with the assay, development is therefore needed. 
 
4. Antibody dependent cellular cytotoxicity (ADCC)/Antibody dependent effector cell 
activation (ADCA) 
Antibodies can drive the killing of infected cells, a key mediator of protection (37).  
Techniques exist to measure this ability of antibodies from the serum (38, 39) and such 
techniques could be used to determine mucosal antibody function. 
• Antibody dependent cytotoxicity assays require target cells to either be infected by 
the pathogen of interest, or to express a target antigen (e.g. a HA variant). The assay 
then determines lysis of these target cells by immune effector cells, in an antibody 
dependent manner (38, 39). 
• Alternatively antibody dependent effector cell activation (ADCA) assays look at 
activation of effector cells, but do not measure lysis of a target cell. Instead they 
measure expression of a surface activation marker, or secretion of an effector 
protein. These assays therefore do not require target cells and can be performed 
with recombinant proteins (38, 39). 
Performance of respiratory samples (nasosorption or nasal wash) unknown: detergent in 
the samples could interfere with the assay, development is therefore needed. 
 
5. Antibody dependent phagocytosis 
Antibodies can also induce the phagocytosis of targets through antibody dependent 
phagocytosis (39). This property may play a role in viral clearance and prevention of re-
infection with influenza (16-18), but has not been studied in respiratory mucosal antibodies.  
Performance of respiratory samples (nasosorption or nasal wash) unknown: detergent in 
the samples could interfere with the assay, development is therefore needed. 
 
Data Analysis: Bioinformatics and Systems Biology 
 
Systemic and mucosal vaccine systems biology 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 27 of 37 
Systems biology approaches have been introduced to looking at seasonal influenza infection 
in humans (21), and also for blood systemic vaccine responses, regarding the vaccination as 
an “experimental challenge model” (22-24). There is now the opportunity to perform 
systems biology on both the blood (systemic) and nasal mucosal (local) immune response to 
LAIV. This is because in this proposed study on LAIV vaccination, we propose to primarily 
study nasal mucosal and serum antibodies, the innate immune response and viral load. 
However, there will also be assessment of the transcriptional gene expression response 
(systemic (blood) and mucosal) to LAIV. Integrating these transcriptomics data with other 
data arising from this study (antibody generation, function, viral load and the innate immune 
response) a complete view of the immune response to influenza can be generated. This 
approach will further the ability to distinguish responses to infection and correlates of 
vaccine efficacy. 
 
Responders and non-responders 
Our primary postulated correlate of influenza protection is the nasal MLF level of influenza-
specific IgG and IgA. Following LAIV we would like to classify our subjects in terms of nasal 
mucosal IgG and IgA geometric mean fold rises (GMFR): or nasal mucosal “responders” and 
“non-responders”. However, since our study is exploratory we will have to assess the kinetics 
and magnitude of the nasal mucosal immune response before we can provide this definition. 
Other important post-LAIV vaccine responses will be the magnitude of the initial mucosal 
innate immune response and the nasal viral load. 
 
Predictors of the Response 
It will be necessary to construct predictive models to then postulate those indices pre- and 
post-vaccination which correlate with protection. These generated predictors of response 
would need to be validated in a subsequent cohort or in the context of influenza live viral 
challenge or natural infection. 
 
Before LAIV:  
We postulate that nasal antibody levels inversely correlate with subsequent local and 
systemic IgG and IgA responses. 
There may be molecular signatures in nasal MLF, nasal curettage and blood that will predict 
the nasal and systemic antibody responses to LAIV. 
 
Following LAIV: 
We postulate that the size of the initial nasal innate immune response and viral load may 
predict the magnitude or functionality of the subsequent nasal antibody response.  
There may be molecular signatures in nasal MLF, nasal curettage and blood following LAIV 
that will predict (1) the nasal and systemic HAI IgG/IgA responses to LAIV (2) protection 
against influenza. 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 28 of 37 
Risks and Benefits to Study Participants 
 
In the USA this vaccine is routinely administered to adults with excellent safety. As such we 
do not anticipate any adverse reactions to vaccination beyond mild symptoms to influenza 
like illness, such as malaise, fever and respiratory tract symptoms. Even such symptoms are 
rare. This study has received written confirmation from the Medicines and Healthcare 
products Regulatory Agency (MHRA) that the study does not require registration as a clinical 
trial of an investigational medicinal product, despite there not being a marketing 
authorisation for this product in the UK.  
 
The participants will benefit through the studies administration of an influenza vaccine, 
which may protect them from infection. However, we will request that the participants do 
not receive any other influenza vaccines until after the final study visit. As LAIV has a lower 
protective efficacy than intramuscular inactivated influenza in this group of young adults, the 
participants may be at higher risk of influenza infection than if they had elected to receive an 
inactivated influenza vaccine. This risk is minimal, as vaccination is not routinely 
recommended to this population. Additionally, at the end of the study we will ensure that 
participants are informed about available influenza vaccines, should they decide that they 
would like an additional vaccination.  
 
Participants will receive remuneration for their time and travel costs associated with the 
study. For the pilot study (single one hour visit) participants will receive £25. For the main 
LAIV study (6 visits, totalling 13 hours) participants will receive £325.  
 
 
 
 
 
 
 
 
 
 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 29 of 37 
Monitoring 
Risk Assessment 
Given the excellent safety profile of LAIV in the target population in other countries, 
including an FDA license for the USA, and the marketing authorisation in the UK for infants, 
the study is considered low risk. As such a minimum level of monitoring will be required by 
the study and will be performed between the study team and the Joint Research Compliance 
Office of Imperial College London. 
Monitoring at ICRRU 
Due to the low risk nature of the trial monitoring visits will not be routinely scheduled, but 
will be available for monitoring as required. 
Regulatory Issues 
 
Clinical Trials Authorisation 
This study has received written confirmation from the Medicines and Healthcare products 
Regulatory Agency (MHRA) that this study does not require registration as a clinical trial of 
an investigational medicinal product. As such, review for approval by the MHRA is not 
required. 
 
Ethics Approval 
The Study Coordination Centre has obtained approval from the London Camberwell St Giles 
Research Ethics Committee (REC) and Health Regulator Authority (HRA). The study will also 
receive confirmation of capacity and capability from the Imperial College NHS Healthcare 
Trust before accepting participants into the study or any research activity is carried out. The 
study will be conducted in accordance with the recommendations for physicians involved in 
research on human subjects adopted by the 18th World Medical Assembly, Helsinki 1964 
and later revisions. 
Consent 
Consent to enter the study will be sought from each participant only after a full explanation 
has been given, an information leaflet offered and time allowed for consideration. Signed 
participant consent will be obtained. The right of the participant to refuse to participate 
without giving reasons will be respected. All participants are free to withdraw at any time 
from the protocol treatment without giving reasons and without prejudicing future 
treatment. 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 30 of 37 
Confidentiality 
Participants’ identification data will be required for the registration process. The Study 
Coordination Centre will preserve the confidentiality of participants taking part in the study 
and is registered under the Data Protection Act. 
Indemnity 
 
Imperial College London holds negligent harm and non-negligent harm insurance policies 
which apply to this study. 
Sponsor 
Imperial College London will act as the main Sponsor for this study. Delegated 
responsibilities will be assigned to the NHS trusts taking part in this study. 
Funding  
The National Institute for Health Research (NIHR) Imperial Biomedical Research Cluster (BRC) 
are funding this study. 
Audits and Inspections 
The study may be subject to inspection and audit by Imperial College London under their 
remit as Sponsor and other regulatory bodies to ensure adherence to GCP. 
Publication Policy 
 
The results of the trial will be published in scientific journals, presented at conferences as 
oral and poster presentations and distributed to collaborators. No patient identifiable 
information 
will 
be 
included 
in 
any 
publication 
or 
disseminated 
material.

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 31 of 37 
APPENDIX 1: Main Study Sampling Schedule 
 
* - See Appendix 2 for further detail on the schedule for study Visit 2 
Study procedures 
Visit 1 
 
Screening 
Visit 2* 
 
Nasal LAIV 
Visit 3 
 
Day 1 (24h) 
Visit 4 
 
Day 3 (72h) 
Visit 5 
 
Day 7 
Visit 6 
 
Day 28 (26-30) 
1 hour visit 
Full day visit 
1 hour visit 
1 hour visit 
1 hour visit 
1 hour visit 
Assessment of 
Contraindications for 
LAIV 
X 
 
 
 
 
 
Vaccination with Nasal 
LAIV 
 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
 
Physical Examination 
X 
X 
X 
X 
X 
X 
ECG 
X 
 
 
 
 
 
Urine pregnancy test 
X 
 
 
 
 
 
Blood Test 
X 
 
X 
X 
X 
X 
Skin Prick Test 
X 
 
 
 
 
 
Nasal Inspiratory Flow 
X 
X 
X 
X 
X 
X 
Nasosorption 
X 
X (x7*) 
X 
X 
X 
X 
Nasal wash 
X 
 
 
 
 
X 
Nasal scrape (nasal 
curettage) 
X 
 
 
X 
 
 
Gum swab 
X 
 
 
 
 
X 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 32 of 37 
APPENDIX 2: Visit 2 (Vaccination day) sampling schedule 
 
 
 
 
Study 
procedures 
Time from arrival at ICRRU 
0h 
3h 
4h 
5h 
6h 
7h 
8h 
Vaccination 
with Nasal LAIV 
X 
 
 
 
 
 
 
Physical 
Examination 
X 
 
X 
 
 
 
X 
Nasal 
Inspiratory Flow 
X 
 
X 
 
 
 
X 
Nasosorption 
X 
X  
X 
X 
X 
X 
X 

 
 
 
Nasal and systemic responses to nasal influenza vaccination      Study Protocol  Page 33 of 37 
 
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