Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection
Phase: PHASE4
Conditions: Influenza
Sponsor: Stanford University

 
 
 ____________________________________________________________________________________________  
 
 
Innate and Acquired Immunity to Influenza 
Infection and Immunization 
NCT03028974 
August 7, 2015 
 
Cornelia L. Dekker, MD, Principal Investigator 
Stanford University 
Stanford, California 94305 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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Adaptive and Innate Immunity, Memory and Repertoire  
in Vaccination and Infection 
  
(U19 CCHI Renewal 2014-2019) 
 
 
Project 2: Innate and Acquired Immunity to Influenza  
Infection and Immunization 
 
DAIT Protocol Number:  NA 
 
DAIT Funding Mechanism: 2U19AI057229-11 
 
Other Identifying Numbers:  SLVP029; IRB Protocol: 31136 
 
U19 CCHI Center Principal Investigator: Mark M. Davis, PhD 
 
Clinical Core Principal Investigator:  Cornelia L. Dekker, MD 
 
Project 2 Leader:  Harry Greenberg, MD 
 
Project 2 Co-Leader:   William H Robinson MD, PhD 
 
 
DAIT Program Officer (Basic Immunology Branch): 
 
 
DAIT Medical Monitor: NA 
 
 
Draft or Version Number: 2.0 
Day Month Year 
07AUG2015 
 
 
 
 
 
 
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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STATEMENT OF COMPLIANCE 
 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following: 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
• 
ICH E6; 62 Federal Register 25691 (1997)  
• 
NIH Clinical Terms of Award 
 
All key personnel (all individuals responsible for the design and conduct of this study) have 
completed Human Subjects Protection Training. 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
Site Investigator: 
 
Signed: 
 
Date: 
 
 
Name: Cornelia L. Dekker, M.D. 
Title: Professor of Pediatrics, Principal Investigator 
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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TABLE OF CONTENTS 
 
 
Page 
Statement of Compliance ........................................................................................................... 2 
Signature Page .......................................................................................................................... 3 
Table of Contents ....................................................................................................................... 4 
Protocol Summary ...................................................................................................................... 9 
1 
Key Roles ........................................................................................................................ 16 
2 
Background Information and Scientific Rationale ............................................................. 18 
2.1 
Background Information ....................................................................................... 18 
2.2 
Rationale .............................................................................................................. 20 
2.3 
Potential Risks and Benefits ................................................................................. 21 
2.3.1 
Potential Risks .......................................................................................... 21 
3 
Objectives ........................................................................................................................ 23 
3.1 
Study Objectives .................................................................................................. 23 
3.2 
Study Outcome Measures .................................................................................... 23 
3.2.1 
Primary Outcome Measures: .................................................................... 23 
• 
mRNA levels, ............................................................................................ 23 
• 
Ig gene sequence ..................................................................................... 23 
3.2.2 
Secondary Outcome Measures: ............................................................... 23 
4 
Study Design ................................................................................................................... 24 
5 
Study Enrollment and Withdrawal .................................................................................... 26 
5.1 
Subject Inclusion Criteria ...................................................................................... 26 
5.2 
Subject Exclusion Criteria for Enrollment and Subsequent Annual Vaccinations .. 26 
5.3 
Treatment Assignment Procedures ...................................................................... 27 
5.3.1 
Randomization Procedures ...................................................................... 27 
5.3.2   Reasons for Withdrawal ............................................................................ 28 
5.3.3   Handling of Withdrawals ............................................................................ 28 
5.3.4   Termination of Study ................................................................................. 28 
6 
Study Intervention/Investigational Product ....................................................................... 29 
6.1 
Study Product Description .................................................................................... 29 
6.1.1 
Acquisition ................................................................................................ 29 
6.1.2 
Formulation, Packaging, and Labeling ...................................................... 29 
6.1.3 
Product Storage and Stability ................................................................... 30 
6.2 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product ................................................................................................................ 30 
6.3 
Accountability Procedures for the Study Intervention/Investigational Product(s) ... 30 
6.4 
Concomitant Medications ..................................................................................... 31 
7 
Study Schedule ............................................................................................................... 32 
7.1 
Screening ............................................................................................................. 32 
7.2 
Day 0; Enrollment/Baseline/Immunization for all Groups; annually for Groups A 
and D. .................................................................................................................. 32 
7.3 
Day 1 post- Dose 1 immunization for Group C and Group F only ......................... 33 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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7.4 
Day 6-8 post-Dose 1 immunization except Group C and vaccine-naïve volunteers 
in Group D and Group E requiring Dose 2; annually for Groups A and D. ............ 33 
7.5 
Day 21 post-Dose 1 immunization for Group C only ............................................. 34 
7.6 
Day 28 post-Dose 1 immunization ........................................................................ 34 
7.6.1 
Day 28-32 for vaccine-naïve volunteers requiring second dose of vaccine 
(Groups A, B, C, D and E) ........................................................................ 34 
7.6.2 
Day 28 ± 4 for Groups F and G ................................................................. 34 
7.7 
Day 6-8 post-Dose 2 immunization for previously vaccine-naïve volunteers in 
Group D and E ..................................................................................................... 35 
7.8 
Early Termination Visit ......................................................................................... 35 
8 
Study Procedures/Evaluations ......................................................................................... 36 
8.1 
Clinical Evaluations .............................................................................................. 36 
8.2 
Laboratory Evaluations ........................................................................................ 36 
8.2.1 
Special Assays or Procedures .................................................................. 36 
8.2.2 
Specimen Preparation, Handling, and Shipping ........................................ 37 
8.3 
Specification of Safety Parameters ...................................................................... 38 
8.4 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters38 
8.4.1 
Serious Adverse Events ........................................................................... 38 
8.5 
Reporting Procedures .......................................................................................... 38 
8.5.1 
Regulatory Reporting for Studies Not Conducted Under an IND ............... 38 
8.5.2 
Type and Duration of Follow-up of Subjects after Adverse Events ............ 39 
8.6 
Safety Oversight .................................................................................................. 39 
9 
Clinical Monitoring ........................................................................................................... 40 
10 
Statistical Considerations................................................................................................. 41 
10.1 
Study Hypotheses ................................................................................................ 41 
10.2 
Sample Size Considerations ................................................................................ 41 
10.3 
Final Analysis Plan ............................................................................................... 41 
11 
Source Documents and Access to Source Data/Documents ............................................ 43 
12 
Quality Control and Quality Assurance ............................................................................ 44 
13 
Ethics/Protection of Human Subjects ............................................................................... 45 
13.1 
Ethical Standard ................................................................................................... 45 
13.2 
Institutional Review Board .................................................................................... 45 
13.3 
Informed Consent Process ................................................................................... 45 
13.3.1 Informed Consent/Assent Process (in Case of a Minor) ............................ 45 
13.4 
Exclusion of Women, Minorities, and Children (Special Populations) ................... 46 
13.5 
Subject Confidentiality .......................................................................................... 46 
13.6 
Study Discontinuation .......................................................................................... 46 
13.7 
Future Use of Stored Specimens ......................................................................... 47 
14 
Data Handling and Record Keeping ................................................................................. 48 
14.1 
Data Management Responsibilities ...................................................................... 48 
14.2 
Data Capture Methods ......................................................................................... 48 
14.3 
Types of Data ....................................................................................................... 48 
14.4 
Timing/Reports ..................................................................................................... 48 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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14.5 
Study Records Retention ..................................................................................... 49 
14.6 
Protocol Deviations .............................................................................................. 49 
15 
Publication Policy ............................................................................................................ 50 
16 
Literature References ...................................................................................................... 51 
SupplementS/Appendices ........................................................................................................ 54 
Appendix A: Schedule of Visits ................................................................................................. 55 
Appendix B: Schedule of Events .............................................................................................. 56 
Appendix C: Flumist QUADRIVALENT Package InsertS 2014-2018 ........................................ 57 
Appendix D: FluZONE QUADRIVALENT Package Inserts 2014-2018 ..................................... 57 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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List of Abbreviations 
AE 
Adverse Event/Adverse Experience 
ACIP 
The CDC Advisory Committee on Immunization Practices 
CDC 
The Centers for Disease Control 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CONSORT 
Consolidated Standards of Reporting Trials 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
DAIT 
Division of Allergy, Immunology and Transplantation 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, NIH, 
DHHS 
DSMB 
Data and Safety Monitoring Board 
DZ 
Dizygotic twin 
eCRF 
Electronic Case Report Form 
FACS 
Fluorescence Activated Cell Sorting 
FDA 
Food and Drug Administration 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IEC 
Independent or Institutional Ethics Committee 
IIV4 
Quadrivalent Inactivated Influenza Vaccine 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
JAMA 
Journal of the American Medical Association 
LAIV 
Trivalent Live, Attenuated Influenza Vaccine 
LAIV4 
Quadrivalent Live, Attenuated Influenza Vaccine  
MedDRA  
Medical Dictionary for Regulatory Activities 
MZ 
Monozygotic twin 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
NCI 
National Cancer Institute, NIH, DHHS 
NDA 
New Drug Application 
NEJM 
New England Journal of Medicine 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, DHHS 
NIH 
National Institutes of Health 
NK cells 
Natural Killer Cells 
OCRA 
Office of Clinical Research Affairs, DMID, NIAID, NIH, DHHS 
OHRP 
Office for Human Research Protections 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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OHSR 
Office for Human Subjects Research 
ORA 
Office of Regulatory Affairs, DMID, NIAID, NIH, DHHS 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SLVP 
Stanford-LPCH Vaccine Program 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
TIV 
Trivalent inactivated influenza vaccine 
US 
United States 
WHO 
World Health Organization 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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PROTOCOL SUMMARY 
 
 
Title: Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization 
 
Number of Sites: 1 
Study Duration: 5 years for enrollment, vaccination, analysis 
Subject Participation Duration: 4-5 weeks 
 
Description of Agent or Intervention:   
Quadrivalent, Live Attenuated Influenza Vaccine (LAIV4) 
Quadrivalent, Inactivated Influenza Vaccine (IIV4)  
 
Objectives: 
 
Primary: 
  
1. To compare the nasal mucosal transcriptional responses to LAIV4 between the 
following age groups: 2-4 year old children, 9-13 year old children, and 18-49 year old 
adults 
 
2. To compare the B cell response to LAIV4 between the following age groups: 2-4 year 
old children, 9-13 year old children, and 18-49 year old adults 
 
3. To investigate the evolution of B cell repertoire in young children after repeated 
LAIV4 immunization 
 
4. To compare the B cell response to IIV between the following age groups: 6-23 month 
old children, 9-13 year old children, and 18-49 year old adults 
 
5. To investigate the evolution of B cell repertoire in young children first immunized with 
IIV followed by repeated LAIV4 immunization. 
 
 
Secondary:  
1.  To compare the B cell repertoire in response to LAIV4 versus IIV in different 
age groups. 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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Description of Study Design:  This is a Phase IV study of healthy children and adults who will receive 
the current seasonal influenza vaccine. There are no exclusions for gender, ethnicity or race.  The 
volunteers will be enrolled into one of 7 groups over a 5-year period. Immunization will be at Day 0. 
Blood and NP swab samples will be collected at various time points based upon group assignment for 
various immunological assays. 
 
Group A (LAIV4/annual return): Up to six 2-4 year old volunteers will be given a quadrivalent live, 
attenuated influenza vaccine (LAIV4). Blood samples to conduct the assays described will be taken at 
Day 0 pre-immunization and Day 6-8 post-Dose 1 for all children irrespective of those who require 1 
dose (prior flu vaccination history) or 2 doses (vaccine-naïve). For children requiring 2 doses of vaccine, 
a second immunization will be given at Day 28-32 after Dose 1. All participants in this group will be 
asked to return annually for repeat immunization per ACIP guidelines and blood sample collection. 
 
Group B (LAIV4/single year): Up to twenty 2-4 year old volunteers will be given a quadrivalent live, 
attenuated influenza vaccine (LAIV4). Blood samples to conduct the assays described will be taken at 
Day 0 pre-immunization and Day 6-8 post-Dose 1 for all children irrespective of those who require 1 
dose (prior flu vaccination history) or 2 doses (vaccine-naïve). For children requiring 2 doses of vaccine, 
a second immunization will be given at Day 28-32 after Dose 1. 
 
Group C (LAIV4/NP swab group/single year):  Up to twenty 2-4 year old volunteers will be given a 
quadrivalent live, attenuated influenza vaccine (LAIV4). NP swabs will be collected at Day 1 and Day 20-
22 post-Dose 1 for all children irrespective of those who require 1 dose or 2 doses of vaccine. For 
children requiring 2 doses of vaccine, a second immunization will be given at least 28 days after Dose 1. 
No blood will be collected for this group.  
 
Group D (IIV4/annual return): Up to six 6 month-23 month old (inclusive) volunteers will be given a 
quadrivalent inactivated influenza vaccine (IIV4). Blood samples to conduct the assays described will be 
taken at Day 0 pre-immunization and Day 6-8 post-Dose 1 for children requiring 1 dose of vaccine (prior 
flu vaccination history).   For children requiring 2 doses of vaccine (vaccine-naïve), a second 
immunization will be given at Day 28-32 after Dose 1. The second blood sample will be collected on Day 
6-8 post-Dose 2. Volunteers will be asked to return annually for repeat immunization per ACIP 
guidelines and blood sample collection. 
 
Group E (IIV4/single year): Up to twenty 6 month–23 month old (inclusive) volunteers will be given a 
quadrivalent inactivated influenza vaccine (IIV4). Blood samples to conduct the assays described will be 
taken at Day 0 pre-immunization and Day 6-8 post-Dose 1 for children requiring 1 dose of vaccine (prior 
flu vaccination history).   For children requiring 2 doses of vaccine (vaccine-naïve), a second 
immunization will be given at Day 28-32 after Dose 1. The second blood sample will be collected on Day 
6-8 post- Dose 2. Volunteers will participate for single year. 
 
Group F (LAIV4/single year) Up to forty 9-13 year old (n= 20) and 18-49 year old (n= 20) volunteers will 
be given a quadrivalent live, attenuated influenza vaccine (LAIV4). Blood samples and NP swabs will be 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029.                                                                  
V2.0_07AUG2015 
 
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collected. Blood samples will be taken at Day 0 pre-immunization, Day 6-8 post-immunization and Day 
28±4 post-immunization. NP swabs will be collected at Day 1 post-immunization and Day 28 post –
immunization. Volunteers will participate for a single year. 
 
Group G (IIV4/single year) Up to forty 9-13 year old (n= 20) and 18-49 year old (n= 20) volunteers will be 
given a quadrivalent inactivated influenza vaccine (IIV4). Blood samples to conduct assays will be taken 
at Day 0 pre-immunization, Day 6-8 post immunization and Day 28±4 post-immunization. Volunteers will 
participate for single year. No NP swabs will be collected for this group. 
 
Estimated Time to Complete Enrollment: Participants will be enrolled Aug-Dec in each of 5 years  
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant ‘Adaptive and Innate Immunity, Memory and 
Repertoire in Vaccination and Infection’); SLVP029.                                                                                                                                              V2.0_07AUG2015 
 
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U19 CCHI Project 2 (SLVP029) 
Innate and Acquired Immunity to Influenza Infection and Immunization 
 
 
 
 
 
 
 
 
 
 
Day 0-------------- 
Visit V01  
 
 
 
                                                                                                                                              
 
Day 6-8 ------------------------------------------------------------------------------------------------- 
Visit V02 
 
 
Day 28-32 ------------------------------------------------------------------------------------------------------------------------------ 
 
 
 
 
(post-Dose 1)  
 
 
 
 
       
Visit V03A                                                            
         
Group A 
LAIV4 
Annual Return 
 
2-4 yo 
n= 6 
Group B 
LAIV4 
Single Year 
 
2-4 yo 
n= 20 
(ONLY VACCINE-NAÏVE) 
Provide LAIV4 Dose 2 
SAE assessment; NO blood draw 
All Volunteers 
Obtain parental informed consent, screen subjects by criteria, meds, and vital signs  
 Collect baseline blood for assays. Administer LAIV4 Dose 1 Vaccine 
  Assessment of Study Outcome 
Measures 
 
Collect blood; SAE assessment 
(ONLY VACCINE-NAÏVE) 
Provide LAIV4 Dose 2 
SAE assessment; NO blood draw  
 
Collect blood; SAE assessment 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant ‘Adaptive and Innate Immunity, Memory and 
Repertoire in Vaccination and Infection’); SLVP029                                                                                                                                                 v2.0_07AUG2015 
 
13 | P a g e  
 
U19 CCHI PROJECT 2 (SLVP029) 
 
 
 
 
 
 
 
 
 
 
Day 0------------ 
 
Visit V01 
 
 
 
 
 
Day 1---------------- 
 
Visit V01A 
 
 
 
 Day 20-22--------------- 
 
Visit V02A 
 
 
 
 
 
Day 28-32  (post-Dose1)------------ 
 
Visit V03A 
 
 
 
 
 
 
Group C 
LAIV4- NP Swab 
Single Year 
 
2-4 yo 
n= 20 
 
Collect NP swab; NO blood draw; SAE assessment 
(ONLY VACCINE-NAÏVE) 
Provide LAIV4 Dose 2 SAE 
assessment; NO blood draw 
All Volunteers 
Obtain parental informed consent, screen subjects by criteria, meds, and vital signs  
Administer LAIV4 Dose 1 Vaccine;  NO blood draw. 
 
Collect NP swab; NO blood draw; SAE assessment 
  Assessment of Study Outcome 
Measures 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant ‘Adaptive and Innate Immunity, Memory and 
Repertoire in Vaccination and Infection’); SLVP029                                                                                                                                                 v2.0_07AUG2015 
 
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U19 CCHI PROJECT 2 (SLVP029) 
 
 
 
 
 
 
 
 
 
 
Day 0 ----------- 
 
Visit V01 
 
 
 
                           Require 1 dose of vaccine                   Requires 2 doses of vaccine         Requires 1 dose of vaccine                  Require 2 doses of vaccine   
                                  (Prior vaccination)                                 (Vaccine-naïve)                            (Prior vaccination)  
                 (Vaccine-naïve) 
 
 
Day 6-8 ------------------------------------------------------------------------------------------ 
(post-Dose1) 
Visit V02 
 
Day 28-32 (post-Dose1)------------------------------------------------------------------------------------------------------------------------------ 
 
 
Visit V03A 
 
Day 6-8 (post-Dose 2)--------------------------------------------------------------------------------------------------- 
 
 
 
 
       
Visit V04 
                                                                
         
 
 
 
All Volunteers 
Obtain parental informed consent, screen subjects by criteria, meds, and vital signs  
 Collect baseline blood for assays. Administer IIV4 Dose 1 vaccine 
  Assessment of Study Outcome 
Measures 
Group E 
IIV4 
Single Year 
 
6 mo – 23 mo 
n= 20 
 
Collect blood; SAE assessment 
Collect blood; SAE assessment 
(ONLY VACCINE NAÏVE) 
Provide IIV4 Dose 2 
SAE assessment; NO blood draw 
Group D 
IIV4 
Annual Return 
  
6 mo – 23 mo 
n= 6 
 
Collect blood; SAE assessment 
Collect blood; SAE assessment 
(ONLY VACCINE NAÏVE) 
Provide IIV4 Dose 2 
SAE assessment; NO blood draw 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant ‘Adaptive and Innate Immunity, Memory and 
Repertoire in Vaccination and Infection’); SLVP029                                                                                                                                                 v2.0_07AUG2015 
 
15 | P a g e  
 
U19 CCHI Project 2 (SLVP029) 
 
 
 
 
 
 
 
 
 
 
Day 0 --------------- 
Visit V01  
 
 
 
Day 1 -------------- 
Visit V01A 
 
 
Day 6-8 -------------------------------------------------------------------------------------- 
Visit V02 
 
 
Day 28±4 ---------------------------------------------------------------------------------------- 
 
 
 
 
 
Visit V03 
 
           
 
 
 
Group F (n=40) 
LAIV4 
Single Year 
 
9-13 yo n= 20 
18-49 yo n= 20 
Group G (n=40) 
IIV4 
Single Year 
 
9-13 yo n= 20 
18-49 yo n= 20 
Collect NP Swab, NO blood draw; SAE 
assessment
All Volunteers 
Obtain parental or adult informed consent. Screen subjects by criteria, meds, and vital signs  
 Collect baseline blood for assays. Administer Vaccine 
  Assessment of Study Outcome 
Measures 
 
Collect blood; SAE assessment 
Collect NP Swab, Blood; SAE Assessment  
 
Collect blood; SAE assessment 
Collect Blood; SAE Assessment  

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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1 
KEY ROLES 
Individuals: 
 
Principal Investigator (Clinical Core):   
Cornelia L. Dekker, MD 
Professor of Pediatrics, Stanford University School of Medicine 
 
Stanford, CA 
 
Tel. 650-724-4437  
 
cdekker@stanford.edu 
 
 
Project Leaders and Co-Investigators: 
Mark M. Davis, PhD 
Professor, Immunology 
Howard Hughes Medical Institute  
  
  
  
 
 
 
Harry B. Greenberg, MD 
Senior Associate Dean for Research 
Joseph D. Grant Professor of Medicine and Microbiology & Immunology  
Stanford University School of Medicine  
  
  
  
  
 
 
 
William Hewitt Robinson, MD, PhD 
Associate Professor of Medicine/Immunology & Rheumatology 
CCSR 
  
  
  
 
 
 
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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Xiaosong He, PhD  
Senior Research Scientist  
VA Palo Alto Medical Center 
  
  
  
  
  
 
 
 
 
 
 
 
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
 
2.1 
Background Information 
 
Influenza virus (flu) is a Category C priority human pathogen with a worldwide 
distribution that is responsible for annual epidemics and intermittent pandemics. The 
spread of the 2009 swine-origin flu A/H1N1 pandemic and the identification of highly 
pathogenic avian A H5N1 and H7N9 strains highlight the need for better vaccines. Two 
types of licensed vaccines are available in the US: trivalent (IIV3, previously TIV) and 
quadrivalent (IIV4) inactivated vaccines both administered by injection, and nasally 
administered quadrivalent live, attenuated influenza vaccines (LAIV4) administered by 
nasal spray. Both vaccines are safe and effective in their approved age groups. LAIV is 
more efficacious than IIV, especially against mismatched flu strains, in children 6 months 
to 18 years of age1-3, as well as in a recent study using ferret model35. In adults 18 to 49 
years of age, LAIV has been either equally effective or somewhat less effective than 
IIV1,4-5. In the elderly, IIV has variable but often modest efficacy6. Despite extensive 
investigation of immune responses in infected patients and vaccine recipients, the basis 
of protective immunity is not fully understood. B cell and antibody (Ab) responses are 
key components of the adaptive immunity induced by natural (or “wild type”, wt) flu 
infection and vaccination, but conventional measures of serum Ab do not always predict 
protective efficacy of flu vaccination, especially of LAIV in healthy adults and of IIV in the 
elderly. A recent study of experimentally infected adults showed that levels of flu-specific 
CD4 T cells correlated with reduced virus shedding and disease severity7, suggesting 
that preexisting cellular immunity plays a role in modulating viral replication and 
pathogenesis. In previous studies we showed that LAIV induced greater T cell 
responses in children than in adults8. 
 
The upper respiratory tract (URT) mucosa is the initial replication site of wt flu and LAIV. 
The nasal mucosa is a complex and well-integrated system that serves as a first line of 
defense against infection9. The URT mucosa contains several types of epithelial cells 
thought to be the targets of initial infection by respiratory viruses including flu10. In a 
recent study, nasal swab specimens from flu patients were successfully used to identify 
lipid mediators that both induce and resolve local inflammation11. Previous studies have 
shown robust innate immune responses in primary human alveolar and bronchial 
epithelial cells infected by flu ex vivo12-14.  Studies in mice and humans suggested that 
early innate immune responses help shape subsequent adaptive responses. This is 
supported by the finding that the magnitude and persistence of Ab responses, as well as 
subsequent recall responses, to a live attenuated yellow fever vaccine correlate with 
early innate immune transcriptional responses15-16 and likely involve multiple toll-like 
receptors that stimulate production of proinflammatory cytokines17-18.  
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
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Animal studies have shown that type I interferon (IFN) responses elicit rapid and 
significant amounts of antigen (Ag)-specific IgG2c predominantly from follicular B cells19, 
and are important for the optimal priming, expansion, and memory formation of T cells 
after flu infection20-21. We recently found that rotavirus (RV) infection of intestinal 
enterocytes in vivo results in distinct transcriptional responses in infected vs. bystander 
uninfected epithelial cells and that most of the early IFN response to infection occurs in a 
nonepithelial immune cell compartment22. We also found that the local IgA B cell 
response to RV infection is promoted by plasmacytoid dendritic cells (DCs), a major 
producer of IFN during the early RV infection in both mice and humans23. These findings 
support the notion that early local innate immune responses, characterized by the robust 
induction of IFN and/or other cytokines, in the flu-infected nasal mucosa are likely to 
have a profound effect on viral replication and on subsequent virus-specific adaptive 
responses. This hypothesis will be tested in proposed experiments to examine the 
crosstalk between nasal epithelial and immune cell subsets, the effects of preexisting 
acquired immunity on this crosstalk, and the modulating effect the crosstalk has on viral 
replication and subsequent acquired immunity, presumably because children have lower 
levels of pre-vaccination immunity, resulting in higher levels of LAIV replication and 
subsequent T cell responses. The exact mechanisms linking preexisting immunity to 
local flu replication and subsequent immune responses are not clear.  
 
The URT mucosa also hosts multiple resident professional Ag presenting cells and Ag-
specific lymphocytes critical for adaptive and innate immune responses. These cells, 
including DCs, natural killer (NK) cells, and memory and effector B and T cells (including 
those specific for flu), can be collected using flocked nasopharyngeal swabs24-25. The 
outcome of LAIV immunization in flu-experienced older children and adults is likely 
strongly influenced by pre-existing flu-specific resident memory T and B cells in addition 
to the local innate responses. However, human URT resident immune cells have not 
been extensively studied and their role in controlling local flu replication as well as their 
relationship with local innate responses are unclear, largely due to difficulties in access 
and isolation of sufficient numbers of nasal cells for analysis. Defining the roles of these 
early adaptive immune cell responders to natural (or “wild-type”, wt) flu and LAIV 
infection in the URT will be an important step toward identification of the modulators of 
local replication and immunity. 
 
Ideally, a flu vaccine should induce protective immunity not only to homologous vaccine 
Ags but also to heterovariant strains or antigenically drifted new flu Ags. LAIV is clearly 
more effective at inducing protection against both antigenically matched and 
mismatched strains than IIV, especially in children1,3. However the basis for the 
enhanced induction of heterovariant protective immunity following infection (vs. 
parenteral vaccination) is not understood. It is not known whether heterovariant Ab 
reactivity is mediated by the same Ab molecules that recognize both homotypic and 
variant epitopes of different strains, or by different Ab molecules each targeting a 
cognate Ag on a specific variant strain. This question cannot be easily addressed with 
serum samples and conventional serologic assays. IIV induces a higher titer serum Ab 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
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response than LAIV in adults and older children, but one year after vaccination with 
either LAIV or IIV the levels of flu-specific circulating memory B cells are similar26.  This 
indicates that convalescent serum Ab titers do not give a complete picture of B cell 
mediated immunity following vaccination.26-32 and infection30, 33-34.  We and others have 
studied the B cell response to flu vaccination by examining the acute circulating 
plasmablast (PB) response. Analysis of this response provides a snapshot of the overall 
B cell response to recent Ag exposure without the confounding effects of preexisting 
circulating Abs. Analysis of the PB Ab repertoires revealed that the B cell response of 
adults to flu vaccination is largely a recall response of pre-existing memory B cells28. In 
preliminary studies, we identified flu-specific PBs that recognized previously circulated 
flu viruses better than current vaccine strains. These observations suggest that both the 
quantitative and qualitative nature of the B cell response to a new flu vaccination or to 
infection in the flu-experienced individuals is fundamentally shaped by the pre-
vaccination memory B cell repertoire. However, factors that initially establish this 
repertoire and the evolution of this repertoire during repeated exposure to influenza 
antigens, especially during repeated immunization with different types of influenza 
vaccines, the mechanism of its regulation, and the impact of initial Ag exposure on 
subsequent flu immunity in different age groups are unknown.  
 
 
2.2 
Rationale 
 
Our previous studies have focused on vaccine-induced immunity to influenza (flu); we 
have examined systemic humoral and cellular responses, as measured by levels of 
circulating and plasmablast-derived antibodies (Abs), T cells, B cells and natural killer 
cells, to identify determinants of immunity following intranasally-administered live 
attenuated (LAIV) and parenterally administered inactivated flu vaccines (IIV). The 
mechanistic basis for variable rates of protective immunity induced by vaccination – 
especially heterotypic immunity induced by LAIV in children and decreased immunity 
observed upon vaccination of the elderly with IIV – remains poorly understood. The 
upper respiratory tract (URT) is the initial site of replication of natural (or “wild type”, wt) 
flu and LAIV strains. We hypothesize that key determinants of flu immunity are regulated 
at the URT level and can be understood by careful examination of virus-host interactions 
at the local level. These local determinants include early host antiviral innate responses, 
tissue-resident immune cells, and viral replication levels. We have initiated studies of 
early innate and acquired immune responses in primary human nasal cells collected 
either directly from patients acutely infected with influenza or subjects who had received 
LAIV, and in primary URT nasal cells acutely infected with wt flu or LAIV strains.  
 
As a second approach to better understanding flu immunity, we have examined B cell 
and Ab responses to flu vaccination with a focus on qualitative and quantitative 
differences in responses between old and young vaccinees, IIV and LAIV recipients, and 
heterovariant vs. homotypic Ab reactivities. We propose to extend these studies using a 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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novel barcoding technology for high-throughput analysis of immunoglobulin (Ig) 
repertoires in flu-specific plasmablasts focusing on the initial responses to IIV vs. LAIV in 
influenza-naïve children. Evolutionary trees of Ab repertoires will be generated allowing 
us to identify – in an unbiased manner – affinity-matured clonal Ig families that are likely 
critical components of the flu Ab response. By expressing representative Ig genes from 
these clonal families, their functional property and homotypic vs. heterovariant specificity 
will be characterized. 
 
Based on these two complementary research goals, we hypothesize the following:  
Infection of the URT with LAIV4 or wt virus triggers hierarchical transcriptional signatures 
in local resident epithelial and immune cells that regulate viral replication and 
subsequent immunity.  We will collect NP swab specimens from recipients of LAIV4 and 
examine selected early transcriptional responses and viral replication levels in epithelial 
and lymphocytic cells (collection of the samples from subjects infected with the wild-type 
influenza virus will be completed by enrolling volunteers under a separate protocol). Host 
transcriptional responses and viral replication will be measured in bulk tissue, purified 
epithelial and lymphocyte cells, and single infected and bystander (non-infected) cells 
using flow cytometry and qRT-PCR. We predict that LAIV4 and wt virus will induce 
overlapping but clearly distinct transcriptional and phosphorylation signatures. In the 
older children and adult subjects from whom both NP swab and blood samples will be 
collected, we will also test whether local antiviral transcriptional responses and viral 
replication levels correlate with subsequent flu specific immune responses and will 
identify early local transcripts that predict subsequent B and T cell immunity in vivo. 
 
We also hypothesize that: The antigen-specific Ig repertoires elicited by infection with wt 
flu virus or LAIV4 vs. immunization with inactivated flu vaccine (IIV4) are quantitatively 
and qualitatively distinct, especially in young children undergoing the first or second 
exposure to flu antigens, and are different from those in flu-experienced adults in terms 
of number, size, Ig gene usage, and isotype of clonally expanded Ig gene families, and 
in levels of somatic hypermutation and heterovariant cross-reactive Ab activity. We 
predict these differences have the potential to affect the evolution of an individual’s 
immune response to novel flu antigens. In this protocol, we will examine these 
characteristics in LAIV4-infected vs. IIV4-exposed young children 6 months to 4 years of 
age who are influenza-naïve (or almost naïve) and compare them to children 9 – 13 
years old and adults using a novel barcoding-based amplification and sequencing 
technology. These differences are predicted to shape the subsequent Ab and memory B 
cell responses, in particular their ability to recognize heterovariant strains. 
 
 
2.3 
Potential Risks and Benefits 
2.3.1 
Potential Risks 
This protocol will immunize children and adult participants with either IIV4 (IM) or LAIV4 
(intranasal) vaccines that are licensed for use in the age groups studied. The 
discomforts of this study are those of receiving IM injection or intranasal application of 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
22 | P a g e  
 
the vaccine, and blood drawn from an arm vein, and possible reactions to the vaccine 
and collection of the mucosal tissue using a nasopharyngeal swab. Drawing blood 
causes transient discomfort and may cause fainting. Infection at the site where blood will 
be drawn or where the vaccination is given is extremely unlikely, but is a potential risk. 
Bruising at the site of blood drawing may occur, but can be prevented or lessened by 
applying pressure for several minutes immediately after the blood draw. Immediate 
allergic reactions to vaccine, including anaphylaxis, are in general extremely rare 
(approximately 1 person in 4,000,000), and might occur as a skin rash such as hives, 
difficulty breathing, fainting, drop in the blood pressure and death. Such reactions can 
usually be stopped by emergency medications administered by study personnel. 
Vaccine recipients may develop systemic reactions such as fever, headaches, body 
aches, and fatigue. These reactions are usually greatest within the first 24 to 72 hours 
after vaccination and last 1 to 2 days. Analgesics (e.g., aspirin or Tylenol) and rest will 
generally relieve or moderate these symptoms. Other hypersensitivity reactions, 
including Arthus reactions resulting in large local swelling reactions, are also possible. 
Although Guillain-Barré syndrome may have been associated with the 1976-77 
inactivated swine influenza vaccine and TIV vaccines used in early 1990's, subsequent 
inactivated vaccines have not been associated with an increased risk of this condition. 
Persons who care for severely immunosuppressed persons who require a protective 
environment should not receive LAIV, or should avoid contact with such persons for 7 
days after receipt, given the theoretical risk for transmission of the live attenuated 
vaccine virus to close contacts. It is not known whether LAIV4 (FluMist Quadrivalent) is 
excreted in human milk. Because some viruses are excreted in human milk, a lactating 
woman is not eligible to be enrolled in Group F. 
 
 
2.3.2   Known Potential Benefits 
 
Participants given the seasonal influenza vaccine are likely to experience decreased 
frequency and severity of subsequent influenza infection.  The beneficial role of 
influenza vaccination has been recognized increasingly over the past several years as 
more information has become available about the high rate of morbidity and mortality 
from this respiratory pathogen.  

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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3 
OBJECTIVES 
 
3.1 
Study Objectives 
 
Primary:  
1. To measure the nasal mucosal transcriptional responses to LAIV4 in the following age 
groups: 2-4 year old children, 9-13 year old children, and 18-49 year old adults. 
2. To measure the B cell response to LAIV4 in the following age groups: 2-4 year old 
children, 9-13 year old children, and 18-49 year old adults. 
3. To measure the B cell response to IIV4 in the following age groups: 6 month-4 year 
old children, 9-13 year old children, and 18-49 year old adults. 
Secondary: 
1. To compare the nasal mucosal transcriptional responses to LAIV4 between the 
different age groups. 
2. To determine the evolution of B cell repertoire in young children after repeated 
immunization with different types of influenza vaccines. 
3. To compare the B cell repertoire in response to LAIV4 versus IIV4 in different age 
groups. 
 
 
3.2 
Study Outcome Measures 
3.2.1 Primary Outcome Measures:  
• mRNA levels,  
• Ig gene sequence 
  
 
3.2.2 Secondary Outcome Measures:  
• 
HAI titers 
• 
PPAb titer 
• 
mAb reactivity 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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4 
STUDY DESIGN 
 
This is a Phase IV study of healthy children and adults who will receive the current 
seasonal influenza vaccine. There are no exclusions for gender, ethnicity or race.  The 
volunteers will be enrolled into one of 7 groups over a 5 year period. Following 
confirmation of written informed consent and assent, immunization will be administered; 
blood samples and NP swabs for immunogenicity assays will be collected at various 
time points based on groups assigned.  All volunteers will be followed for serious 
adverse events (SAEs).  
 
Group A (LAIV4/annual return): Up to six 2-4 year old volunteers will be given a 
quadrivalent live, attenuated influenza vaccine (LAIV4). Blood samples to conduct the 
assays described will be taken at Day 0 pre-immunization and Day 6-8 post-Dose 1 for 
all children irrespective of those who require 1 dose (prior flu vaccination history) or 2 
doses (vaccine-naïve). For children requiring 2 doses of vaccine, a second immunization 
will be given at Day 28-32  after Dose 1. All participants in this group will be asked to 
return annually for flu immunization and blood samples. 
 
Group B (LAIV4/ single year): Up to twenty 2-4 year old volunteers will be given a 
quadrivalent live, attenuated influenza vaccine (LAIV4). Blood samples to conduct the 
assays described will be taken at Day 0 pre-immunization and Day 6-8 post-Dose 1 for 
all children irrespective of those who require 1 dose (prior flu vaccination history) or 2 
doses (vaccine-naïve). For children requiring 2 doses of vaccine, a second immunization 
will be given at Day 28-32 after Dose 1. 
 
Group C (LAIV4/NP swab group):  Up to twenty 2-4 year old volunteers will be given a 
quadrivalent live, attenuated influenza vaccine (LAIV4). NP swabs will be collected at 
Day 1 and Day 21 post-Dose 1 for all children irrespective of those who require 1 dose 
or 2 doses of vaccine. For children requiring 2 doses of vaccine, a second immunization 
will be given at Day 28-32 after Dose 1. No blood will be collected for this group.  
 
Group D (IIV4/annual return): Up to six 6 month-23 month old (inclusive) volunteers will 
be given a quadrivalent inactivated influenza vaccine (IIV4). Blood samples to conduct 
the assays described will be taken at Day 0 pre-immunization and Day 6-8 post-Dose 1 
for children requiring 1 dose of vaccine (prior history of flu immunization).  For children 
requiring 2 doses of vaccine (vaccine-naïve), a second immunization will be given at Day 
28-32 after Dose 1. The second blood sample will be collected on Day 6-8 post-Dose 2. 
Volunteers will be asked to return annually for flu immunization and blood samples. 
 
Group E (IIV4/single year): Up to twenty 6 month–23 month old (inclusive) volunteers will 
be given a quadrivalent inactivated influenza vaccine (IIV4). Blood samples to conduct 
the assays described will be taken at Day 0 pre-immunization and Day 6-8 post-Dose 1 
for children requiring 1 dose of vaccine (prior history of flu immunization).   For children 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
25 | P a g e  
 
requiring 2 doses of vaccine (vaccine-naïve), a second immunization will be given at Day 
28-32 after Dose 1. The second blood sample will be collected on Day 6-8 post- Dose 2. 
Volunteers will participate for single year. 
 
Group F (LAIV4/single year) Up to forty 9-13 year old (n= 20) and 18-49  year old (n= 
20) volunteers will be given a quadrivalent live, attenuated influenza vaccine (LAIV4). 
Blood samples and NP swabs will be collected. Blood samples will be taken at Day 0 
pre-immunization, Day 6-8 post-immunization and Day 28±4 post-immunization. NP 
swabs will be collected at Day 1 post-immunization and Day 28 post –immunization. 
Children 9 yrs and older, and adults do not require a second dose of flu vaccine if 
previously unimmunized. Volunteers will participate for single year. 
 
Group G (IIV4/single year) Up to forty, 9-13 year old (n= 20) and 18-49   year old (n= 20) 
volunteers will be given a quadrivalent inactivated influenza vaccine (IIV4). Blood 
samples to conduct assays will be taken at Day 0 pre-immunization, Day 6-8 post 
immunization and Day 28±4 post-immunization. No NP swabs will be collected for this 
group. Children 9 yrs and older, and adults do not require a second dose of flu vaccine if 
previously unimmunized. Volunteers will participate for single year. 
 
 
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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5 
STUDY ENROLLMENT AND WITHDRAWAL 
 
5.1 
Subject Inclusion Criteria 
 
1. Otherwise healthy, 6 mo-49 years old volunteers 
2. Willing to complete the informed consent process (including assent for minors 7-17 
years old). 
3. Availability for follow-up for the planned duration of the study. 
4. For parents of children 6 months – 4 years of age:  Willing to participate in the study 
annually for up to 5 years (if yes, consider for annual return groups). 
5. Acceptable medical history by review of inclusion/exclusion criteria and vital signs. 
6. Influenza vaccine-naïve or only one prior season of flu immunization with IIV (does 
not apply to Groups F and G). 
 
5.2 
Subject Exclusion Criteria for Enrollment and Subsequent 
Annual Vaccinations 
 
1. Prior off-study vaccination with the current year’s seasonal influenza vaccine 
2. Receipt of LAIV in the prior season (does not apply to Groups F and G) 
3. Received flu immunizations in 2 or more prior flu seasons (does not apply to Groups 
F and G) 
4. Allergy to egg or egg products, or to vaccine components, including gentamicin, 
gelatin, arginine or MSG (if will be given LAIV4) 
5. Life-threatening reactions to previous influenza vaccinations 
6. Asthma in adults.  Children aged 2 through 4 years who have asthma or who have 
had a wheezing episode noted in the medical record within the past 12 months, or for 
whom parents report that a health care provider stated that they had wheezing or 
asthma within the last 12 months  [If yes, not eligible for LAIV Groups A, B, C, & F].  
7. Active systemic or serious concurrent illness, including febrile illness on the day of 
vaccination                                                                                                
8. History of immunodeficiency (including HIV infection)  
9. For children or adolescents through 17 years of age: receiving aspirin therapy or 
aspirin-containing products [If yes, not eligible for LAIV Groups A, B, C, and F]. 
10. Known or suspected impairment of immunologic function, including, but not limited 
to, clinically significant liver disease, diabetes mellitus treated with insulin, moderate 
to severe renal disease, or any other chronic disorder which, in the opinion of the 
investigator, might jeopardize volunteer safety or compliance with the protocol.   
11. Blood pressure >150 systolic or >95 diastolic at first study visit and the day of 
vaccination (for children 12 yrs and older, and adults). 
12. Hospitalization in the past year for congestive heart failure or emphysema.   
13. Chronic Hepatitis B or C                                     
14. Recent or current use of immunosuppressive medication, including systemic 
glucocorticoids (corticosteroid nasal sprays and topical steroids are permissible in all 
groups; inhaled steroid use is not permissible) 
15. Persons who care for severely immunosuppressed persons who require a protective 
environment should not receive LAIV, or should avoid contact with such persons for 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
27 | P a g e  
 
7 days after receipt, given the theoretical risk for transmission of the live attenuated 
vaccine virus to close contacts. [If yes, may be ineligible for Groups A,B, C and F].  
16. Malignancy, other than squamous cell or basal cell skin cancer (includes solid 
tumors such as breast cancer or prostate cancer with recurrence in the past year, 
and any hematologic cancer such as leukemia).                                 
17. Autoimmune disease (including rheumatoid arthritis treated with immunosuppressive 
medication such as Plaquenil, methotrexate, prednisone, Enbrel) which, in the 
opinion of the investigator, might jeopardize volunteer safety or compliance with the 
protocol 
18. History of blood dyscrasias, renal disease, or hemoglobinopathies requiring regular 
medical follow up or hospitalization during the preceding year 
19. Use of any anti-coagulation medication such as Coumadin or Lovenox, or anti-
platelet agents such as aspirin (except up to 325 mg. per day), Plavix, or Aggrenox 
must be reviewed by investigator to determine if this would affect the volunteer’s 
safety.  
20. Has taken an influenza antiviral medication within 48 hours prior to study vaccination 
[If yes, may not eligible for LAIV Groups A, B, C and F if unable to reschedule study 
vaccination]. 
21. Receipt of blood or blood products within the past 6 months or planned used during 
the study 
22. Medical or psychiatric condition or occupational responsibilities that preclude 
participant compliance with the protocol                                                                        
23. Receipt of inactivated vaccine 14 days prior to study enrollment, or planned 
vaccinations prior to completion of last study visit  
24. Receipt of live, attenuated vaccine within 60 days prior to enrollment of planned 
vaccination prior to completion of last study visit  
25. Need for allergy immunization (that cannot be postponed) during the study period 
26. History of Guillain–Barré syndrome                         
27. Pregnant woman 
28. Breastfeeding [If yes, not eligible for LAIV Group F]  
29. Use of investigational agents within 30 days prior to enrollment or planned use 
during the study                             
30. Donation of the equivalent of a unit of blood within 6 weeks prior to enrollment or 
planned donation prior to completion of the last visit 
31. Any condition which, in the opinion of the investigator, might interfere with volunteer 
safety, study objectives or the ability of the participant to understand or comply with 
the study protocol. 
 
5.3 
Treatment Assignment Procedures 
 
5.3.1 Randomization Procedures 
Volunteers will not be randomized to study treatment. All eligible volunteers will receive 
either quadrivalent live, attenuated influenza vaccine (LAIV4) or quadrivalent inactivated 
influenza vaccine (IIV4) based upon eligibility and group assigned.  
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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5.3.2   Reasons for Withdrawal 
 
A study participant will not receive the study product at the first visit if a significant 
intercurrent illness, or other medical condition or situation occurs that meets the 
exclusion criteria. Volunteers will be withdrawn from the study if participation in the study 
would not be in the best interest of the participant, or if doing so would harm the 
participant in the opinion of the investigator. Participants may withdraw from the study 
voluntarily. 
 
5.3.3   Handling of Withdrawals 
 
If, for safety reasons a participant is deemed by the investigators to not be eligible to 
receive the study product as per protocol, he/she will be terminated from the study.  If a 
participant voluntarily withdraws from the study or is withdrawn by the investigator after 
immunization, the participant will be followed for safety whenever possible until Day 28 
post-immunization or until any serious adverse events are resolved or considered stable. 
 
 
5.3.4   Termination of Study  
 
The study may be terminated for administrative reasons or other unanticipated 
circumstances. 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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6 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
 
6.1 
Study Product Description 
 
6.1.1 Acquisition 
 
The study product will be shipped from the manufacturer to the investigational 
pharmacy at the study site.  The quadrivalent IIV4 will be supplied by Sanofi-Pasteur 
as Fluzone® Quadrivalent.  The LAIV4 will be supplied by MedImmune as FluMist® 
Intranasal spray, Quadrivalent Influenza Vaccine. 
 
 
6.1.2 Formulation, Packaging, and Labeling 
 
FDA’s Vaccines and Related Biological Products Advisory Committee (VRBPAC) will 
make annual recommendations regarding the composition of the U.S. seasonal 
influenza vaccines based on global influenza virus surveillance data related to 
epidemiology and antigenic characteristics, serological responses to trivalent and 
quadrivalent seasonal vaccines, and the availability of candidate strains and reagents.   
 
The season-specific manufacturer package inserts for each study year will be 
attached in the IRB protocol submissions when available. 
 
Fluzone® Quadrivalent: Each 0.5 mL dose of Fluzone Quadrivalent (IIV4) will contain 
a total of 60 µg (15 μg of each strain) of influenza virus hemagglutinin of each of the 4 
strains selected for the seasonal formulation. Each 0.25 mL dose contains a total of 
30 μg (7.5 μg of each strain).   The vaccine will be supplied in a prefilled, single dose 
syringe 0.25 mL (no preservative) 0.5 mL (no preservative), single dose vial 0.5 mL 
(no preservative) or multi-dose vial, 5 mL (with preservative). Each multi-dose vial 
contains ten 0.5 mL doses. 
 
FluMist® (Influenza Virus Vaccine) Live, Intranasal Spray. 
Each pre-filled refrigerated FluMist® Quadrivalent sprayer contains a single 0.2 mL 
dose. Each 0.2 mL dose contains 106.5-7.5 FFU (fluorescent focus units) of live 
attenuated influenza virus reassortants of each of the selected strains. Each 0.2 mL 
dose may also contains 0.188 mg/dose monosodium glutamate, 2.00 mg/dose 
hydrolyzed porcine gelatin, 2.42 mg/dose arginine, 13.68 mg/dose sucrose, 2.26 
mg/dose dibasic potassium phosphate, and 0.96 mg/dose monobasic potassium 
phosphate. Each dose contains residual amounts of ovalbumin (< 0.24 mcg/dose), 
and may also contain residual amounts of gentamicin sulfate (< 0.015 mcg/mL), and 
ethylenediaminetetraacetic acid (EDTA) (< 0.37 mcg/dose).  FluMist® Quadrivalent 
contains no preservatives. It is supplied in a package of 10 pre-filled, single-dose (0.2 
mL) intranasal sprayers.  
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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6.1.3 Product Storage and Stability 
 
Fluzone® Quadrivalent vaccine presentations should be refrigerated at 2° to 8°C (35° to 
46°F). Vaccine that has been frozen will be discarded. Between uses, multi-dose vials 
will be returned to the recommended storage conditions at 2° to 8°C (35° to 46°F). 
Should not use after the expiration date shown on the label.   
 
FluMist® Quadrivalent should be stored in a refrigerator between 2 to 8°C (35-46°F) 
upon receipt and until use.  The product must be used before the expiration date on the 
sprayer label and must not be frozen. The cold chain (2 to 8°C) must be maintained 
when transporting FluMist® Quadrivalent. Once FluMist® Quadrivalent has been 
administered; the sprayer should be disposed of according to the standard procedures 
for medical waste (e.g., sharps container or biohazard container). One should not use 
after the expiration date shown on the label. 
 
6.2 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
 
Fluzone® Quadrivalent: 0.25 mL for children 6 months to 35 months of age and 0.5 mL 
for 36 months – adults. IIV4 vaccine will be administered with a sterile, disposable 
syringe and needle by IM injection into the thigh or deltoid muscle. The 
participant/clinician will choose whether the injection will be administered into the thigh 
or right or left deltoid. Previously unvaccinated children through 8 years of age should 
receive two doses of influenza vaccine - one on day 0, followed by a second dose at 
least 28 days later.  
 
LAIV4 vaccine dosage is 0.2 mL. Vaccine will be administered as an intranasal spray. 
Each sprayer contains a single dose of FluMist® Quadrivalent; approximately one-half of 
the contents should be administered into each nostril. 0.1 mL (i.e., half of the dose from 
a single FluMist sprayer) is administered into each nostril while the recipient is in an 
upright position. Insert the tip of the sprayer just inside the nose and rapidly depress the 
plunger until the dose-divider clip stops the plunger. The dose divider clip is removed 
from the sprayer to administer the second half of the dose (0.1 mL) into the other nostril. 
Previously unvaccinated children through 8 years of age should receive two doses of 
influenza vaccine - one on day 0, followed by a second dose at least 28 days later.  
 
 
6.3 
Accountability Procedures for the Study 
Intervention/Investigational Product(s) 
 
The study article will be shipped by the manufacturer to the investigational pharmacy at 
the study site. Use and disposition of each unit of vaccine will be appropriately 
documented by the Clinical Core staff in accordance with ICH GCP. Unused products 
will be returned to the investigational pharmacy for use or destruction. 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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6.4 
Concomitant Medications 
 
At each study visit, the participant will be questioned about any vaccinations they have 
received or concomitant medication use, and the information will be recorded. 
Medication history will include medications taken within 28 days prior to enrollment and 
currently taken prescription and over-the-counter medications used throughout the study 
period.  The investigator should be consulted regarding eligibility if the participant is 
taking oral steroid medications or medications for treatment of autoimmune disease 
(such as Plaquenil, methotrexate, prednisone, Enbrel), anti-coagulation/anti-platelet 
medications such as Coumadin, Lovenox, aspirin (except aspirin up to 325 mg. per day), 
Plavix, Aggrenox, or any other medications which might indicate a condition that 
precludes participant compliance with the protocol. 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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7 
STUDY SCHEDULE 
 
7.1 
Screening  
Volunteers will be contacted by phone prior to the first study visit.  Study staff will briefly 
review the eligibility criteria, participant availability and the study procedures. If the 
participant is eligible and interested, the volunteers will be assigned to groups based on, 
age, inclusion exclusion criteria and availability for annual return vs single year 
participation. 
 
7.2 
Day 0; Enrollment/Baseline/Immunization for all Groups; 
annually for Groups A and D. 
• 
Adult participants or parents of the volunteer will be provided with a verbal 
description of the study (purpose and study schedule and procedures). They then will 
be asked for any questions and to read/sign the consent form (in the first year only). 
Minor children ages 7-17 years will be given an assent at enrollment. The consent 
form will be signed prior to the performance of any study procedures. The study staff 
will discuss with the volunteer or volunteer’s parent of their study eligibility criteria 
and concomitant medication use. 
• 
Obtain vital signs (oral temperature, pulse, and respiratory rate), height and weight. 
Obtain blood pressure for volunteers 12 years and older. 
• 
Collect blood samples (except Group C) as follows: 
 
For blood volumes > 20 mL, collect one 5 mL gold top tube (serum), one 2.5 mL 
PAXgene tube and one 2.5 mL heparinized, green top tube (CyTOF sample).  The 
remainder of the blood sample will be collected in heparinized, green top tubes. 
Blood volumes <20 mL will be collected in heparinized, green top tubes only.  
o For children weighing more than 100 lb., and from adults, total volume not to 
exceed 70 mL   
o From children weighing 66-100 lb:  total volume not to exceed 30 mL 
o From children weighing 61-65 lb:  total volume not to exceed 25 mL  
o From children weighing 41-60 lbs:  total volume not to exceed 20 mL 
o From children weighing 16-40 lbs:  total volume not to exceed 10 mL 
o From children 6 months through 35 months old, regardless of weight: Obtain 
a 4 mL blood sample into a heparin (green top) tube. 
• 
All volunteers will be administered the seasonal flu vaccine according to the group 
assignment. Unless there is a different recommendation from the ACIP in a given 
year, Group A, B, C and F will receive intranasal spray of LAIV4. Group D, E and G 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
33 | P a g e  
 
will be vaccinated with IIV4. Children who are vaccine-naïve will receive their Dose 1 
of their vaccine at this visit. Volunteers will be observed in clinic for 15 minutes for 
any immediate serious reactions. Vaccine-naïve volunteers requiring Dose 2 will 
return at least 28 days post-Dose 1. 
• 
A Memory Aid will be given to the volunteers/volunteer’s parent with written and oral 
instructions for collection of information about serious adverse events. The memory 
aid is a simple diary for volunteer to record change in a health status and 
medications between study visits.  
• 
The schedule for subsequent study visits will be reviewed. 
 
7.3 
Day 1 post- Dose 1 immunization for Group C and Group F only  
• 
Review of medications and memory aid for serious adverse events 
• 
Collect nasopharyngeal swab specimen; no blood sample will be collected at this 
visit. 
• 
The schedule for subsequent study visits will be reviewed. 
 
7.4 
Day 6-8 post-Dose 1 immunization except Group C and vaccine-
naïve volunteers in Group D and Group E requiring Dose 2; 
annually for Groups A and D. 
• 
Review of medications and memory aid for serious adverse events 
• 
Collect blood samples as follows: 
All blood samples will be collected in heparinized, green top tubes to be picked up by 
the Greenberg lab for processing and analysis. For sample volumes >30 mL, one 
heparinized, green top tube will be sent to the HIMC if available (Greenberg lab 
requires at least 30 mL in heparinized, green top tubes at Day 6-8). 
o For children weighing more than 100 lb., and from adults:  total volume not to 
exceed 70 mL   
o From children weighing 66-100 lb:  total volume not to exceed 30 mL 
o From children weighing 61-65 lb:  total volume not to exceed 25 mL  
o From children weighing 41-60 lbs:  total volume not to exceed 20 mL 
o From children weighing 16-40 lbs:   total volume not to exceed 10 mL 
o From children 6 months through 35 months old, regardless of weight: Obtain 
a 4 mL blood sample into a heparin (green top) tube. 
• 
The schedule for subsequent study visits will be reviewed for, Group F and G 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
34 | P a g e  
 
• 
This will be the final visit for volunteers with prior influenza vaccination history in 
Group A, B, D and E. Complete Study Status form and Study Reimbursement form 
for these volunteers. 
 
7.5 
Day 21 post-Dose 1 immunization for Group C only 
• 
Review of medications and memory aid for serious adverse events 
• 
Collect nasopharyngeal swab specimen. 
• 
No blood sample will be collected at this visit. 
• 
This will be the final visit for volunteers with prior influenza vaccination history. 
Complete Study Status form and Study Reimbursement form for these volunteers. 
 
7.6 
Day 28 post-Dose 1 immunization 
7.6.1 Day 28-32 for vaccine-naïve volunteers requiring second dose of 
vaccine (Groups A, B, C, D and E)  
• 
Review of medications and memory aid for serious adverse events 
• 
Obtain temperature.  
• 
No blood samples will be collected at this visit 
• 
Volunteers will be vaccinated with the same type of vaccine as Dose 1 and observed 
in clinic for at least 15 minutes for any immediate serious adverse events. 
• 
Complete Study Status form and Study Reimbursement form for Groups A, B and C. 
• 
For Groups D and E Memory aid will be given with written and oral instructions for 
collection of information about serious adverse events.   
• 
The schedule for subsequent study visit will be reviewed for Group D and E 
 
7.6.2 Day 28 ± 4 for Groups F and G 
• 
Review of medications and memory aid for serious adverse events 
• 
Collect nasopharyngeal swab specimen for Group F only 
• 
Collect blood samples for both group as follows: 
For blood volumes > 20 mL, collect one 5 mL gold top tube (serum).  The remainder 
of the blood sample will be collected in heparinized, green top tubes. Blood volumes 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
35 | P a g e  
 
<20 mL will be collected in heparinized, green top tubes only. 
o For children weighing more than 100 lb., and from adults, Volume not to 
exceed 70 mL   
o From children weighing 66-100 lb., total volume not to exceed 30 mL 
o From children weighing 61-65 lb., total volume not to exceed 25 mL  
o From children 6 months through 35 months old, regardless of weight: Obtain 
a 4 mL blood sample into a heparin (green top) tube. 
• 
Complete Study Status form and Study Reimbursement form 
 
 
7.7 
Day 6-8 post-Dose 2 immunization for previously vaccine-naïve 
volunteers in Group D and E 
• 
Review of medications and memory aid for serious adverse events 
• 
Collect blood samples for both group as follows: 
All blood samples will be collected in heparinized, green top tubes to be picked up by 
the Greenberg lab for processing.  
o From children weighing 41-60 lbs. total volume not to exceed 20 mL 
o From children weighing 16-40 lbs.  total volume not to exceed 10 mL 
o From children 6 months through 35 months old, regardless of weight: Obtain 
a 4 mL blood sample into a heparin (green top) tube. 
• 
Complete Study Status form and Study Reimbursement form 
 
7.8 
Early Termination Visit 
If a volunteer is terminated from the study early, every effort should be made to perform 
the following procedures: 
• 
Review current health status and note any changes since the last visit.  Solicit 
information regarding SAEs and record all concomitant medications.  Any ongoing 
related SAEs will be followed to resolution or until a stable chronic condition has 
been established.  Volunteers will be encouraged to permit continued follow-up of 
SAEs if necessary 
• 
Obtain remaining samples, if possible. 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
36 | P a g e  
 
8 
STUDY PROCEDURES/EVALUATIONS 
 
8.1 
Clinical Evaluations 
 
Clinical Evaluation will be obtained by interview with a research nurse, and will include: 
• 
Review eligibility and past medical history including  influenza vaccine history. 
• 
Medication history of medications used within 28 days prior to enrollment and 
currently taken prescription and over-the-counter medications used throughout 
the study period. 
• 
Height, weight, temperature, pulse, BP on adolescents and adults.   
• 
Review of memory aid for serious adverse events occurring during the 28 day 
study period. 
 
8.2 
Laboratory Evaluations 
 
8.2.1 Special Assays or Procedures 
 
Subject Analysis – We will use CyTOF mass cytometry to determine the precise 
number of each white blood cell type in a given patient sample and their activation state 
and function (B cells, T cells, NK cells, T cells, monocytes, dendritic cells).  
 
Gene Expression Analysis– We will collect part of the blood sample in a PAXgene 
tube and prepare probes from the mRNA for gene expression analysis using Agilent 
microarrays that survey global gene expression.  This will tell us if there is a particular 
gene expression signature. 
 
Serum Cytokine Analysis – As there have been reports of changes in the serum 
cytokine repertoire in older people, we will survey serum samples for different cytokines 
using the Panoics/Luminex system now running the Human Immune Monitoring Center 
at Stanford.  
 
Responses to Cytokine Stimulation – We will stimulate PBMCs with various cytokines 
and will analyze the phosphorylation status of different STATs and other key mediators 
of cellular activation in B cells, T cells and monocytes by flow cytometry.  
 
HAI Antibody Titer – The standard HAI antibody assay will be run on samples at 
baseline and Day 28 or other study visit to evaluate the duration of response to 
vaccination.  
 
Sequencing of TCRαβ and Immunoglobulin Genes– We will sort B and T cells and 
sequence their antibody repertoires. 
 
Host transcriptional response - We will carry out quantitative real time PCR analysis 
to determine the levels of specific transcripts in bulk and sorted nasal cell populations. 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
37 | P a g e  
 
 
Antibody reactivity – We will generate plasmablast-derived polyclonal antibodies 
(PPAb) and recombinant mAbs and determine their reactivity against influenza antigens 
of the vaccine and variant strains by ELISA and SPR assays.  
 
 
8.2.2 Specimen Preparation, Handling, and Shipping 
 
Serum  
Red top or Gold Top serum separator tube: 
Procedural recommendations:  
•  Slowly invert tubes 8-10 times immediately after collection  
•  Store upright at room temperature until centrifugation  
• Centrifuge within 2 hrs. of collection at 1100 RCF for 15 minutes at room temperature  
• Aliquot and freeze at -20C within 2 hrs. of collection, and transfer to -80C within 2 
weeks . 
•  An aliquot of serum may be sent to Hana Golding at the FDA for analysis in the future. 
 
PBMCs 
Collect whole blood into heparinized green top tubes for flow cytometric analysis of 
PBMC subsets and phospho-proteins, stimulation assays (cytokine production and gene 
expression), MHC tetramer arrays.   
 
Tilt tubes immediately after filling and deliver to CTRU or HIMC lab for processing 
  
RNA  
2.5 ml in a PAXgene Blood RNA Tube for transcriptional (mRNA) profiling using 
microarrays, MicroRNA analysis, quantitative PCR (qPCR) analysis of mRNA expressed 
in peripheral blood.   
Recommendations: 
• Should be drawn as 2nd or later tube (not first) 
•  Invert 3 – 5 times immediately following collection 
•  Freeze within 1 hour at -20°C or -70°C, long term storage at -70°C prior to processing 
• Do not freeze PAXgene Blood RNA Tubes standing upright; instead place the tubes 
horizontally in a plastic bag or tray for freezing.  
 
NP swabs 
NP swab specimens will be collected using standard flocked swabs, following the 
manufacturer’s instruction.  
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
38 | P a g e  
 
8.3 
Specification of Safety Parameters 
Serious adverse events will be assessed through the last visit and followed to resolution 
or stability. 
All SAEs occurring while the volunteer is on study will be collected and appropriately 
reported. 
 
8.4 
Methods and Timing for Assessing, Recording, and Analyzing 
Safety Parameters 
 
8.4.1 Serious Adverse Events 
 
Serious Adverse Event (SAE): An SAE is defined as an adverse event (AE) that meets 
one of the following conditions: 
• Death during the period of protocol defined surveillance 
• Life-threatening event (defined as a participant at immediate risk of death at the time 
of the event) 
• An event requiring inpatient hospitalization or prolongation of existing hospitalization 
during the period of protocol defined surveillance  
• Results in congenital anomaly or birth defect 
• Results in a persistent or significant disability/incapacity 
• Any other important medical event that may not result in death, be life threatening, or 
require hospitalization, may be considered a serious adverse experience when, based 
upon appropriate medical judgment, the event may jeopardize the participant and 
may require medical or surgical intervention to prevent one of the outcomes listed 
above. Examples of such medical events include allergic bronchospasm requiring 
intensive treatment in an emergency room or at home, blood dyscrasias or 
convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. 
 
All SAEs will be: 
• Reviewed and evaluated by a study clinician 
• Recorded on the appropriate AE reporting form  
• Reported to the Stanford IRB as required 
• Followed through resolution by a study clinician 
 
8.5 
Reporting Procedures 
 
8.5.1 Regulatory Reporting for Studies Not Conducted Under an IND 
 
For those events meeting the previously described definition of Serious Adverse Events, 
the completion of an SAE report form is required. For SAEs related to vaccine, a VAERS 
form will be filled out and submitted to the Vaccine Adverse Event Reporting System 
(VAERS) per federal regulations. The VAERS form will simultaneously be sent to the 
sponsor.  SAEs and events that meet the Prompt Reporting guidelines (events meeting 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
39 | P a g e  
 
the criteria for Unanticipated Problems) will also be reported to the Stanford IRB as 
required.  Unexpected deaths or life-threatening experiences related to the research will 
be reported to the sponsor and to the IRB within 5 working days from when the 
investigator learns of event.  SAEs not related to vaccine will be reported to the IRB on 
an annual basis. 
 
8.5.2 Type and Duration of Follow-up of Subjects after Adverse Events 
 
All SAEs will be followed until satisfactory resolution or until the PI or Sub-investigator 
deems the event to be chronic or the patient to be stable.  
 
8.6 
Safety Oversight  
 
The Clinical Core Principal Investigator will oversee compliance with the protocol, the 
participant’s safety and any unanticipated problems involving risks to participants and 
will report these events as described above.  Unanticipated problems and serious 
adverse events will be reported to the Stanford IRB as required.  

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
40 | P a g e  
 
9 
CLINICAL MONITORING 
 
This study will not be monitored by the sponsor.  This is an exploratory study using 
licensed products.  The investigator is responsible to ensure compliance with the 
protocol, and the accuracy, completeness, legibility, and timeliness of the data reported. 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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10 
STATISTICAL CONSIDERATIONS 
 
10.1 Study Hypotheses 
1. Infection of the URT with LAIV4 or wt virus triggers hierarchical transcriptional 
signatures in local resident epithelial and immune cells that regulate viral replication and 
subsequent immunity. 
2. The antigen-specific Ig repertoires elicited by IIV4 are quantitatively and qualitatively 
distinct, especially in young children undergoing the first or second exposure to flu 
antigens, and are different from those in flu-experienced adults in terms of number, size, 
Ig gene usage, and isotype of clonally expanded Ig gene families, and in levels of 
somatic hypermutation and heterovariant cross-reactive Ab activity. 
This is an exploratory study aimed at providing initial estimates for subsequent studies 
design aimed at identifying age-specific characteristics in B cell response to influenza 
vaccine.  
 
 
10.2 Sample Size Considerations 
This is an exploratory study using a strategy that has not been employed previously to 
investigate the nasal mucosal transcriptional responses and the Ig repertoire of B cell 
response to influenza vaccination. We plan to enroll a total of 20 volunteers in each age 
group.  This projected sample size is comparable or larger to previous studies on Ig 
repertoire in human or zebra fish, which in general used relatively small numbers of 
participants to generate large numbers (10 – 50) of clones from each individual. 
 
 
10.3 Final Analysis Plan 
In this proposal, the host innate immune responses to influenza virus infection will be 
examined within flow cytometry defined subsets of primary infected and bystander cells, 
as well as in bulk nasal mucosa RNA preparations. We have recently developed 
statistical models and methods that are appropriate to analyze transcriptional profiles 
following viral infection in high dimensional datasets.  The basic approach that we 
propose is to obtain bulk measures at different hierarchical levels of the human nasal 
mucosa, including from sorted bulk populations and single cells from selected 
populations, and to identify significant innate response transcriptional signatures in the 
different age groups. Detailed procedures and analysis routines for analysis have been 
published recently and will be followed36  

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
42 | P a g e  
 
We will obtain sequences of cognate heavy- and light-chain pairs and determine their 
V(D)J usage by analyzing them with IMGT HighV-QUEST. The heavy and light chains 
will be grouped according to their V-gene usage to clonal families of antibodies that use 
the same heavy-chain V(D)J and light-chain VJ sequences.  Antibody phylogenetic trees 
will be generated based on the Ig sequences.  The number, size, Ig gene usage, and 
isotype of clonally expanded Ig gene families, and in levels of somatic hypermutation 
and heterovariant cross-reactive Ab activity will be compared between the different 
vaccines and age groups. 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
43 | P a g e  
 
11 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
 
All participant information will be obtained by the investigators and their support staff and 
will remain confidential.  Specimens for research laboratory testing will be coded by a 
unique participant ID number and data for individual participants will be coded for data 
analysis.  A database containing a code key will be kept on a computer that is password 
secured or a locked cabinets and only available to clinical research staff.  Participants 
will not be identified in any reports or publications that may result from the study.  
Personal identifierswill be removed for analyses and publications.   
 
Participant confidentiality is held strictly in trust by the participating investigators, their 
staff, and their agents. This confidentiality extends to genetic and biological sample 
tests, in addition to the clinical information relating to participating volunteers.  The study 
protocol, documentation, data, and all other information generated will be held in strict 
confidence.   
 
The clinical study site will permit access to all documents and records that may require 
inspection by the sponsor or its authorized representatives, including but not limited to, 
medical records (office, clinic or hospital) and pharmacy records for the participants in 
this study.   

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
44 | P a g e  
 
12 
QUALITY CONTROL AND QUALITY ASSURANCE 
 
Quality Control and Quality Assurance activities will generally be completed as outlined 
in the current version of the Stanford Quality Management Plan.  Chart audits for the trial 
will be conducted for research participant records utilizing the SLVP Chart Audit Tool.  
Audits will be conducted on a random sampling of participant charts in accordance with 
the low risk to volunteers participating in the trial. Charts will be randomly selected from 
among those not previously audited.  Results of these audits will be summarized using 
the SLVP QM_QA Summary Report tool, and shared with research staff at staff 
meetings, as necessary. 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
45 | P a g e  
 
13 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
13.1 Ethical Standard 
 
The investigator will ensure that this study is conducted in full conformity with the 
principles set forth in The Belmont Report: Ethical Principles and Guidelines for the 
Protection of Human Subjects of Research of the US National Commission for the 
Protection of Human Subjects of Biomedical and Behavioral Research (April 18, 1979) 
and codified in 45 CFR Part 46 and/or the ICH E6; 62 Federal Regulations 25691 
(1997). 
   
13.2 Institutional Review Board 
 
Prior to enrollment of participants into this trial, the protocol and the informed consent 
form will be reviewed and approved by the appropriate IRB. The responsible official for 
the IRB will sign the IRB letter of approval of the protocol prior to the start of this trial and 
a copy will be provided to DAIT.  Notification of the IRB's composition and the institutions 
Federal Wide Assurance number will be provided to DAIT as needed. 
Should amendments to the protocol be required, the amendments will be reviewed by 
the sponsor and/or the investigators and submitted to the IRB. 
Volunteers will be compensated for their participation in this study. Compensation will be 
in accordance with the local IRB’s policies and procedures, and requires IRB approval. 
 
13.3 Informed Consent Process 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to 
participate in the study and continuing throughout the individual’s study participation.  
Extensive discussion of risks and possible benefits of participation in this study will be 
provided to the participants and their families.  Consent forms describing in detail the 
study procedures and risks are given to the participant and written documentation of 
informed consent is required prior to enrolling in the study.  Consent forms will be IRB-
approved and the participant or parent will be asked to read and review the document. 
After reviewing the document, the investigator or authorized study staff will explain the 
research study to the participant or parent and answer any questions that may arise.   
 
The participant or parent will sign the informed consent document prior to being enrolled 
in the study.  The participant or parent should have the opportunity to discuss the study 
with their surrogates or think about it prior to agreeing to participate.  The participant or 
parent may withdraw consent at any time throughout the course of the study.  A copy of 
the informed consent document will be given to the participant or parent for their records.  
The rights and welfare of the participants will be protected by emphasizing to them that 
the quality of their medical care will not be adversely affected if they decline to 
participate in this study. 
 
13.3.1 
Informed Consent/Assent Process (in Case of a Minor) 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
46 | P a g e  
 
An assent form approved by the IRB will be discussed with minors and signed by 
minor participants who are 7 years of age or older (up to age 18 years) after the 
parent provides consent. 
 
 
13.4 Exclusion of Women, Minorities, and Children (Special 
Populations) 
 
There will be children 6 months – 13 years of age who will be recruited for this study as 
well as adults of differing ages. There will be no exclusions based on gender, race or 
ethnicity. 
 
13.5 Subject Confidentiality 
 
All participant information will be obtained by the investigators and their support staff and 
will remain confidential.  Specimens for laboratory testing will be coded by participant 
number and data for individual participants will be coded for data analysis.  A database 
containing a code key will be kept on a computer that is password secured and available 
only to study staff.  Participants will not be identified (except for age) in any reports or 
publications that may result from the study.  Personal identifiers, except for age, will be 
removed for publications. Upon completion of the study, data containing PHI will be 
retained for 50 years from the time of informed consent.  
 
Participant confidentiality is held strictly in trust by the participating investigators, their 
staff, and their agents. This confidentiality extends to genetic and biological sample 
tests, in addition to the clinical information relating to participating volunteers. 
The study protocol, documentation, data, and all other information generated will be held 
in strict confidence.   
 
The clinical study site will permit access to all documents and records that may require 
inspection by the sponsor or its authorized representatives, including but not limited to, 
medical records (office, clinic or hospital) and pharmacy records for the participants in 
this study.   
 
13.6 Study Discontinuation 
 
The investigators have the right to terminate this study at any time.  Reasons for 
terminating the study may include, but are not limited to, the following: 
 
Incidence or severity of serious adverse events indicating a potential health 
hazard 
 
Data recording is inaccurate or incomplete 
 
Study staff does not adhere to the protocol or applicable regulatory guidelines in 
conducting the study. 
 
A participant may withdraw or may be withdrawn from the study for the following 
reasons: 
 
Participant withdraws consent 
 
Development of serious adverse event warranting withdrawal  

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
47 | P a g e  
 
 
Trial termination 
 
Any reason that, in the opinion of the investigator, precludes the participant’s 
participation in the study. 
 
 
13.7 Future Use of Stored Specimens 
 
After the study is complete, residual specimens will be stored for future research.  As 
new scientific discoveries identify technologies or mediators that might be useful in 
studying the immune response, stored samples may be used to explore new information 
that could be made available with these advanced methods.  Volunteer specimens will 
be stored under a unique identifier. The volunteer’s name or other personal identifiers 
will not be available in any data shared with outside investigators.  New studies using 
stored samples will be reviewed by the IRB as required. 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
48 | P a g e  
 
14 
DATA HANDLING AND RECORD KEEPING 
 
The investigator is responsible to ensure the accuracy, completeness, legibility, and 
timeliness of the data reported. All source documents and data collection forms should 
be completed in a neat, legible manner to ensure accurate interpretation of data. Black 
or dark blue ink is required to ensure clarity of reproduced copies. When making 
changes or corrections, the original entry will be crossed out with a single line, and 
initialed and dated to indicate the change. DO NOT ERASE, OVERWRITE, OR USE 
CORRECTION FLUID OR TAPE ON THE ORIGINAL. 
 
Data entered into the data entry system that is derived from source documents or data 
collection forms should be consistent with the source documents and data collection 
forms or the discrepancies should be explained. 
 
14.1 Data Management Responsibilities 
 
All source documents will be reviewed by the clinical team and data entry staff, who will 
ensure that they are accurate and complete. Serious adverse events will be assessed 
for causality, and reviewed by the site PI or designee. 
 
Data collection is the responsibility of the clinical trial staff at the site under the 
supervision of the site PI. During the study, the investigator must maintain complete and 
accurate documentation for the study. 
 
The PI (or designee) will be responsible for data management, quality review, analysis, 
and reporting of the study data. 
 
14.2 Data Capture Methods 
 
Clinical data (including SAEs) will be entered into a 21 CFR Part 11-compliant data entry 
system. The data system includes password protection and internal quality checks, such 
as automatic range checks, to identify data that appear inconsistent, incomplete, or 
inaccurate. Clinical data will be entered directly from the source documents or data 
collection forms.  
 
14.3 Types of Data 
 
Data for this study will include clinical, safety and outcome measures. 
 
14.4 Timing/Reports 
 
 
Data will be reviewed on an ongoing basis according to the site Quality Management 
Plan.  Data analysis will begin once all clinical data have been collected and verified for 
accuracy. Preliminary analyses of outcome measures will begin as soon as laboratory 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
49 | P a g e  
 
data are available. Participants will receive a unique study ID at enrollment.  All data for 
study analysis will be coded by study ID number and will be password-protected.  Coded 
protected health information will be provided only as needed for data analysis of study 
outcome measures.  Personal identifierswill be removed for publications. 
 
Protected health information may be disclosed as requested by The Office for Human 
Research Protections in the U.S. Department of Health and Human Services, the 
sponsor, or Stanford University Administrative Panel on Human Subjects in Medical 
Research and any other unit of Stanford University as necessary. 
 
14.5 Study Records Retention 
 
Records and documents pertaining to the conduct of this study, including CRFs, source 
documents, data collection forms, consent forms, and medication inventory records, 
must be retained by the investigator for at least 3 years and in accordance with Stanford 
University and Stanford IRB requirements and until the sponsor authorizes transfer or 
destruction of study records.   
 
14.6 Protocol Deviations 
 
A protocol deviation is any noncompliance with the clinical trial protocol or Good Clinical 
Practice (GCP).  The noncompliance may be either on the part of the participant, the 
investigator, or the study site staff.  As a result of deviations, corrective actions are to be 
developed by the site and implemented promptly.  
 
It is the responsibility of the site to use continuous vigilance to identify and report 
deviations. All deviations from the Protocol will be addressed in a study subject data 
collection form,   Protocol deviations will be sent to the local IRB/IEC per their guidelines.  
The site PI/study staff is responsible for knowing and adhering to their IRB requirements.  

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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15 
  PUBLICATION POLICY 
 
Following completion of the study, the investigator may publish the results of this 
research in a scientific journal. The International Committee of Medical Journal Editors 
(ICMJE) member journals have adopted a trials-registration policy as a condition for 
publication. This policy requires that all clinical trials be registered in a public trials 
registry such as ClinicalTrials.gov, which is sponsored by the National Library of 
Medicine (NLM). Other biomedical journals are considering adopting similar policies. Any 
clinical trial starting enrollment after 27SEP2007 must be registered either on or before 
the onset of participant enrollment.  
 
The ICMJE defines a clinical trial as any research project that prospectively assigns 
human subjects to intervention or comparison groups to study the cause-and-effect 
relationship between a medical intervention and a health outcome. Studies designed for 
other purposes, such as to study pharmacokinetics or major toxicity (e.g., Phase 1 
trials), would be exempt from this policy.  
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
51 | P a g e  
 
16 
LITERATURE REFERENCES 
 
 
1. Ambrose CS, Levin MJ, Belshe RB. The relative efficacy of trivalent live attenuated 
and inactivated influenza vaccines in children and adults. Influenza Other Respi Viruses 
2011; 5:67-75. PMCID: PMC3151550. 
2. Ambrose CS, Wu X, Belshe RB. The efficacy of live attenuated and inactivated 
influenza vaccines in children as a function of time postvaccination. Pediatr Infect Dis J 
2010; 29:806-811. PMID: 20458256. 
3. Belshe RB, Edwards KM, Vesikari T, Black SV, Walker RE, Hultquist M, Kemble G, 
Connor EM. Live attenuated versus inactivated influenza vaccine in infants and y oung 
children. N Engl J Med 2007; 356:685-696. PMID: 17301299. 
4. Monto AS, Ohmit SE, Petrie JG, Johnson E, Truscon R, Teich E, Rotthoff J, 
Boulton M, Victor JC. Comparative efficacy of inactivated and live attenuated influenza 
vaccines. N Engl J Med 2009; 361:1260- 1267. PMID: 19776407. 
5. Ohmit SE, Victor JC, Rotthoff JR, Teich ER, Truscon RK, Baum LL, Rangarajan B, 
Newton DW, Boulton ML, Monto AS. Prevention of antigenically drifted influenza by 
inactivated and live attenuated vaccines. N Engl J Med 2006; 355:2513-2522.  PMCID: 
PMC2614682. 
6. Goodwin K, Viboud C, Simonsen L. Antibody response to influenza vaccination in the 
elderly: a quantitative review. Vaccine 2006; 24:1159-1169. PMID: 16213065. 
7. Wilkinson TM, Li CK, Chui CS, Huang AK, Perkins M, Liebner JC, Lambkin-Williams R, 
Gilbert A, Oxford J, Nicholas B, Staples KJ, Dong T, Douek DC, McMichael AJ, Xu XN. 
Preexisting influenza-specific CD4+ T cells correlate with disease protection against 
influenza challenge in humans. Nat Med 2012; 18:274-280. PMID: 22286307. 
8. He XS, Holmes TH, Zhang C, Mahmood K, Kemble GW, Lewis DB, Dekker CL, 
Greenberg HB, Arvin AM. Cellular immune responses in children and a dults receiving 
inactivated or live attenuated influenza vaccines. J Virol 2006; 80:11756-11766.  PMCID: 
PMC1642596. 
9. Sanders CJ, Doherty PC, Thomas PG. Respiratory epithelial cells in innate immunity 
to influenza virus infection. Cell Tissue Res 2011; 343:13-21. PMID: 20848130. 
10. Holt PG, Strickland DH, Wikstrom ME, Jahnsen FL. Regulation of immunological 
homeostasis in the respiratory tract. Nat Rev Immunol 2008; 8:142-152. PMID: 
18204469. 
11. Tam VC, Quehenberger O, Oshansky CM, Suen R, Armando AM, Treuting PM, Thomas 
PG, Dennis EA, Aderem A. Lipidomic Profiling of Influenza Infection Identifies 
Mediators that Induce and Resolve Inflammation. Cell 2013; 154:213-227. PMID: 
23827684. 
12. Chan MC, Cheung CY, Chui WH, Tsao SW, Nicholls JM, Chan YO, Chan RW, Long HT, 
Poon LL, Guan Y, Peiris JS. Proinflammatory cytokine responses induced by influenza 
A (H5N1) viruses in primary human alveolar and bronchial epithelial cells. Respir Res 
2005; 6:135.  PMCID: PMC1318487. 
13. Chan RW, Yuen KM, Yu WC, Ho CC, Nicholls JM, Peiris JS, Chan MC. Influenza H5N1 
and H 1N1 virus replication and i nnate immune responses in bronchial epithelial cells 
are influenced by the state of differentiation. PLoS One 2010; 5:e8713. PMCID: 
PMC2806912. 
14. Lee SM, Chan RW, Gardy JL, Lo CK, Sihoe AD, Kang SS, Cheung TK, Guan YI, Chan 
MC, Hancock RE, Peiris MJ. Systems-level comparison of host responses induced by 
pandemic and s easonal influenza A H1N1 viruses in primary human type I-like 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
52 | P a g e  
 
alveolar epithelial cells in vitro. Respir Res 2010; 11:147. PMCID: PMC2988725. 
15. Gaucher D, Therrien R, Kettaf N, Angermann BR, Boucher G, Filali-Mouhim A, Moser 
JM, Mehta RS, Drake DR, 3rd, Castro E, Akondy R, Rinfret A, Yassine-Diab B, Said EA, 
Chouikh Y, Cameron MJ, Clum R, Kelvin D, Somogyi R, Greller LD, Balderas RS, 
Wilkinson P, Pantaleo G, Tartaglia J, Haddad EK, Sekaly RP. Yellow fever vaccine 
induces integrated multilineage and polyfunctional immune responses. J Exp Med 
2008; 205:3119-3131.  PMCID: PMC2605227. 
16. Querec TD, Akondy RS, Lee EK, Cao W, Nakaya HI, Teuwen D, Pirani A, Gernert K, 
Deng J, Marzolf B, Kennedy K, Wu H, Bennouna S, Oluoch H, Miller J, Vencio RZ, 
Mulligan M, Aderem A, Ahmed R, Pulendran B. Systems biology approach predicts 
immunogenicity of the yellow fever vaccine in humans. Nat Immunol 2009; 10:116-125. 
PMID: 19029902 
17. Querec T, Bennouna S, Alkan S, Laouar Y, Gorden K, Flavell R, Akira S, Ahmed R, 
Pulendran B. Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via 
TLR2, 7, 8, and 9 to stimulate polyvalent immunity. J Exp Med 2006; 203:413-424.  
PMCID: PMC2118210. Kasturi SP, Skountzou I, Albrecht RA, Koutsonanos D, Hua T, 
Nakaya HI, Ravindran R, Stewart S, Alam M, Kwissa M, Villinger F, Murthy N, Steel J, 
Jacob J, Hogan RJ, Garcia-Sastre A, Compans R, Pulendran 
18. B. Programming the magnitude and persistence of antibody responses with innate 
immunity. Nature 2011; 470:543-547.  PMCID: PMC3057367. 
19. Swanson CL, Wilson TJ, Strauch P, Colonna M, Pelanda R, Torres RM. Type I IFN 
enhances follicular B cell contribution to the T c ell-independent antibody response. J Exp 
Med 2010; 207:1485-1500. PMCID: PMC2901065. 
20. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. Type I interferons act 
directly on CD8 T cells to allow clonal expansion and m emory formation in response to 
viral infection. J Exp Med 2005; 202:637-650.  PMCID: PMC2212878. 
21. Curtsinger JM, Valenzuela JO, Agarwal P, Lins D, Mescher MF. Type I IFNs provide a 
third signal to CD8 T cells to stimulate clonal expansion and differentiation. J Immunol 
2005; 174:4465-4469. PMID: 15814665. 
22. Sen A, Rothenberg ME, Mukherjee G, Feng N, Kalisky T, Nair N, Johnstone IM, Clarke 
MF, Greenberg HB. Innate immune response to homologous rotavirus infection in the 
small intestinal villous epithelium at single-cell resolution. Proc Natl Acad Sci U S A 2012; 
109:20667-20672. PMCID: PMC3528539. 
23. Deal EM, Lahl K, Narvaez CF, Butcher EC, Greenberg HB. Plasmacytoid dendritic cells 
promote rotavirus- induced human and murine B cell responses. J Clin Invest 2013. 
PMCID:PMC3668833. 
24. Daley P, Castriciano S, Chernesky M, Smieja M. Comparison of flocked and rayon swabs 
for collection of respiratory epithelial cells from uninfected volunteers and symptomatic 
patients. J Clin Microbiol 2006; 44:2265-2267.  PMCID: PMC1489401. 
25. Gill MA, Long K, Kwon T, Muniz L, Mejias A, Connolly J, Roy L, Banchereau J, Ramilo O. 
Differential recruitment of dendritic cells and monocytes to respiratory mucosal sites in 
children with influenza virus or respiratory syncytial virus infection. J Infect Dis 2008; 
198:1667-1676.  PMCID: PMC2696361. 
26. Sasaki S, Jaimes MC, Holmes TH, Dekker CL, Mahmood K, Kemble GW, Arvin AM, 
Greenberg HB. Comparison of the influenza virus-specific effector and memory  B-cell 
responses  to immunization of children and adults with live attenuated or inactivated 
influenza virus vaccines. J Virol 2007; 81:215-228. PMCID: PMC1797237. 
27. Sasaki S, He XS, Holmes TH, Dekker CL, Kemble GW, Arvin AM, Greenberg HB. 
Influence of prior influenza vaccination on antibody and B-cell responses. PLoS One 2008; 
3:e2975.  PMCID: PMC2500171. 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
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28. Wrammert J, Smith K, Miller J, Langley WA, Kokko K, Larsen C, Zheng NY, Mays I, 
Garman L, Helms C, James J, Air GM, Capra JD, Ahmed R, Wilson PC. Rapid cloning of 
high-affinity human monoclonal antibodies against influenza virus. Nature 2008; 453:667-
671. PMCID: PMC2515609. 
29. Sasaki S, Sullivan M, Narvaez CF, Holmes TH, Furman D, Zheng NY, Nishtala M, 
Wrammert J, Smith K, James JA, Dekker CL, Davis MM, Wilson PC, Greenberg HB, He 
XS. Limited efficacy of inactivated influenza vaccine in elderly individuals is associated 
with decreased production of vaccine-specific antibodies. J Clin Invest 2011; 121:3109-
3119.  PMCID: PMC3148747. 
30. Thomson CA, Wang Y, Jackson LM, Olson M, Wang W, Liavonchanka A, Keleta L, Silva6 
V, Diederich S, Jones7 RB, Gubbay J, Pasick J, Petric M, Jean F, Allen VG, Brown EG, 
Rini JM, Schrader JW. Pandemic H1N1 influenza infection and vaccination in humans 
induces cross-protective antibodies that target the hemagglutinin stem. Front Immun 2012; 
3:Article 87. PMCID: PMC3347682. 
31. He XS, Sasaki S, Baer J, Khurana S, Golding H, Treanor JJ, Topham DJ, Sangster MY, 
Jin H, Dekker CL, Subbarao K, Greenberg HB. Heterovariant cross-reactive B-cell 
responses induced by the 2009 pandemic influenza virus A subtype H1N1 vaccine. J Infect 
Dis 2013; 207:288-296. PMCID: PMC3532823. 
32. Li GM, Chiu C, Wrammert J, McCausland M, Andrews SF, Zheng NY, Lee JH, Huang M, 
Qu X, Edupuganti S, Mulligan M, Das SR, Yewdell JW, Mehtad AK, Wilson PC, Ahmed R. 
Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly 
cross-reactive memory B cells. Proc Natl Acad Sci U S A 2012; 109:9047-52. 
PMCID:PMC3384143. 
33. Wrammert J, Koutsonanos D, Li GM, Edupuganti S, Sui J, Morrissey M, McCausland M, 
Skountzou I, Hornig M, Lipkin WI, Mehta A, Razavi B, Del Rio C, Zheng NY, Lee JH, Huang 
M, Ali Z, Kaur K, Andrews S, Amara RR, Wang Y, Das SR, O'Donnell CD, Yewdell JW, 
Subbarao K, Marasco WA, Mulligan MJ, Compans R, Ahmed R, Wilson PC. Broadly cross-
reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 
influenza virus infection. J Exp Med 2011; 208:181-193. PMCID: PMC3023136. 
34. Li Y, Myers JL, Bostick DL, Sullivan CB, Madara J, Linderman SL, Liu Q, Carter DM, 
Wrammert J, Esposito S, Principi N, Plotkin JB, Ross TM, Ahmed R, Wilson PC, Hensley 
SE. Immune history shapes specificity of pandemic H1N1 influenza antibody responses. J 
Exp Med 2013 Jul 15. [Epub ahead of print] PMID:23857983. 
35. Cheng X, Zengel JR, Suguitan AL Jr, Xu Q, Wang W, Lin J, Jin H. Evaluation of the 
Humoral and Cellular Immune Responses Elicited by the Live Attenuated and Inactivated 
Influenza Vaccines and Their Roles in Heterologous Protection in Ferrets. J Infect Dis 2013 
[Epub ahead of print]; PMID: 23656978. 
36.   Sen A, Rothenberg ME, Mukherjee G, Feng N, Kalisky T, Nair N, Johnstone IM, Clarke 
MF, Greenberg HB. Innate immune response to homologous rotavirus infection in the 
small intestinal villous epithelium at single-cell resolution. Proc Natl Acad Sci U S A. 
2012;109(50):20667-72 .PMID: 23188796. 
 
 
 
 
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
54 | P a g e  
 
SUPPLEMENTS/APPENDICES 
 
Appendix  A.  Schedule of visits 
 
Appendix B.  Schedule of events 
 
 
Appendix C.  Season-specific FluMist Quadrivalent package inserts 2014-2018 
 
Appendix D.  Season-specific Fluzone Quadrivalent package insert 2014-2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
55 | P a g e  
 
APPENDIX A: SCHEDULE OF VISITS 
 
* When these children turn 2 yrs old in annual f/u, they will be switched to receive LAIV4 vaccine 
unless they develop a contraindication. 
 
Groups 
 
 
 
Naïve 
/prior 
vacc 
Day 0 
 
 
 
 
Day 1 
Post- 
Dose 
1 
 
Day 6-8 
Post-
Dose1 
 
 
 
 
Day 21 
Post-
Dose1 
Day 28-32 post-
Dose 1 (for 
Dose 2) 
 
Day 28 + 4 
Post-Dose 1 
(blood draw, 
NP Swab) 
 
 
 
 
Day 6-8 
Post-
Dose 2 
 
 
Group A 
2-4 yo 
Annuals 
naive 
LAIV4 Dose 
1/ 
Blood 
-- 
Blood 
-- 
LAIV4 Dose 2 
-- 
Prior 
vacc 
LAIV4/Blood 
-- 
Blood 
-- 
-- 
-- 
Group B 
2-4 yo 
Single yr 
Naive 
LAIV4 Dose 
1/ 
Blood, 
-- 
Blood 
-- 
LAIV4 Dose 2 
-- 
Prior 
vacc 
LAIV4/Blood 
-- 
Blood 
-- 
-- 
-- 
Group C 
2-4 yo 
Single yr 
Naive 
LAIV4 Dose 1 
NP 
Swab 
-- 
NP 
Swab 
LAIV4 Dose 2 
-- 
Prior 
vacc 
LAIV4 
NP 
Swab 
-- 
NP 
Swab 
-- 
-- 
Group D 
6-23mo 
Annual 
Naive 
IIV4* Dose 1/ 
Blood 
-- 
-- 
-- 
IIV4* Dose 2 
Blood 
Prior 
vacc 
IIV4*/ Blood 
-- 
Blood 
-- 
-- 
-- 
Group E 
6-23mo 
Single yr 
Naive 
IIV4 Dose 1/ 
Blood 
-- 
-- 
-- 
IIV4 Dose 2 
Blood 
Prior 
vacc 
IIV4/ 
Blood 
-- 
Blood 
-- 
-- 
-- 
Group F 
9-13 yo 
18-49 yo 
Prior 
vacc 
LAIV4, blood 
NP 
Swab 
Blood 
-- 
Blood, NP 
Swab 
-- 
Group G 
9-13 yo 
18-49 yo 
 
Prior 
vacc 
IIV4, blood 
-- 
Blood 
-- 
Blood 
-- 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
56 | P a g e  
 
APPENDIX B: SCHEDULE OF EVENTS  
 
 
 
 
 
Day 0 
Day 1 
 
Day 6-8 
post-Dose 1 
Day 21 post-
Dose 1 
Day 28-32 post-
Dose 1 (give 
Dose 2) 
OR 
Day 24 - 32 
Post-Dose 1 
(blood draw) 
 
 
Day 6-8 
post-Dose 2 
 
 
Obtain Informed 
Consent/Assent 
X 
 
 
 
 
 
Eligibility 
X 
 
 
 
 
 
Concomitant 
meds 
X 
 
X 
 
X 
 
Vital Signs1 
X 
 
X2 
 
X2 
 
Blood Draw 
 
X 
 
 
X 
 
 
X7 
 
X6 
NP Swab 
 
X3, 4 
 
X3 
X4 
 
Immunization 
 
X 
 
 
 
X5 
 
Clinical 
Assessment 
X2 
X2 
X2 
X2 
X2 
X2 
Review of SAEs 
X 
X 
X 
X 
X 
X 
1 Vital Signs: temperature, pulse, respiration; height and weight (blood pressure for 
adolescents as indicated and adults) 
2 As indicated 
3 Group C only 
4 Group F only 
5 Dose 2 vaccine (at least 28 days post-Dose 1), Vaccine-naïve volunteers in   
Groups A-E only 
6 Groups D and E vaccine-naïve volunteers only 
7 Groups F and G volunteers only 
 
 

Stanford U19 CCHI. Project 2: Innate and Acquired Immunity to Influenza Infection and Immunization (Under grant 
‘Adaptive and Innate Immunity, Memory and Repertoire in Vaccination and Infection’); SLVP029                                                                
v2.0_07AUG2015 
 
57 | P a g e  
 
 
 
 
APPENDIX C: FLUMIST QUADRIVALENT PACKAGE INSERTS 2014-2018 
Attached in eProtocol section 16  
 
 
APPENDIX D: FLUZONE QUADRIVALENT PACKAGE INSERTS 2014-2018 
Attached in eProtocol section 16