A Phase 1b, Double-Blind, Randomized, Dose-Escalating, Age De-Escalating, Placebo-Controlled Study to Assess the Safety and Immunogenicity of One or Two Doses of Sing2016 M2SR H3N2 Influenza Vaccine Delivered Intranasally In a Healthy Pediatric Population 6 Months Through 17 Years of Age.
Phase: PHASE1
Conditions: H3N2 Influenza
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

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A Phase 1b, double-blind, randomized, dose-escalating, age de-escalating, placebo-
controlled study to assess the safety and immunogenicity of one or two doses of Sing2016 
M2SR H3N2 influenza vaccine delivered intranasally in a healthy pediatric population 6 
months through 17 years of age.  
   
 
 
DMID Protocol Number: 20-0009 
 
DMID Funding Mechanism: Collaborative Influenza Vaccine Innovation Centers 
 
Pharmaceutical Support: FluGen, Inc 
 
IND Sponsor: DMID, NIAID 
 
Principal Investigator: James D. Campbell, MD, MS 
DMID Clinical Project Manager: Sonja Crandon, BSN, PMP 
DMID Medical Officer: Mamodikoe Makhene, MD, MPH 
DMID Scientific Lead: Catherine Luke, PhD 
 
Version Number: 5.0 
 
Date: 6 Dec 2023 
 
 
 

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STATEMENT OF COMPLIANCE 
Each institution engaged in this research will hold a current FederalWide Assurance (FWA) 
issued by the Office of Human Research Protection (OHRP) for federally funded research. The 
IRB/IEC must be registered with OHRP as applicable to the research.   
 
The study will be carried out in accordance with the following as applicable: 
• United States Code of Federal Regulations (CFR) 45 CFR Part 46: Protection of Human 
Subjects 
• Food and Drug Administration (FDA) Regulations: 21 CFR Part 50 (Protection of 
Human Subjects), 21 CFR Part 54 (Financial Disclosure by Clinical Investigators), 21 
CFR Part 56 (Institutional Review Boards), 21 CFR Part 11, and 21 CFR Part 312 
(Investigational New Drug Application), and/or 21 CFR 812 (Investigational Device 
Exemptions) 
• The International Council for Harmonization of Technical Requirements for Registration 
of Pharmaceuticals for Human Use (ICH) E6(R2) Good Clinical Practice, and the 
Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects 
of Research, Report of the National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research 
• The policies and procedures of National Institutes of Health (NIH) Office of Extramural 
Research and DMID 
• The National Institute of Allergy and Infectious Diseases (NIAID) Terms of Award 
• Any additional Federal, State, and Local Regulations and Guidance 
 
The signature below provides the necessary assurance that this study will be conducted 
according to all stipulations of the protocol including statements regarding confidentiality, and 
according to local legal and regulatory requirements, US federal regulations, and ICH E6(R2) 
Good Clinical Practice (GCP) guidelines. 
 
Site Investigator Signature: 
 
 
Signed: 
 
Date: 
 
 
Name 
Title 
 
 
 
 
 
 

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TABLE OF CONTENTS 
STATEMENT OF COMPLIANCE ................................................................................................ 2 
TABLE OF CONTENTS ................................................................................................................ 3 
LIST OF TABLES .......................................................................................................................... 7 
LIST OF FIGURES ........................................................................................................................ 8 
1. 
PROTOCOL SUMMARY ...................................................................................................... 9 
1.1 
Synopsis .......................................................................................................................... 9 
1.2 
Schedule of Activities (SoA) ........................................................................................ 22 
1.3 
Study Schema ................................................................................................................ 26 
2. 
INTRODUCTION ................................................................................................................ 27 
2.1 
Study Rationale ............................................................................................................. 28 
2.2 
Background ................................................................................................................... 29 
2.2.1 
Purpose of Study ................................................................................................... 29 
2.3 
Risk/Benefit Assessment .............................................................................................. 29 
2.3.1 
Known and Unknown Potential Risks .................................................................. 29 
2.3.1.1 
Potential Risks Associated with Receipt of the Vaccine or Placebo ................ 29 
2.3.1.2 
Potential Risks Associated with Drawing Blood or Receipt of an Intramuscular 
Injection or Intranasal Spray ............................................................................................. 31 
2.3.1.3 
Potential Risks Associated with Nasal Washes and Nasal Swabs .................... 32 
2.3.1.4 
Potential Risks Associated with Pregnancy ...................................................... 32 
2.3.1.5 
Risk of Loss of Confidentiality ......................................................................... 33 
2.3.1.6 
Unknown Risks ................................................................................................. 33 
2.3.2 
Known Potential Benefits ..................................................................................... 33 
2.3.3 
Assessment of Potential Risks and Benefits ......................................................... 33 
3. 
OBJECTIVES AND ENDPOINTS ...................................................................................... 33 
4. 
STUDY DESIGN.................................................................................................................. 38 
4.1 
Overall Design .............................................................................................................. 39 
4.2 
Scientific Rationale for Study Design ........................................................................... 41 
4.3 
Justification for Dose .................................................................................................... 42 
5. 
STUDY POPULATION ....................................................................................................... 44 
5.1 
Inclusion Criteria .......................................................................................................... 44 
5.2 
Exclusion Criteria ......................................................................................................... 45 
5.2.1 
Exclusion of Specific Populations ........................................................................ 46 
5.3 
Inclusion of Vulnerable Participants ............................................................................. 46 
5.4 
Lifestyle Considerations ............................................................................................... 46 
5.5 
Screen Failures .............................................................................................................. 46 
5.6 
Strategies for Recruitment and Retention ..................................................................... 47 
5.6.1 
Recruitment ........................................................................................................... 47 
5.6.2 
Retention ............................................................................................................... 47 
5.6.3 
Compensation Plan for Participants ...................................................................... 47 

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5.6.4 
Costs ...................................................................................................................... 47 
6. 
STUDY PRODUCT ............................................................................................................. 47 
6.1 
Study Product(s) and Administration ............................................................................ 47 
6.1.1 
Study Product Description .................................................................................... 47 
6.1.1.1 
Product 1: Sing2016 M2SR H3N2 Intranasal Influenza Vaccine .................... 47 
6.1.1.2 
Product 2: SPG buffer to be used as the diluent for Product 1 ......................... 48 
6.1.1.3 
Product 3: Placebo ............................................................................................ 48 
6.1.1.4 
Product 4: Seasonal Inactivated Influenza Vaccine (IIV4) ............................... 48 
6.1.1.5 
Product 5: MAD300™ Sprayer Device ............................................................ 48 
6.1.2 
Dosing and Administration ................................................................................... 48 
6.1.3 
Dose Escalation ..................................................................................................... 49 
6.1.4 
Dose Modifications ............................................................................................... 49 
6.2 
Preparation/Handling/Storage/Accountability .............................................................. 49 
6.2.1 
Acquisition and Accountability ............................................................................ 49 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ............................................ 51 
6.2.2.1 
Product 1: Sing2016 M2SR H3N2 Intranasal Influenza Vaccine .................... 51 
6.2.2.2 
Product 2: SPG-NaCl Buffer ............................................................................ 51 
6.2.2.3 
Product 3: Saline placebo .................................................................................. 51 
6.2.2.4 
Product 4: Seasonal IIV4 .................................................................................. 52 
6.2.2.5 
Product 5: MAD300™ Nasal Device ............................................................... 52 
6.2.3 
Product Storage and Stability ................................................................................ 52 
6.2.3.1 
Product 1: Sing2016 M2SR H3N2 Intranasal Influenza Vaccine .................... 52 
6.2.3.2 
Product 2: SPG-NaCl Diluent ........................................................................... 52 
6.2.3.3 
Product 3: Saline placebo .................................................................................. 52 
6.2.3.4 
Product 4: Licensed Seasonal IIV ..................................................................... 52 
6.2.4 
Preparation ............................................................................................................ 53 
6.3 
Measures to Minimize Bias: Randomization and Blinding .......................................... 53 
6.3.1 
Treatment Assignment Procedures ....................................................................... 53 
6.3.2 
Randomization and Blinding ................................................................................ 54 
6.3.3 
Blinding and Masking Procedures ........................................................................ 54 
6.4 
Study Intervention Compliance .................................................................................... 54 
6.5 
Concomitant Therapy .................................................................................................... 54 
6.5.1 
Rescue Medicine ................................................................................................... 55 
7. 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL ..................................................................................... 55 
7.1 
Halting Criteria and Discontinuation of Study Intervention ......................................... 55 
7.1.1 
Study Halting Criteria Study-Wide ....................................................................... 55 
7.1.2 
Study Halting Criteria for “Lead-In” Participants ................................................ 56 
7.1.3 
Individual Halting Criteria .................................................................................... 56 
7.1.4 
Criteria for Redosing ............................................................................................. 57 
7.1.5 
Follow up for Participants who Discontinued Study Intervention ........................ 57 
7.2 
Participant Withdrawal from the Study and Replacement ............................................ 58 
7.3 
Lost to Follow-Up ......................................................................................................... 58 
8. 
STUDY ASSESSMENTS AND PROCEDURES ................................................................ 59 

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8.1 
Screening Procedures (Day -28 to -1, or Day 1): .......................................................... 59 
8.1.1 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings .................................................................................................. 59 
8.2 
Efficacy / Immunogenicity Assessments ...................................................................... 59 
8.2.1 
Humoral Immune Evaluations After Sing2016 M2SR H3N2 Vaccine ................ 60 
8.2.2 
Cellular Immune Evaluations After Sing2016 M2SR H3N2 Vaccine ................. 60 
8.2.3 
Mucosal Immune Evaluations After Sing2016 M2SR H3N2 Vaccine ................ 60 
8.2.4 
Immune Evaluations After Seasonal Influenza Vaccine (IIV4) ........................... 60 
8.3 
Safety and Other Assessments ...................................................................................... 60 
8.3.1 
Solicited AEs (Reactogenicity) ............................................................................. 60 
8.3.2 
Unsolicited Non-Serious AEs ............................................................................... 61 
8.3.2.1 
Suspected Unexpected Serious Adverse Reactions (SUSAR) .......................... 61 
8.3.3 
Adverse Events of Special Interest (AESIs). ........................................................ 61 
8.3.4 
New Onset Chronic Medical Conditions (NOCMCs). ......................................... 61 
8.3.5 
Serious Adverse Events (SAEs). ........................................................................... 61 
8.3.6 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings .................................................................................................. 61 
8.4 
Adverse Events and Serious Adverse Events ............................................................... 61 
8.4.1 
Definition of Adverse Events (AE) ....................................................................... 61 
8.4.2 
Solicited Adverse Events ...................................................................................... 62 
8.4.3 
Local Reactogenicity ............................................................................................ 62 
8.4.4 
Unsolicited Events ................................................................................................ 70 
8.4.5 
Special Reporting of Adverse Events ................................................................... 70 
8.4.6 
Definition of Serious Adverse Events ................................................................... 71 
8.4.7 
Classification of an Adverse Event ....................................................................... 71 
8.4.7.1 
Severity of Event ............................................................................................... 72 
8.4.7.2 
Relationship to Study Intervention ................................................................... 72 
8.4.8 
Time Period and Frequency for Event Assessment and Follow-Up ..................... 72 
8.4.9 
Adverse Event Reporting ...................................................................................... 73 
8.4.9.1 
Investigators Reporting of AEs ......................................................................... 73 
8.4.10 
Serious Adverse Event Reporting ......................................................................... 73 
8.4.10.1 
Investigators Reporting of SAEs .................................................................. 73 
8.4.10.2 
Regulatory Reporting of SAEs ..................................................................... 73 
8.4.11 
Reporting Events to Participants ........................................................................... 74 
8.4.12 
Adverse Events of Special Interest ....................................................................... 74 
8.4.13 
Reporting of Pregnancy ........................................................................................ 74 
8.5 
Unanticipated Problems ................................................................................................ 75 
8.5.1 
Definition of Unanticipated Problems (UP) ......................................................... 75 
8.5.2 
Unanticipated Problem Reporting ......................................................................... 75 
8.5.3 
Reporting Unanticipated Problems to Participants ............................................... 75 
9. 
STATISTICAL CONSIDERATIONS.................................................................................. 76 
9.1 
Statistical Hypotheses ................................................................................................... 76 
9.2 
Sample Size Considerations .......................................................................................... 76 
9.3 
Populations for Analyses .............................................................................................. 79 
9.4 
Statistical Analyses ....................................................................................................... 80 
9.4.1 
General Approach ................................................................................................. 81 

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9.4.2 
Analysis of the Primary Endpoint(s) ..................................................................... 81 
9.4.3 
Baseline Descriptive Statistics .............................................................................. 82 
9.4.4 
Analysis of the Secondary Endpoint(s) ................................................................. 82 
9.4.5 
Analysis of the Exploratory Immunogenicity Endpoints ...................................... 83 
9.4.6 
Planned Interim Analyses ..................................................................................... 83 
9.4.6.1 
Interim Safety Analyses .................................................................................... 83 
9.4.6.2 
Interim Analyses ............................................................................................... 84 
9.4.7 
Sub-Group Analyses ............................................................................................. 84 
9.4.8 
Tabulation of Individual Participant Data ............................................................. 84 
10. SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS ....... 84 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ............................................ 84 
10.1.1 
Informed Consent Process .................................................................................... 84 
10.1.1.1 
Requirements for Permission by Parents/Guardians and Assent by Children 
(in case of a minor) ........................................................................................................... 85 
10.1.1.2 
Other Informed Consent Procedures ............................................................. 86 
10.1.2 
Study Termination and Closure ............................................................................ 86 
10.1.3 
Confidentiality and Privacy .................................................................................. 86 
10.1.4 
Future Use/Secondary Use of Stored Specimens and Data .................................. 87 
10.1.4.1 
Samples for Secondary Research .................................................................. 88 
10.1.4.2 
Data Sharing for Secondary Research .......................................................... 89 
10.1.5 
Key Roles and Study Governance ........................................................................ 89 
10.1.6 
Safety Oversight .................................................................................................... 89 
10.1.7 
Clinical Monitoring ............................................................................................... 90 
10.1.8 
Quality Assurance and Quality Control ................................................................ 90 
10.1.9 
Data Handling and Record Keeping ..................................................................... 91 
10.1.9.1 
Data Collection and Management Responsibilities ...................................... 91 
10.1.9.2 
Study Record Retention ................................................................................ 91 
10.1.9.3 
Source Records ............................................................................................. 92 
10.1.10 Protocol Deviations ............................................................................................... 92 
10.1.11 Publication and Data Sharing Policy .................................................................... 92 
10.1.12 Human Data Sharing Plan ..................................................................................... 92 
10.1.13 Genomic Data Sharing Plan .................................................................................. 92 
10.1.14 Publication ............................................................................................................ 92 
10.1.15 Conflict of Interest Policy ..................................................................................... 93 
10.2 
Additional Considerations ............................................................................................ 93 
10.2.1 
Research Related Injuries ..................................................................................... 93 
10.3 
Abbreviations ................................................................................................................ 93 
10.4 
Protocol Amendment History ....................................................................................... 94 
11. APPENDIX ........................................................................................................................... 96 
11.1 
Appendix A – Blood and Fluid Collection Volumes by age group .............................. 96 
11.2 
Appendix B – Evaluation Criteria for Acute Wheezing ............................................... 97 
12. REFERENCES ................................................................................................................... 100 
 

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LIST OF TABLES 
Table 1: Schedule of Activities: .................................................................................................... 23 
Table 2. Cohorts ............................................................................................................................ 40 
Table 3: Age and Dose Cohorts .................................................................................................... 49 
Table 4: Grades of Local & Systemic Reactogenicity Assessments, By Age Group ................... 62 
Table 5: Probability (%) of observing at least one safety event, given varying underlying event 
probabilities and sample sizes ........................................................................................ 76 
Table 6: Estimated event probabilities (%) and associated exact 95% Clopper-Pearson 
confidence intervals resulting from varying numbers of events observed in groups of 
different sizes. ................................................................................................................. 78 
Table 7: Detectable probabilities (%) with 80% or 90% power when comparing binary event 
probabilities between groups with two-sided Barnard’s exact tests with α=0.05. .......... 79 
Table 8: Blood and Fluid Collection volumes by Age ................................................................. 97 
 
 

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LIST OF FIGURES 
Figure 1– The overall study design is captured in the schema below: ......................................... 26 
 
 
 

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1. 
PROTOCOL SUMMARY 
 
1.1 
Synopsis 
 
Rationale for Proposed Clinical Study: 
The influenza virus circulates widely in the United States with peaks usually between December 
and February of each year. Young children (≤ 2 years), older adults (≥ 65 years), and persons 
with underlying medical problems are especially vulnerable to more severe disease and may 
suffer dire consequences, including hospitalization and even death. Prior to widespread 
vaccinations, influenza globally claimed 40 -70 million lives annually due to influenza-related 
complications [1]. Today, in the United States alone, despite wider spread vaccination efforts, 
influenza continues to claim 30,000 to 49,000 lives annually with approximately 200,000 
hospitalizations. Although vaccines are available and recommended for nearly the entire US 
population, not all people receive annual influenza vaccination and, according to the US Flu-VE 
Network, the effectiveness of our currently licensed vaccines is typically only 40-60% [2]. The 
overall effectiveness of currently licensed influenza vaccines against H3N2 influenza strains 
during influenza seasons with a mismatch between the vaccine and the circulating strain can be 
as low as 0% with an average of only 23% (95% Confidence Interval [CI] 2% to 40%) [3]. 
Improved vaccines against influenza are needed. FluGen, Inc. has developed a novel influenza 
vaccine which is designed to provide broader-spectrum immunity against multiple influenza type 
A viruses, especially in pediatric and geriatric patients. 
 
The FluGen vaccine that is being evaluated in this study is a live vaccine product called 
Sing2016 M2SR H3N2. It has the ability to infect respiratory epithelial cells in a manner similar 
to wild-type influenza virus and to induce an immune response against a broad spectrum of type 
A influenza viral strains [4]. Given that Sing2016 M2SR H3N2 has a defective M2 gene, unlike 
wild-type viruses, it is unable to replicate and generate progeny in animals and in humans, 
making its infectivity self-limiting. This attenuation is expected to improve safety while 
maintaining the ability to elicit an immune response. Furthermore, like FluMist®, the US-
licensed live attenuated intranasal influenza vaccine, the Sing2016 M2SR H3N2 vaccine induces 
both a mucosal and cell-mediated immune response and is administered intranasally. However, 
unlike FluMist®, the Sing2016 M2SR H3N2 vaccine has been demonstrated to induce immune 
responses in animal models even in the presence of pre-existing antibodies.[5] Furthermore, 
Sing2016 M2SR H3N2 induces antibodies in animal models against the highly conserved stem 
region of hemagglutinin (HA) as well as against neuraminidase (NA) surface proteins on the 
surface of the influenza virus.[6] These features may improve its ability to induce immune 
responses against a broader spectrum of influenza type A strains and potentially may prolong 
immunity despite on-going drift mutations in the influenza type A virus.[4] Early stage clinical 
trials with the M2SR vaccine platform vaccines have included 3 studies conducted in the US and 
an influenza challenge study conducted in Belgium. Please refer to the Investigator’s Brochure 
(IB) for more information about these trials. The 3 US studies included two dose-ranging studies 
in adults and one study in pediatric participants aged 9 to 17 years. The DMID 17-0012 study, 
entitled “A Phase I Trial to Evaluate the Safety and Immunogenicity of an Influenza Vaccination 
Strategy Including a H3N2 M2SR Prime followed by a Seasonal Quadrivalent Inactivated 
Vaccine Boost in a Pediatric Population 9-17 years old” (BB-IND 18170; sponsored by DMID, 
NIAID), enrolled 43 males and non-pregnant females ages 9 to 17 years old, inclusive. 

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Participants were randomly allocated in a 1:1 ratio to receive one 108 TCID50 dose of Bris10 
M2SR vaccine or placebo administered intranasally (IN) followed by one dose of licensed 
quadrivalent influenza vaccine (IIV4) administered intramuscularly 3 months later. Enrollment 
was completed on September 20, 2019 with 43 participants enrolled. Twenty-eight participants 
were in the 9–14-year-old age range and 15 participants in the 15–17-year-old age range. There 
were no reported serious adverse events (SAEs), adverse events (AEs) of special interest, or 
halting rules met. Preliminary results for this study are posted on clinicaltrials.gov, study 
identifier NCT03553940. 
 
The M2SR vaccine platform vaccines have been found to be safe and well-tolerated during these 
4 studies, including administration of doses as high as 109 TCID50 in the second adult dose 
ranging study.[4]  In adults, M2SR platform vaccines induce T-cell responses as well as serum 
and mucosal antibody responses to both matched and drifted influenza strains. Protection against 
a highly drifted H3N2 influenza challenge strain was observed in a subset of volunteers who 
received a single intranasal dose of 108 TCID50 of monovalent H3N2 M2SR one month prior to 
challenge.  
 
In this study, we propose to perform a Phase 1b, double-blind, dose-escalating, age de-escalating 
study of the Sing2016 M2SR H3N2 vaccine in children ages 6 months to 17 years. The purpose 
of the study is to assess the safety, tolerability/reactogenicity, and immunogenicity of the 
vaccine.  
 
Study Design:  
This is a Phase 1b, randomized, double-blind, dose-escalating, age de-escalating, placebo-
controlled study of 200 children, ages 6 months to 17 years. To allow for simultaneous 
enrollment at the four research sites, the number of children in each cohort and the total number 
of children in the entire study may slightly deviate from the targeted numbers mentioned 
throughout the protocol. The study will enroll seven cohorts of children. The study design 
includes pre-planned Safety Review Committee (SRC) reviews.  
 
The first two groups to be vaccinated will be Cohorts 1 and 2. Cohort 1 consists of 45 
children 9-17 years old. Thirty of them will receive one dose of the vaccine at a dose of 109 
TCID50, and 15 will receive one dose of placebo. Cohort 2 comprises 45 children 2-8 years old. 
Thirty of them will receive one dose of the vaccine at a dose of 108 TCID50 and 15 will receive 
one dose of placebo. Sites will enroll participants into Cohorts 1 and 2 simultaneously. Once 25 
or more participants in each of the first 2 cohorts (Cohorts 1 and 2) have completed Day 8, the 
SRC will evaluate if any halting rules are met and if it is deemed safe, enrollment in Cohort 3 
may open. Enrollment within each cohort will not halt during the SRC review. Cohort 2 must 
fully enroll before enrollment in Cohort 3 may begin. If any halting rules are met or if the SRC 
raises any safety concerns other than halting rules that should be independently reviewed, the 
external Safety Monitoring Committee (SMC) will meet to discuss the data prior to progression 
to the next cohort.  
 
Cohort 3 consists of 25 children 2-8 years old. Fifteen of them will receive one dose of vaccine 
at 109 TCID50 and 10 will receive one dose of placebo. Once all 25 participants in Cohort 3 have 
completed Day 8 of follow-up, similar to Cohorts 1 and 2, the SRC will review to ensure no 

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halting rules are met and if no rules are met and the SRC determines it is safe to proceed, 
simultaneous enrollment into Cohorts 4 and 5 can begin. If any halting rules are met or any 
concerns are raised by the SRC, an external SMC may meet to discuss the data for 
recommendations on either progression or clinical trial modification before progression to the 
next cohort. 
 
Cohort 4 consists of 25 children 2-8 years old; 15 of them will receive two doses of vaccine at 
109 TCID50 and 10 will receive two doses of placebo, with a 28-day interval between the first 
and second doses. Cohort 5 will enroll 8 influenza naïve children (defined as children without 
receipt of influenza vaccine and without previous documented influenza infection) who are 6-23 
months old who will be randomly assigned to receive two doses of 107 TCID50 Sing2016 M2SR 
(n=6) or two doses of placebo (n=2) with a 28-day interval between the first and second dose. 
Once all 8 participants in Cohort 5 have completed Day 8 of follow-up after the first dose, the 
SRC will conduct a safety assessment before beginning enrollment in Cohort 6. Once 6 of the 8 
participants in Cohort 5 have completed Day 4 after the second dose (e.g., Study Day 32), the 
following process will be implemented prior to receipt of the second dose for any participant in 
Cohort 6: 
 
1. An email will be sent from the site to the SDMCC attesting that all data entry through 
Day 4 after the second dose (e.g., Study Day 32) for at least 6 participants in Cohort 5 has 
been completed. 
2. The SDMCC will perform data cleaning as it pertains to assessment of the halting rules. 
3. The SDMCC will assess if there were any halting rules met based on data entered in the 
data system and email DMID indicating what halting rules, if any, were met based on 
data entered in the data system. 
a. If any of the halting rules were met, the study will halt and a review by the SMC, 
sponsor, and investigators will convene as specified in Section 7.1.  
b. If none of the halting rules were met, DMID will confirm that the study may 
proceed as per-protocol. 
 
Cohorts 6 and 7 will also enroll 6 to 23-month-olds who are influenza-naïve. Cohort 6 will 
consist of 26 children. A lead-in group of 8 children will be randomly assigned to receive either 
two doses of 108 TCID50 of the Sing2016 M2SR (n=6) or two doses of placebo (n=2). Once the 
first 8 children have completed Day 8 of follow up after the first dose, the SRC will review the 
safety and determine if Cohort 7 may open to enrollment. Cohort 7 enrollment will not begin 
until Cohort 6 is fully enrolled. Similar to other cohorts, the additional 18 children in Cohort 6 
may continue to enroll during the SRC review.  
 
Cohort 7 will be the final cohort. 26 children will be randomly allocated to receive two doses of 
either 109 TCID50 Sing2016 M2SR (n=18) or placebo (n=8). This cohort does not have a “lead-
in” group since data from such a group are not needed to allow the enrollment in a subsequent 
cohort. However, the study-wide and individual halting rules still apply. 
 
For all cohorts, a minimally acceptable pre-vaccination blood volume is set at one 5-mL tube of 
blood (SST for serum), regardless of age (Appendix A – Blood and Fluid Collection Volumes by 

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age group). Should the site not be able to collect that blood before the first vaccination, the child 
will not be permitted to continue.  
 
After the administration of the above noted vaccine doses, safety will be assessed by the 
evaluation of reactogenicity from Days 1-8 for participants in Cohorts 1-3, who are receiving a 
single dose of vaccine or placebo. For Cohort 4 – 7, who receive two doses of vaccine or 
placebo, safety assessment will be from Day 1-8 and approximately Days 29-36. All participants 
will be followed for AEs, AESIs, NOCMCs, and SAEs in the time periods described in the 
protocol.  Safety will be reviewed by the internal SRC and an external independent SMC, as 
applicable. The responsibility of the SRC will be to review if any halting rules are met and to 
determine if safety data available permit progression from cohort to cohort. The responsibility of 
the SMC will be to review safety outcomes if criteria for progression to a subsequent cohort are 
not met, a halting rule occurs, or as otherwise determined in their charter. The SMC may propose 
to the sponsor and investigators to make study product dose adjustments or other modifications. 
The roles of the SRC, and SMC are described in detail below. All participants will be evaluated 
for immune responses as described below.  
 
All participants will be vaccinated with the first dose of investigational product or placebo in the 
influenza off-season for the Northern Hemisphere. In the past, we could reliably predict that the 
influenza off-season would begin by May in the regions where this study will be conducted. 
Since the start of the COVID-19 pandemic, the seasonality of influenza in the US has become 
less predictable, and the influenza off-season may begin as early as March 1. The decision for 
each site’s start either on or after March 1 will be determined by the protocol team and sponsor, 
using data from local (e.g., site-affiliated hospital/healthcare), state, or regional microbiological 
surveillance systems representative of the recruitment catchment area of each site. The 
decision(s) for determination of influenza off-season also may consider data from larger 
surveillance programs, such as CDC [7]. Each site may begin enrolling as early as March 1, if 
influenza virus is reported in less than 5% of respiratory specimens, and absolute numbers of 
influenza cases are consistent with off-season status. Study activities may be halted if the 
proportion of local or regional respiratory specimens positive for influenza virus exceeds 10% 
and the local or regional ILI activity exceeds low. Sites may re-initiate enrollment activities if the 
enrollment criteria are met once again for two consecutive weeks.  
 
During the Fall (September-November), participants will receive CDC recommended seasonal 
influenza vaccine (IIV4) administered at least 28 days after receipt of the final investigational 
vaccine administration. The final study visit for each participant will occur in April of the 
following calendar year.   
 
Due to the limited availability of product, and funding to support additional years of enrollment 
into Cohorts 5, 6, and 7, the decision was made to stop enrollment after the final participant was 
enrolled into Cohort 4. 
 
Study Objectives:  
Primary 

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1. To assess the safety and tolerability of one and two administrations of the Sing2016 
M2SR H3N2 influenza vaccine at 108 or 109 TCID50 delivered intranasally to healthy 
participants, 2 to 17 years of age  
 
Secondary 
 
2. To assess the humoral immunogenicity (serum antibody and mucosal antibody responses) 
directed against homologous viral strains after one or two administrations of Sing2016 
M2SR H3N2 influenza vaccine at 108 or 109 TCID50 delivered intranasally to healthy 
participants, 2 to 17 years of age  
 
Exploratory  
3. To assess the cellular immunogenicity (T-cell immune responses) against Sing2016 
M2SR H3N2 influenza vaccine following one or two administrations of Sing2016 M2SR 
H3N2 influenza vaccine at 108 or 109 TCID50 delivered intranasally to healthy 
participants, 2 to 17 years of age 
4. To assess participant immunological response to neuraminidase (NA) following one or 
two administrations of Sing2016 M2SR H3N2 influenza vaccine at 108 or 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age 
5. To assess the humoral immunogenicity (serum and/or plasma antibody) and mucosal 
antibody responses and cellular immunogenicity (T-cell responses) after seasonal 
influenza vaccine (IIV4) following one or two administrations of Sing2016 M2SR H3N2 
influenza vaccine at 108 or 109 TCID50 delivered intranasally to healthy participants, 2 to 
17 years of age   
6. To assess mucosal antibody responses directed against homologous viral strains after one 
or two administrations of Sing2016 M2SR H3N2 influenza vaccine at 108or 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age   
7. To assess humoral immunogenicity (serum and/or plasma antibody), and mucosal 
antibody responses directed against heterologous viral strains of one or two 
administrations of Sing2016 M2SR H3N2 influenza vaccine at 108 or 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age 
8. To conduct additional characterization of humoral immunity against homologous and/or 
heterologous viral strains (e.g., extra-neutralizing antibody function) following one or 
two administrations of Sing2016 M2SR H3N2 influenza vaccine at 108 or 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age   
9. To assess the effects of age, dose level, sex, prior receipt of seasonal influenza vaccine(s), 
and other variables on humoral immunogenicity (serum antibody and mucosal antibody 
responses) directed against homologous viral strains of one or two administrations of 
Sing2016 M2SR H3N2 influenza vaccine at 108 or 109 TCID50 delivered intranasally to 
healthy participants, 2 to 17 years of age   
 
Study Endpoints: 
Primary 
1. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience solicited local reactogenicity events, of all severity grades and by 
grade, in the 7 days following administration of each dose of vaccine 

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2. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience solicited systemic reactogenicity events, of all severity grades and by 
grade, in the 7 days following administration of each dose of vaccine  
3. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience unsolicited non-serious adverse events, of all severity grades and by 
grade, in the 28 days following administration of each dose of vaccine     
4. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience adverse events of special interest (AESIs) the time of first 
vaccination through the end of the study period (final visit in the month of April of the 
calendar year following enrollment)  
5. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience Serious Adverse Events (SAEs) from the time of first vaccination 
through the end of the study period (final visit in the month of April of the calendar year 
following enrollment)  
6. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience new-onset chronic medical conditions (NOCMCs) from the time of 
the first study vaccination through the end of the study period (final visit in the month of 
April of the calendar year following enrollment)  
 
Secondary 
7. The number and percentage of participants in Cohorts 1, 2, and 3 with putative 
seroprotection at baseline (Day 1) and on approximately Day 29, defined as an HAI titer 
≥1:40 in serum against an H3N2 M2SR-like virus  
8. The number and percentage of participants in Cohort 4 with putative seroprotection at 
baseline (Day 1) and on approximately Day 57, defined as an HAI titer ≥1:40 in serum 
against an H3N2 M2SR-like virus 
9. The number and percentage of participants in Cohorts 1, 2, 3 with neutralization titer 
≥1:40 in serium against an H3N2 M2SR-like virus at baseline (Day 1) and on 
approximately Day 29 
10. The number and percentage of participants in Cohort 4 with neutralization titer ≥1:40 in 
serum against an H3N2 M2SR-like virus at baseline (Day 1) and on approximately Day 
57 
11. Geometric Mean Titers (GMTs) of serum HAI against an H3N2 M2SR-like virus at 
baseline (Day 1) and on approximately Day 29 for participants in Cohorts 1, 2, and 3 
12. Geometric Mean Titers (GMTs) of serum HAI against an H3N2 M2SR-like virus at 
baseline (Day 1) and approximately Day 57 for participants in Cohorts 4 
13. Geometric Mean Titers (GMTs) of serum neutralizing antibodies against an H3N2 
M2SR-like virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1) and on 
approximately Day 29  
14. Geometric Mean Titers (GMTs) of serum neutralizing antibodies against an H3N2 
M2SR-like virus for participants in Cohorts 4 at baseline (Day 1) and on approximately 
Day 57  
15. Geometric Mean Fold Rise (GMFR) and proportions in serum with greater than or equal 
to 2- and 4-fold rises from baseline (Day 1) in HAI titers against an H3N2 M2SR-like 
virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1) and on approximately Day 
29  

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16. Geometric Mean Fold Rise (GMFR) and proportions in serum with greater than or equal 
to 2- and 4-fold rises from baseline (Day 1) in HAI titers against an H3N2 M2SR-like 
virus for participants in Cohorts 4 at baseline (Day 1) and on approximately Day 57 
17. Geometric Mean Fold Rise (GMFR) and proportions in serum with greater than or equal 
to 2- and 4-fold rises from baseline (Day 1) in neutralization titers against an H3N2 
M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29  
18. Geometric Mean Fold Rise (GMFR) and proportions in serum with greater than or equal 
to 2- and 4-fold rises from baseline (Day 1) in neutralization titers against an H3N2 
M2SR-like virus for participants in Cohorts 4 on approximately Day 57  
19. Mean secretory IgA (sIgA) response, as measured by binding antibody multiplex 
(BAMA) in nasal lavage specimens, against an H3N2 M2SR-like virus for participants in 
Cohorts 1, 2, and 3 before vaccination and on approximately Day 29 
20. Mean secretory IgA (sIgA) responses, as measured by binding antibody multiplex 
(BAMA) in nasal lavage specimens, against an H3N2 M2SR-like virus for participants in 
Cohorts 4 before vaccination and on approximately Day 57  
21. Mean change (difference) from baseline in secretory IgA (sIgA) response, as measured 
by the binding antibody multiplex assay (BAMA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29  
22. Mean change (difference) from baseline in secretory IgA (sIgA) response, as measured 
by the binding antibody multiplex assay (BAMA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohorts 4 on approximately Day 57 
Exploratory 
23. Antigen-specific T-cell responses to vaccine immunogens, at baseline (Day 1) and 
following one or two doses of vaccine 
24. Humoral antibody, as detected by one or more assays that measure responses to NA, at 
baseline (Day 1) and following one or two doses of vaccine or IIV4 
25. Serum antibody, as detected by HAI and neutralization, directed against homologous 
H3N2 strains measured at baseline (Day 1) and after IIV4 administration 
26. Antigen-specific T-cell responses to vaccine immunogens, at baseline (Day 1) and 
measured after IIV4 administration 
27. Serum antibody to additional influenza antigens, as detected by multiplex ELISA assays 
and/or protein microarrays with subsequent evaluation of HAI and neutralization when 
multiplex ELISA screen is positive, directed against heterologous strains at baseline (Day 
1) and following one or two doses of Sing2016 M2SR H3N2 vaccine 
28. Humoral and/or mucosal antibody neutralizing and extra-neutralizing function against 
homologous and heterologous strains based on positive responses detected by methods 
described above.  
29. Associations between serum antibody responses and age, dose level, sex, prior receipt of 
seasonal influenza vaccine(s) and other variables over time. 
30. Associations between mucosal antibody responses and age, dose level, sex, prior receipt 
of seasonal influenza vaccine(s) and other variables over time. 
31. Geometric mean titer (GMTs) of plasma HAI against an H3N2 M2SR-like virus at 
baseline (Day 1) and on approximately Day 29 for participants in Cohorts 1, 2 and 3  
32. GMTs of plasma HAI against an H3N2 M2SR-like at baseline (Day 1) and on 
approximately D57 for participants in all Cohorts 4 
33. Geometric Mean Titers (GMTs) of plasma neutralizing antibodies against an H3N2 

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M2SR-like virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1) and on 
approximately Day 29  
34. Geometric Mean Titers (GMTs) of plasma neutralizing antibodies against an H3N2 
M2SR-like virus for participants in Cohort 4 at baseline (Day 1) and on approximately 
Day 57 
35. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma HAI titers against an H3N2 M2SR-like virus 
for participants in Cohorts 1, 2, and 3 on approximately Day 29 
36. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma HAI titers against an H3N2 M2SR-like virus 
for participants in Cohort 4 on approximately Day 57 
37. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma neutralization titers against an H3N2 M2SR-
like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29 
38. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma neutralization titers against an H3N2 M2SR-
like virus for participants in Cohort 4 on approximately Day 57  
39. The number and percentage of participants in Cohorts 1, 2, and 3 with putative 
seroprotection at baseline (Day 1) and on approximately Day 29, defined as a plasma 
HAI titer ≥1:40 against an H3N2 M2SR-like virus 
40. The number and percentage of participants in Cohort 4 with putative seroprotection at 
baseline (Day 1) and on approximately Day 57, defined as a plasma HAI titer ≥1:40 
against an H3N2 M2SR-like virus 
41. The number and percentage of participants in Cohorts 1, 2, 3 with plasma neutralization 
titer ≥1:40 against an H3N2 M2SR-like virus at baseline (Day 1) and on approximately 
Day 29 
42. The number and percentage of participants in Cohort 4 with plasma neutralization titer 
≥1:40 against an H3N2 M2SR-like virus at baseline (Day 1) and on approximately Day 
57 
43. Geometric mean titers (GMTs) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1, before 
vaccination) and on approximately Day 29 
44. Geometric mean titers (GMTs) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohort 4 at baseline (Day 1, before 
vaccination) and on approximately Day 57 
45. Geometric mean fold rise (GMFR) and proportions with greater than or equal to 2- and 4-
fold rises from baseline (Day 1) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29 
46. Geometric mean fold rise (GMFR) and proportions with greater than or equal to 2- and 4-
fold rises from baseline (Day 1) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohort 4 on approximately Day 57 

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47. Mean total secretory IgA (sIgA), as measured by the enzyme-linked immunosorbent 
assay (ELISA) in nasal lavage specimens , against an H3N2 M2SR-like virus for 
participants in Cohorts 1, 2, and 3 at baseline (Day 1, before vaccination) and on 
approximately Day 29 
48. Mean total secretory IgA (sIgA), as measured by the enzyme-linked immunosorbent 
assay (ELISA) in nasal lavage specimens, against an H3N2 M2SR-like virus for 
participants in Cohort 4 at baseline (Day 1, before vaccination) and on approximately 
Day 57 
49. Mean change (difference) from baseline in total secretory IgA (sIgA), as measured by the 
enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29 
50. Mean change (difference) from baseline in total secretory IgA (sIgA), as measured by the 
enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohort 4 on approximately Day 57 
51. Mean normalized secretory IgA (sIgA), as measured by the enzyme-linked 
immunosorbent assay (ELISA) in nasal lavage specimens , against an H3N2 M2SR-like 
virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1, before vaccination) and on 
approximately Day 29 
52. Mean normalized secretory IgA (sIgA), as measured by the enzyme-linked 
immunosorbent assay (ELISA) in nasal lavage specimens , against an H3N2 M2SR-like 
virus for participants in Cohort 4 at baseline (Day 1, before vaccination) and on 
approximately Day 57 
53. Mean change (difference) from baseline in normalized secretory IgA (sIgA), as measured 
by the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against 
an H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 
29 
54. Mean change (difference) from baseline in normalized secretory IgA (sIgA), as measured 
by the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against 
an H3N2 M2SR-like virus for participants in Cohort 4 on approximately Day 57 
55. Geometric Mean Titers (GMTs) of plasma or serum NA antibodies against an H3N2 NA 
for participants in Cohorts 1, 2, and 3 at baseline (Day 1) and on approximately Day 29 
56. Geometric Mean Titers (GMTs) of plasma or serum NA antibodies against an H3N2 NA 
for participants in Cohort 4 at baseline (Day 1) and on approximately Day 57 
57. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma or serum NA antibodies against an H3N2 
NA for participants in Cohorts 1, 2, and 3 on approximately Day 29 
58. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma or serum NA antibodies against an H3N2 
NA for participants in Cohort 4 on approximately Day 57 
59. The number and percentage of participants in Cohorts 1, 2, 3 with NA antibody titer 
≥1:40 against an H3N2 NA at baseline (Day 1) and on approximately Day 29 
60. The number and percentage of participants in Cohort 4 with NA antibody titer  ≥1:40 
against an H3N2 NA-like virus at baseline (Day 1) and on approximately Day 57 
 
Eligibility: 

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A potential participant is eligible to join this trial if he/she meets all the following inclusion 
criteria and none of the following exclusion criteria: 
 
Inclusion Criteria: 
1. Participant is a male or female child aged 6 months to 17 years inclusive at time of 
enrollment (each cohort has its own age upper and lower limits1) 
1Cohort 1: 9-17 years (on or after the 9th birthday and before the 18th birthday at the time of the first dose); 
Cohorts 2, 3, and 4: 2-8 years (on or after the 2nd birthday and before the 9th birthday at the time of the first 
dose); Cohorts 5, 6, and 7: 6 months to 23 months (on or after the 6th month of life based on calendar day 
and before the second birthday at the time of the first dose)  
2. For Cohorts 1 to 4, receipt of at least 2 doses of seasonal influenza vaccine in the past.  
3. For Cohorts 5 to 7, receipt of no seasonal influenza vaccines in the past and no documented 
history of laboratory-confirmed influenza illness 
4. Parent/guardian of the participating child provides written informed permission and 
participating child provides assent2 prior to initiation of any study procedures   
2as appropriate by age or development and approved by the IRB 
5. Parent/guardian and participant, as appropriate, are able to understand and comply with 
planned study procedures and are available for all study visits 
6. Participant is in good health as assessed by the principal investigator or other designated 
study investigator3  
3based on medical history and physical examination (physical examination may be done as part of routine 
medical care or specifically for eligibility screening) 
7. Parent/guardian of the participating child agrees not to allow the participant to join another 
clinical trial that includes an investigational agent or device during the study period   
8. A female participant of child-bearing potential4 agrees to abstain from sexual intercourse or 
to correctly use an acceptable method of contraception5  
4A female of child-bearing potential is defined as a female who is post-menarchal and not sterilized via 
tubal ligation, bilateral oophorectomy, salpingectomy, hysterectomy, or successful Essure® placement 
(permanent, non-surgical, non-hormonal sterilization) with documented radiological confirmation test at 
least 90 days after the procedure. This applies only to participants in Cohort 1.  
5Acceptable methods of contraception must be used from 30 days prior to vaccination until 60 days after 
the last study vaccination (not IIV4) and include full abstinence from sexual intercourse with a male 
partner, monogamous relationship with vasectomized partner who has been vasectomized for 180 days or 
more or shown to be azoospermic prior to the participant receiving the study vaccination, barrier methods 
such as condoms or diaphragms/cervical cap, intrauterine devices, NuvaRing®, and licensed hormonal 
methods such as implants, injectables, or oral contraceptives (“the pill”).  
9. A female participant of child-bearing potential4 must have a negative urine pregnancy test 
within 24 hours prior to each study product 
10. A male who is sexually active with a female of childbearing potential4 must agree to use an 
acceptable method of contraception6  
6From the time of the first dose of study vaccine until 60 days after receipt of the last dose study vaccine, 
only in Cohort 1. The only acceptable method of contraception for males who are sexually active with 
females of childbearing potential is condoms.  
 
Exclusion Criteria: 
 
 
 
 

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1. Has a body temperature of 38.0°C/100.4°F (oral or axillary) or greater 
or another acute illness7 within the 72 hours prior to study vaccination   
7Potential participants who are recovering from an acute illness and have residual minimal symptoms, 
which, in the opinion of the site principal investigator or appropriate sub-investigator, will not likely 
affect the evaluation of outcome measures are not ineligible. Temperature evaluation will not be performed 
as a study procedure on participants prior to administration of seasonal influenza vaccine  
2. Has any medical or mental health disease or condition8 that would render study participation 
unsafe, or would interfere with the evaluation of the responses 
8in the opinion of the site principal investigator or appropriate sub-investigator 
3. Has a history of provider-diagnosed asthma requiring the use of medications at any age, or 
has had a wheezing episode or use of medications to treat asthma in the 12 months prior to 
screening. 
4. Has immunosuppression as a result of an underlying illness or treatment, a recent history 
or current use of immunosuppressive or immunomodulating disease therapy  
5. Has a diagnosis of or history of malignant neoplastic disease   
6. Has taken oral, parenteral (intramuscular or intravenous), inhaled, or nasal corticosteroids of 
any dose within 30 days prior to study vaccination   
7. Has known HIV, hepatitis B, or hepatitis C infection  
8. Has known hypersensitivity or allergy to any components of the study vaccine or material in 
the nasal delivery device9  
9vaccine components: sucrose, sodium chloride, phosphate, glutamate; delivery device material: 
polycarbonate, polypropylene, synthetic rubber  
9. Has a history of severe reactions following previous immunization with licensed or 
unlicensed influenza vaccines   
10. Has a history of an anatomic disorder of the nares or nasopharynx   
11. Has a history of chronic sinus infections  
12. Has a history of or currently smokes or vapes  
13. Has a history of Guillain-Barré syndrome   
14. Use of aspirin- or salicylate-containing products in the 30 days prior to or intends to use these 
products in the 30 days following administration of the investigational vaccine  
15. Has a history of documented influenza or receipt of influenza antiviral treatment in the 4 
months prior to the first vaccination 
16. Receipt of any antiviral drug within the week prior to or following the investigational vaccine 
17. Receipt of a licensed live vaccine within 30 days prior to the first study vaccination, or plans 
to receive a licensed live vaccine within the 30 days after the last study vaccination   
18. Receipt of licensed inactivated non-influenza vaccine10 within 14 days prior to the 1st study 
vaccination, or plans to receive licensed, inactivated vaccine within the 30 days after the 
last study vaccination 
10 Participants will be asked to avoid receipt of any routine licensed vaccines or vaccines under emergency 
use authorization during the periods described.  
19. Receipt of an influenza vaccine within the 4 months prior to the first study vaccination or 
plans to receive an influenza vaccine following the last study vaccination. Seasonal IIV4 will 
be received by participants as part of this trial.   
20. Receipt of immunoglobulin or other blood products within the 6 months prior to the first 
study vaccination or plans to receive during the period of study participation. 
21. Receipt of an experimental11 agent or device within the 6 months prior to the first 
study vaccination or expects to receive an experimental agent or device during the study 
period   

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11Products for treatment or prevention of COVID-19, when received under Emergency Use Authorization 
[EUA] or full FDA approval and not as part of a clinical trial, will not be deemed “experimental” for the 
purposes of this criterion and will not make an otherwise eligible prospective participant ineligible.  
22. Is a family member of study personnel or personnel directly involved in the conduct or 
monitoring of the study 
23. Receipt of an approved or experimental product for treatment or prevention of COVID-19 
within the 10 days12,13 prior to study enrollment 
12Participants may enroll if greater than 10 days after receipt of the COVID-19 treatment or prevention. 
13Participants who are receiving COVID-19 vaccines around the time of dosing of the investigational 
product will be asked to avoid COVID-19 vaccination within the 10 days before any vaccination in the 
study and within any reactogenicity period (the day of and 7 days following each intranasal vaccination).  
24. Inability of the study team to collect 5 mL of blood from the participant before the first 
vaccination (pre-vaccination blood).  
  
Study Phase: Phase 1b 
 
Study Population: Healthy 6 month to 17-year-old children, inclusive   
 
Sites: 4 US sites   
 
Study Intervention: 
The study intervention is intranasal vaccination with Sing2016 M2SR H3N2 influenza vaccine 
supplied by FluGen, Inc. Commercially available saline will be used as the corresponding 
placebo. The Sing2016 M2SR H3N2 influenza vaccine made by FluGen is a live vaccine product 
with a defective M2 gene which renders the virus unable to replicate and generate progeny in 
animals and in humans, making its infectivity self-limiting. This attenuation is expected to 
improve safety while maintaining the ability to elicit an immune response. Furthermore, like 
FluMist®, the Sing2016 M2SR H3N2 vaccine induces both a mucosal and cell-mediated 
immune response and is administered intranasally. However, unlike FluMist®, the Sing2016 
M2SR H3N2 vaccine has been demonstrated to induce Immune responses in animal models even 
in the presence of pre-existing antibodies. Early studies of this study intervention have reported 
no serious adverse events (SAEs), adverse events (AEs) of significance or any halting rules. 
Further, Sing2016 M2SR H3N2 induces antibodies in animal models against the highly 
conserved stem region of hemagglutinin (HA) as well as against NA glycoproteins on the surface 
of the influenza virus. In this study we evaluate the safety and reactogenicity of the Sing2016 
M2SR H3N2 influenza vaccine in a dose-escalating and age de-escalating double-blinded 
randomized manner.  
 
All enrolled children will also receive seasonal influenza vaccine with licensed IIV4 at the dose 
and schedule recommended for the individual participant with first dose administered once 
seasonal influenza vaccine is available usually between September through November but no 
later than end of November of the same calendar year.  
 
The study is designed as a Phase 1b, randomized, double-blind, dose-escalating, age de-
escalating, placebo-controlled study of approximately 200 children ages 6 months to 17 years. 
Cohort 1 to Cohort 4 will enroll non-naïve children.  Cohorts 5-7 will enroll 6- to 23-month-olds 
who are influenza-naïve (no previous influenza vaccine and no previous documented influenza 

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infection). Safety reviews may be performed by the SRC, and the SMC. Details on those reviews 
are found the protocol.  
 
Total Study Duration: Up to 4 years  
 
Participant Duration: Up to 14 months 
 
Safety: The study has three sets of halting criteria.  
 
Halting Criteria Study-wide:  
The study-wide halting criteria apply to all enrolled participants study-wide and serve to pause 
enrollment in the study until the data are reviewed by the SMC, sponsor, and investigators. The 
criteria are defined in the protocol. 
 
Halting Criteria for “Lead-In” Participants: 
The 8 participants in Cohort 5 and the first 8 participants in Cohort 6 are termed “lead-ins” 
because data on safety and tolerability from Days 1 to 8 from these participants will inform 
enrollment into subsequent cohorts. For progression to Cohort 6, safety data must be reviewed 
for Cohort 5 participants through Day 8 of follow-up after the first dose. Given that the lead-ins 
are young (6-23 months), influenza-naïve, and only 8 participants in each of those cohorts, the 
halting criteria are slightly more stringent with lower thresholds for tolerability of AEs and 
SAEs. The criteria are defined in the protocol. 
 
Halting Criteria for Individual Participants: 
These halting criteria will apply to any individual participant within the study. If any of these 
halting criteria are met, the individual will not receive any additional doses of investigational 
product.  
 
Safety Committee Reviews: 
The study has three safety review persons or groups, as defined below. 
 
SRC:  
The Safety Review Committee (SRC) will review the blinded data required to allow progression 
from cohort to cohort. The review and the composition of this committee are detailed in the 
protocol. Ad hoc reviews may also be called for by the investigators or sponsor. Decisions are 
dependent on the failure to meet halting criteria when the required follow-up data are available.   
 
SMC:  
The Safety Monitoring Committee (SMC) is an independent group that will provide safety 
oversight for the study. They will meet to review safety data as delineated in their charter. They 
will meet and make recommendations if any halting/pausing rules, as listed above, occur. They 
will also meet if the SRC decides not to permit progression to a subsequent cohort or requests the 
SMC meet to make that determination. The SMC may also call ad hoc meetings. The SMC 
deliberations may occur by teleconference, videoconference, or email. 
 
 

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Detailed description of the role of each of the levels of safety review can be found in Section 
10.1.6.  
 
1.2 
Schedule of Activities (SoA) 
 
 

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Table 1: Schedule of Activities: 
Color legend: Gray= all cohorts, Orange = only Cohorts 1, 2, and 3 (cohorts receiving single dose of IP); Blue= only Cohorts 4, 
5, 6, 7 (cohorts receiving 2 doses of IP); Green = only Cohort 1 
 
1Screening and enrollment may occur on the same day (Day 1) or up to 28 days apart. Consent is 
obtained either on the screening day (when there is a separate screening day), or on the 
enrollment day (if there is no separate screening day). If there is a separate screening day, then 
all the procedures noted for that day will be performed on the screening day and the following 
will be done on the enrollment day (Day 1): update medical history and update concomitant 
medications. A physical will be performed either on the screening day or the enrollment day but 
is not required on both days. If performed on the screening day, a targeted physical exam may 
occur on the enrollment day, if needed, but is not required. Pregnancy test, final eligibility 
determination, randomization, and administration of study product occur on Day 1.  
2Visits 4A, 4B, and 4C will not be done for Cohorts 1, 2, and 3 because participants in these 
cohorts receive a single dose of vaccine or placebo. Participants in Cohorts 4, 5, 6, and 7 are 
scheduled to receive 2 doses, but if they discontinue participation prior to the second dose, these 
participants will not complete Visits 4A, 4B, and 4C rather will follow the visit schedule of 
Cohort 1,2 and 3. 
3Prior to any study related procedures, families will be provided information about the study and 
informed consent will be obtained from the parent/guardian and assent obtained from the 
participant, when required by the IRB. Note that the screening and enrollment may occur on the 
same day (Day 1) or up to 28 days apart. Consent is obtained either on the screening day (when 
there is a separate screening day), or on the enrollment day (if there is no separate screening 
day).  
4The investigator or appropriately delegated study staff will obtain demographic data. 
Visit number 
01 
11 
2 
3 
4 
4A2 
4B2 
4C2 
5 
6 
7 
Target day 
- 
1 
3 
8 
29 
V4+3d V4+7d V4+28d - 
V5+28d April 
First day of window 
-28 
1 
3 
8 
26 
V4+3d V4+7d V4+25d 09.01 
V5+25d 04.01 
Final day of window 
-1 
1 
4 
11 
32 
V4+4d V4+10d V4+31d 11.30 
V5+31d 04.30 
Visit Type 
Screen Vaccine1Follow Follow Vaccine/ 
Follow 
Follow Follow Follow 
IIV4 
Follow 
Follow 
Visit Venue 
Clinic   Clinic   Phone 
Phone  Clinic   
Phone 
Phone   Clinic   Clinic Clinic  
Phone 
Procedures 
 
 
 
 
 
 
 
 
 
 
 
Informed consent3 
 
 
 
 
 
 
 
 
 
 
 
Collect demographics4 
 
 
 
 
 
 
 
 
 
 
 
Medical history, eligibility5 
 
 
 
 
 
 
 
 
 
 
 
Concomitant med review6 
 
 
 
 
 
 
 
 
 
 
 
Physical exam7 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test8 
 
 
 
 
 
 
 
 
 
 
 
Randomization9 
 
 
 
 
 
 
 
 
 
 
 
Administer study product10 
 
 
 
 
 
 
 
 
 
 
 
Administer IIV411 
 
 
 
 
 
 
 
 
 
 
 
Solicited AE12 
 
 
 
 
 
 
 
 
 
 
 
Non-serious unsolicited AEs13 
 
 
 
 
 
 
 
 
 
 
 
SAEs/AESIs/NOCMCs14 
 
 
 
 
 
 
 
 
 
 
 
Blood for immune response15 
 
 
 
 
 
 
 
 
 
 
 
Nasal wash immune response16 
 
 
 
 
 
 
 
 
 
 
 

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5The investigator or appropriately delegated study staff will obtain medical history and 
information necessary to determine eligibility. Since screening may occur up to 28 days before 
enrollment, if eligibility and medical history are taken at a screening visit, they will be updated 
on the day of enrollment, before randomization. Demography will not be updated. Review of 
eligibility criteria will occur before second dose of the study product. 
6Medications taken by the participant in the 30 days before enrollment will be recorded (while all 
medications taken from 30 days prior to screening, whether on the same day as enrollment or 
not, will be solicited). At subsequent visits, until Visit 4 (Day 29) for Cohorts 1, 2, and 3 and 
until Visit 4C (target Day 57, or 28 days after second dose), for Cohorts 4, 5, 6, and 7, updates to 
previous concomitant medications and additional medications taken in the interim will be 
recorded. No concomitant medications will be recorded after Day 57.  
7 Physical examination will occur for all participants at either Visit 0 or Visit 1. If they receive it 
at Visit 0, they may get a targeted physical exam on Visit 1 if necessary, to determine eligibility. 
Participants may receive an optional targeted physical exam at any other clinic visits at 
investigator discretion.  
8Urine pregnancy test will be performed for females of childbearing potential and recorded 
negative within the 24 hours prior to each study product administration. No pregnancy test will 
be done before the seasonal influenza vaccine administration. 
9Participants will be randomly allocated to receive either vaccine or placebo. For those receiving 
2 doses (Cohorts 4, 5, 6, and 7), they will receive the same product (vaccine) at the specified 
dose or placebo, at both time points.  
10Participants in Cohorts 1, 2, and 3 will be randomly allocated to receive on Day 1 a single dose 
of intranasal 2016 SingM2SR H3N2 vaccine or placebo. Participants in Cohorts 4, 5, 6, and 7 
will be randomly allocated to receive 2 doses of intranasal 2016 SingM2SR H3N2 vaccine or 
placebo. The first dose will be given on Day 1 and the second, when applicable, on 
approximately Day 29. In order to receive the second dose, participants in Cohorts 4, 5, 6, and 7 
must meet the eligibility requirements for subsequent dosing, as described in the protocol.  
11All participants in Cohorts 1, 2, 3, and 4 (non-naives) will be provided a single injection of 
licensed, seasonal influenza vaccine (IIV4) in the period from September to November in the 
same year but at least 28 days after they receive the investigational vaccine or placebo. If vaccine 
is available earlier than September 1, and the participant is at least 28 days since the last 
intranasal vaccine, he/she may receive it earlier than September 1. Participants in Cohorts 5, 6, 
and 7 (naives) will receive 2 doses of IIV4, as currently recommended per standard of care. 
(Should the standard recommendations change, participants will receive the updated standard of 
care.) This vaccine will be given no earlier than 28 days following the final administration of the 
experimental vaccine or placebo. No safety or reactogenicity data related to IIV4 will be 
collected. Participants in Cohorts 1-3 (who receive a single dose of intranasal 2016 SingM2SR 
H3N2 vaccine or placebo) may receive their IIV4 at Visit 4 and participants in Cohorts 4-7 (who 
receive 2 doses of intranasal 2016 SingM2SR H3N2 vaccine or placebo) may receive their IIV4 
at Visit 4C. IIV4 will not be given until after the completion of other study procedures. 
Participants may also receive their COVID-19 study product on the same day as their IIV4.  
12Solicited AEs will first be collected 20 minutes after administration of vaccine or placebo 
(immediate reactogenicity). Additional solicited AEs will be collected by memory aid (eDiary) 
on Days 1-8 for participants in all cohorts and additionally on the 7 days following the second 
vaccination (e.g., Days 29-36 for those receiving the second vaccination on the target Day 29). 
Solicited AEs consist of immediate reactogenicity, local reactogenicity (upper respiratory 

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symptoms as defined in the protocol) and systemic reactogenicity (constitutional signs and 
symptoms as defined in the protocol). Reactogenicity assessments are based on age at 
enrollment. 
13Non-serious unsolicited AEs will be collected and recorded from Day 1 after administration of 
vaccine or placebo for all participants until Day 29 and for an additional 28 days after the second 
vaccination (Day 57 for those who receive the second vaccination on Day 29) for participants in 
Cohorts 4, 5, 6, and 7.  
14SAEs, AESIs, and NOCMCs, as defined in the protocol, will be collected from Day 1, 
following vaccination, until the end of study follow-up, the date in April of the calendar year 
following enrollment at which Visit 7 occurs. The only AESI is wheezing. Participants will be 
evaluated for wheezing episodes in 2 phases. For the day of each vaccination and the next 28 
days, active surveillance will occur, and each episode will require a standardized clinical 
evaluation as defined in Appendix B – Evaluation Criteria for Acute Wheezing. After 28 days 
from the last receipt of investigational product, parents will be instructed to notify the research 
team and recording of the relevant information may be completed by medical record review or 
parental reported outcomes.   
15All participants will have blood drawn for immune responses at 3 times: on Day 1, before 
vaccination, for all participants; on Day 29, for participants in Cohorts 1, 2, and 3; 28 days after 
second vaccination (Day 57 for participants who receive their second vaccination on Day 29), for 
participants in Cohorts 4, 5, 6, and 7; and approximately 28 days after receipt of the licensed, 
seasonal influenza vaccine (IIV4), for all participants. The assays performed and blood volumes 
and tubes required are found in the protocol and MOP. The volumes and number of tubes 
collected are noted in Appendix A – Blood and Fluid Collection Volumes by age group. 
16All participants will have nasal washes collected for mucosal immune responses at 3 times: on 
Day 1, before vaccination, for all participants; on Day 29, for participants in Cohorts 1, 2, and 3; 
approximately 28 days after second vaccination (Day 57 for participants who receive their 
second vaccination on Day 29), for participants in Cohorts 4, 5, 6, and 7; and approximately 28 
days after receipt of the licensed, seasonal influenza vaccine (IIV4), for all participants. The 
assays performed and samples required are found in the protocol and MOP. The volumes and 
number of tubes collected are noted in Appendix A – Blood and Fluid Collection Volumes by 
age group. 
 
 
 
 

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1.3 
Study Schema 
Figure 1– The overall study design is captured in the schema below:  
 
Note: Due to the limited availability of product, and funding to support additional years of 
enrollment into Cohorts 5, 6, and 7, the decision was made to stop enrollment after the final 
participant was enrolled into Cohort 4. 

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2. 
INTRODUCTION 

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2.1 
Study Rationale 
The CDC estimates 7,000 to 26,000 children are hospitalized each influenza season in the US 
due to illness caused by influenza viruses. Deaths associated with influenza in US persons from 
birth to 17 years of age were first reportable in 2004. In the 17 years since then, the frequency of 
reported influenza-associated deaths in US children has ranged from 37, in the 2011-2012 
season, to 196 deaths, in the 2019-2020 season (The final reports for the 2020-2021 season are 
not available at the time the protocol was written, but as of March 18, 2021, there was only 1 
reported influenza-related pediatric death in the US). Because not all influenza-associated deaths 
in children are reported, CDC also uses models to estimate the actual numbers of influenza-
related deaths. They estimate as high as 1200 pediatric deaths per year, using these models [8, 9].  
 
Vaccination remains the best strategy for preventing influenza and its associated morbidity and 
mortality in young children[10]. Currently available inactivated and recombinant influenza 
vaccines primarily aim to induce neutralizing antibodies directed towards the hemagglutinin 
(HA) region of the influenza virus. No cellular immune response or broad cross-reactive 
(heterosubtypic) immunity against divergent strains is induced by current vaccines. Therefore, 
current influenza vaccine seasonal efficacy is strongly dependent on a close match between 
circulating viruses and neutralizing antibodies. This phenomenon was demonstrated by an 
efficacy of just 13% in the 2014-2015 influenza season, a year when the frequency of pediatric 
mortality was estimated to be 600 in the US[11].  
 
FluGen, Inc. has developed a novel M2SR (M2-deficient Single Replication) intranasal live 
attenuated influenza vaccine platform. Unlike the currently licensed live attenuated influenza 
vaccine platform, the M2SR vaccine platform does not express one of the essential viral matrix 
proteins (influenza M2), thus restricting it to a single replication cycle in the host. Pre-clinical 
testing of the M2SR platform-based vaccines has demonstrated broader reactivity against 
multiple influenza subtypes and heterosubtypes. Furthermore, even in the presence of pre-
existing anti-influenza immunity, a systemic, cellular, and mucosal immune response has been 
noted following a single intranasal dose of an M2SR-platfomr vaccine [5, 12]. In a pre-clinical 
proof-of-concept study, a FluGen vaccine, Bris10 M2SR H3N2 vaccine (clade 1), which uses the 
same platform but different source for HA and NA than found in Sing2016 M2SR H3N2 
vaccine, provided protection to ferrets against a drifted H3N2 challenge virus, 
A/Alaska/140/2015, a virus belonging to clade 3c.2a1[5]. 
 
Further, some studies indicate that horizontal transmission of vaccine viruses may, very 
infrequently, occur with the live, attenuated influenza vaccine (LAIV) currently licensed in the 
US (FluMist®). This property has led to a preference for IIV in persons who care for or are close 
contacts with severely immunosuppressed persons who require a protective environment and that 
those who receive LAIV and have such contacts should avoid contact with such persons for 7 
days after receipt of LAIV. Given the M2 protein defect in the Sing2016 M2SR H3N2 vaccine, 
viral progeny are not generated, lending an additional benefit to this approach [4]. This has been 
confirmed in early in vitro and in vivo studies in ferrets and subsequently in healthy adults (total 
of ~280 participants in three studies).[13] Consequently, no special precautions will need to be 
taken in any subsequent trial of an M2SR-based vaccine to exclude participants who have 
household or other close contacts with immunosuppressive conditions. 

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Lastly, an additional potential benefit of vaccines in the M2SR-platform is that they may, due to 
their inability to replicate in humans, lead to a reduced risk of post-vaccination wheezing when 
compared to live attenuated influenza vaccines. If true, the indications for vaccines using this 
platform may potentially allow for children with asthma. 
 
The above noted early successes of the M2SR vaccine platform and potential improvements over 
current influenza vaccines warrant further investigation of Sing2016 M2SR H3N2 vaccine in 
participants 6 months to 17 years of age. 
 
2.2 
Background 
 
2.2.1 
Purpose of Study 
The purpose of this study is to assess the safety, tolerability/reactogenicity, and immunogenicity 
of the FluGen Sing2016 M2SR H3N2 vaccine in children aged 6 months to 17 years. This will 
be done in a Phase 1b, double blinded, age de-escalating, dose-escalating fashion where in each 
cohort, participants will be randomly assigned to receive either vaccine or placebo, as described 
in detail under study design. All participants will be followed for tolerability or reactogenicity, 
safety, and immune responses.  
 
2.3 
Risk/Benefit Assessment 
 
2.3.1 
Known and Unknown Potential Risks 
The potential risks of participation in this trial are those associated with administration of 
Sing2016 M2SR H3N2 vaccine, placebo, and the licensed inactivated influenza vaccine (IIV4), 
having blood drawn, receiving an intramuscular injection or intranasal spray, having a nasal 
wash or swab procedure performed, the potential risks to a fetus of a participant who 
inadvertently becomes pregnant, breach of confidentiality, and unknown risks. 
 
2.3.1.1 
Potential Risks Associated with Receipt of the Vaccine or Placebo 
Sing2016 M2SR H3N2 Vaccine: 
The risks of receiving the intranasal live attenuated influenza vaccines include risks at the site of 
administration (local, in and around the nose) and systemically (other places in the body). 
 
Based on available safety data, the safety profile of Sing2016 M2SR is similar to other LAIV 
vaccines, such as FluMist®. Possible local side effects of the Sing2016 M2SR vaccine include 
edema, dryness, pain/irritation, congestion, rhinorrhea, sneezing, and nasal bleeding. In other 
local tissues, they may be associated with sore throat (pharyngitis), difficulty swallowing, ear 
infections, conjunctivitis, itchy eyes, and sinusitis. Similar to other LAIVs, these effects may be 
more pronounced in seronegative individuals. Systemic side effects may include fatigue and 
headaches and seronegative individuals may also present with low-grade fever. No SAEs have 
been reported previously from administration of the Sing2016 M2SR vaccine. Following 
administration of other LAIVs, pulmonary adverse effects, including wheezing, have been 
reported. Given that Sing2016 M2SR H3N2 influenza vaccine cannot replicate to spread cell to 
cell, adverse events are not expected to be more severe or frequent than replicating LAIV 
vaccine.  

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Horizontal transmission of the live, attenuated replicating influenza vaccine virus found in 
FluMist® may infrequently occur. However, given that M2SR is replication deficient, horizontal 
transmission is not expected and has not occurred to date. In vitro and in vivo studies in ferrets 
and healthy adults have confirmed these predictions, to date. Therefore, no special precautions 
will be needed for administration to participants who may have household members or other 
close contacts with immunosuppressive conditions.  
 
Acute and potentially life-threatening allergic reactions are also possible. Very rarely, occurring 
in about 1 in 4 million people given an injectable vaccination, there can be a serious allergic 
reaction. These reactions can manifest as skin rash (hives), swelling around the mouth, throat or 
eyes (angioedema), difficulty breathing (bronchospasm), a fast pulse (tachycardia), or loss of 
blood pressure (hypotension). If these reactions occur, they can usually be stopped by the 
administration of emergency medications by the study personnel. As with any vaccine or 
medication, there is a very small chance of a fatal reaction (death), although researchers do not 
expect this to occur. Anaphylactic reactions to intranasal vaccine are not likely.  
 
Placebo: 
The placebo is intranasally administered physiological saline, i.e., 0.9% sodium chloride for 
injection, which is given by spray in the nares. Side effects do not typically occur from 
administration of this solution to the nasal passages. If the participant’s nasal passages are dry or 
irritated, it is possible he or she may experience a stinging sensation or have some mild rhinitis. 
It is possible that the administration itself could lead to mild bleeding in those with dry or 
edematous nasal mucosa. 
 
Licensed IIV4: 
Licensed, injectable inactivated seasonal influenza vaccine will be given to all participants for 
whom it is not contraindicated. It is not expected that the risks will be different in frequency or 
magnitude, when compared to those encountered among children receiving IIV4 as part of 
standard care and described below.  
 
Local and Systemic Reactions to IIV4: Recipients of unadjuvanted licensed IIVs may 
develop influenza-like reactions, such as fever, feverishness (chills/shivering/sweating), 
fatigue (tiredness), malaise (general unwell feeling), myalgia (body aches/muscular pain), 
arthralgia (joint pain), headache, and/or nausea. Some persons may develop reactions at 
the injection site, including pruritus (itching), ecchymosis (bruising), erythema (redness), 
induration (hardness)/swelling, pain, and/or tenderness. Most of these reactions peak in 
intensity in the first 24 hours after vaccination and disappear without treatment within 1 
or 2 days. Analgesics (e.g., acetaminophen or ibuprofen or similar non-steroidal anti-
inflammatory drugs [NSAIDs]) and rest may generally relieve or lessen these reactions. 
Bruising can sometimes occur due to the vaccination procedure. 
 
Potentially Immune Mediated Medical Conditions (PIMMCs) Associated with IIV4: In 
post-marketing surveillance, potentially autoimmune, auto-inflammatory, and immune-
mediated diseases have been reported after receipt of seasonal influenza vaccines. These 
disorders include neuritis, convulsions, severe allergic reactions, syncope, encephalitis, 

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thrombocytopenia, and vasculitis. [14] Reports of these reactions are rare and may or 
may not exceed the rates in unvaccinated persons; exact incidence rates cannot be 
precisely calculated. There has been no consistent reporting of individual immune-
mediated diseases or the aggregate of all these diseases in persons receiving influenza 
vaccines or vaccines directed against any pathogen.  
 
Guillain-Barré Syndrome (GBS) Associated with IIV4: During a 1976 vaccine campaign 
directed against swine influenza, which was caused by a type A H1N1 influenza virus, 
some recipients developed a paralytic illness called Guillain-Barré Syndrome (GBS). 
GBS is an acute inflammatory neuropathy characterized by weakness, hyporeflexia or 
areflexia, and elevated protein concentrations in cerebrospinal fluid. The rate of GBS was 
significantly increased in individuals receiving the 1976 swine influenza vaccine with a 
frequency of about 1 per 100,000 vaccine recipients. This syndrome has not been seen 
consistently with other influenza vaccines.  
 
Most persons who develop GBS recover completely. The recovery period may be as little 
as a few weeks or as long as a few years. About 30% of those with GBS still have 
residual weakness after 3 years and about 3% may suffer a relapse of muscle weakness 
and tingling sensations many years after the initial attack.  
 
Intensive surveillance for GBS after administration of inactivated influenza vaccines 
since 1976 has shown a slight increase in risk over background cases (more than one 
additional case of GBS per million persons) following vaccination, typically with onset 
within 6 weeks after vaccination [6]. Interestingly, although vaccination rates have 
increased in the last 10 years, the numbers of reported cases of vaccine associated GBS 
have declined [7]. A recent study in Canada showed that the 2009 H1N1 vaccine was 
associated with a small but significant risk of GBS in persons 50 years and older [3]. An 
active, population-based surveillance study conducted in the United States during the 
2009-2010 influenza season found less than 1 excess GBS case per million doses of 2009 
H1N1 vaccine administered – a rate similar to that associated with some previously 
administered annual influenza vaccines [9-11]. Another study, using the Medicare 
system, showed an elevated risk of GBS with 2009 monovalent H1N1 vaccination 
(incidence rate ratio = 2.41, 95% confidence interval: 1.14, 5.11; attributable risk = 2.84 
per million doses administered, 95% confidence interval: 0.21, 5.48) [12]. An 
international collaborative study also supported a conclusion of an association between 
2009 H1N1 vaccination and GBS [13].  
 
It is unknown if the administration of the Sing2016 M2SR H3N2 vaccine and seasonal 
vaccines to be used in this study will result in an incidence of GBS that exceeds the 
baseline rate. GBS has not been associated with LAIV.  
 
2.3.1.2 
Potential Risks Associated with Drawing Blood or Receipt of an Intramuscular 
Injection or Intranasal Spray 
 
Phlebotomy Risks: 

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Drawing blood is typically well tolerated and uneventful. It often causes transient mild 
discomfort. Approximately 2% of persons who have blood drawn will experience “near 
syncope”, in which they feel lightheaded or have other symptoms, but do not lose consciousness. 
Syncope (fainting) is a transient autonomic (vasovagal) response to phlebotomy that occurs in 
approximately 1% of blood donors and fewer than 1% of those having diagnostic 
phlebotomy.[15] It is managed by having the participant lie down, loosening his or her clothes, 
monitoring vital signs, reassuring the participant, and giving fluids. Minor bruising at the blood 
draw site may occur in up to 12% and a hematoma in 2-3%. These events can be prevented or 
decreased in frequency by employing experienced phlebotomists and can be ameliorated, when 
they occur, by applying pressure to the draw site for several minutes. Rarely, following 
phlebotomy, persons may have nerve injury (typically lateral antebrachial nerve) causing pain, 
paresthesia, and motor or sensory loss. This phenomenon is uncommon and typically resolves 
quickly with watchful waiting only.[16] Infection at the site of the blood draw is exceedingly 
rare and can be avoided by using antiseptics and other appropriate techniques. In a study of over 
4000 persons undergoing routine venipuncture for insurance applications, there were no serious 
local reactions such as cellulitis or phlebitis.[15] 
 
Intramuscular (IM) Injection Risks: 
The injection of a vaccine into the deltoid or anterolateral aspect of the upper thigh muscle, as 
will be done in this study when participants are given IIV4, is typically well tolerated. However, 
it often causes transient discomfort. Intramuscular injections may cause near syncope or syncope, 
although less commonly than phlebotomy. They can also cause an abscess, a hematoma, injury 
to blood vessels and peripheral nerves, or tingling or numbness. Injection too proximally on the 
lateral aspect of the deltoid can lead to injury of the bursa, which can cause Shoulder Injury 
Related to Vaccine Administration, or SIRVA.[17] Proper education and technique reduce the 
risk of adverse events after IM injection. 
 
Intranasal Administration Risk: 
Regardless of the product administered, giving an intranasal spray may lead to sneezing, nasal 
bleeding, dryness, pain/irritation, and edema.  
 
2.3.1.3 
Potential Risks Associated with Nasal Washes and Nasal Swabs 
Obtaining a nasal wash (for collection of specimens for immunologic analysis) or the obtaining a 
nasal swab (for diagnosis of a respiratory pathogen) may cause mild discomfort and on rare 
occasions may cause a nosebleed. Nosebleeds due to a nasal wash are transient and can be 
managed by applying pressure for a short duration of time.  
 
2.3.1.4 
Potential Risks Associated with Pregnancy 
It is unknown if this vaccine poses any risks to a pregnant woman or her unborn child. As such, 
female participants of childbearing potential, as defined in the Eligibility Criteria, must agree to 
use an effective method of birth control for at least 30 days prior to the study vaccination and for 
at least 60 days following the study vaccination. Males who are sexually active with a female of 
childbearing potential must agree not to father a child for 60 days after receipt of the study 
vaccination. Participants in all cohorts except Cohort 1 are too young (under age 9) to be of 
childbearing potential or to father a child. 
 

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2.3.1.5 
Risk of Loss of Confidentiality 
Parents/guardians of participants will be asked to provide the personal/protected health 
information (PHI) of the participating child. All attempts will be made to keep this PHI 
confidential within the limits of the law. However, there is a chance that unauthorized persons 
will gain access to this PHI. All hard copy study records will be kept in a locked file cabinet or 
maintained in a locked room at the participating site. Electronic files will be password protected. 
Only people who are involved in the conduct, oversight, monitoring, or auditing of this trial will 
be allowed access to the PHI that is collected. Any publications from this trial will not use 
information that will identify participants by name. Organizations that may inspect and/or copy 
research records maintained at the participating site for quality assurance and data analysis, 
include groups such as the local Institutional Review Board (IRB), NIAID, and the FDA. 
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by US law. This web site will not include information that can identify participants. At most, this 
web site will include a summary of the results. 
 
2.3.1.6 
Unknown Risks 
There may be other risks, discomforts, or side effects that are unknown at this time. 
 
2.3.2 
Known Potential Benefits 
Participants in this study may have no personal, direct benefits from participation. Even without 
any personal direct benefits, participants may contribute to a societal benefit through the 
improvement of our understanding of these vaccines. For those participants allocated to the 
vaccine groups, they may receive additional protection against influenza, beyond the protection 
afforded by receipt of seasonal influenza vaccine. All participants will be given routine licensed 
influenza vaccine (IIV4) and will be afforded the associated benefits. And each participant will 
have careful follow-up through the respiratory viral season that may provide some additional 
benefit.  
 
2.3.3 
Assessment of Potential Risks and Benefits 
Individual participants take on the minor risks delineated in the protocol, which are necessary to 
provide the scientific information that could allow for use of this vaccine in the future, in 
children. Although data on the safety and immunogenicity of the vaccine in adults is informative, 
it does not substitute for data collected from studies that enroll children. The potential risks are 
minimized by providing study product prior to the availability of seasonal influenza vaccine, 
carefully designing the study with defined safety oversight measures, performing the study at 
sites with experience in studying influenza vaccines, and by having experienced study personnel 
perform study related procedures. The value of the information to be gained outweighs the risks 
of participation.  
 
3. 
OBJECTIVES AND ENDPOINTS 
 
Primary 
1. To assess the safety and tolerability of one or two administrations of the Sing2016 M2SR 
H3N2 influenza vaccine at 108, or 109 TCID50 delivered intranasally to healthy 
participants, 2 to 17 years of age  

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Secondary 
2. To assess the humoral immunogenicity (serum antibody and mucosal antibody responses) 
directed against homologous viral strains after one or two administrations of Sing2016 
M2SR H3N2 influenza vaccine at 108, or 109 TCID50 delivered intranasally to healthy 
participants, 2 to 17 years of age  
 
Exploratory  
3. To assess the cellular immunogenicity (T-cell immune responses) against Sing2016 
M2SR H3N2 influenza vaccine following one or two administrations of Sing2016 M2SR 
H3N2 influenza vaccine at 108, and 109 TCID50 delivered intranasally to healthy 
participants, 2 to 17 years of age 
4. To assess participant immunological response to neurominidase (NA) following one and 
two administrations of Sing2016 M2SR H3N2 influenza vaccine at 108, or 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age 
5. To assess the humoral immunogenicity (serum and/or plasma  antibody) and mucosal 
antibody  responses, and cellular immunogenicity (T-cell responses) after seasonal 
influenza vaccine (IIV4) following one or two administrations of Sing2016 M2SR H3N2 
influenza vaccine at 108, and 109 TCID50 delivered intranasally to healthy participants, 2 
to 17 years of age   
6. To assess mucosal antibody responsesdirected against homologous viral strains after one 
or two administrations of Sing2016 M2SR H3N2 influenza vaccine at 108, or 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age   
7. To assess humoral immunogenicity (serum and/or plasma antibody), and mucosal 
antibody responses) directed against heterologous   viral strains of one or two 
administrations of Sing2016 M2SR H3N2 influenza vaccine at 108 or 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age 
8. To conduct additional characterization of humoral immunity against homologous and/or 
heterologous viral strains (e.g., extra-neutralizing antibody function) following one or 
two administrations of Sing2016 M2SR H3N2 influenza vaccine at 108, and 109 TCID50 
delivered intranasally to healthy participants, 2 to 17 years of age   
9. To assess the effects of age, dose level, sex, prior receipt of seasonal influenza vaccine(s), 
and other variables on humoral immunogenicity (serum antibody and mucosal antibody 
responses) directed against homologous viral strains of one or two administrations of 
Sing2016 M2SR H3N2 influenza vaccine at 108, and 109 TCID50 delivered intranasally to 
healthy participants, 2 to 17 years of age   
 
Study Endpoints 
Primary 
1. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience solicited local reactogenicity events, of all severity grades and by 
grade, in the 7 days following administration of each dose of vaccine 
2. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience solicited systemic reactogenicity events, of all severity grades and by 
grade, in the 7 days following administration of each dose of vaccine  

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3. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience unsolicited non-serious adverse events, of all severity grades and by 
grade, in the 28 days following administration of each dose of vaccine     
4. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience adverse events of special interest (AESIs) the time of first 
vaccination through the end of the study period (final visit in the month of April of the 
calendar year following enrollment)  
5. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience Serious Adverse Events (SAEs) from the time of first vaccination 
through the end of the study period (final visit in the month of April of the calendar year 
following enrollment)  
6. The number and percentage of study participants in Cohorts 1-4 each and across Cohorts 
1-4 who experience new-onset chronic medical conditions (NOCMCs) from the time of 
the first study vaccination through the end of the study period (final visit in the month of 
April of the calendar year following enrollment)  
 
Secondary 
7. The number and percentage of participants in Cohorts 1, 2, and 3 with putative 
seroprotection at baseline (Day 1) and on approximately Day 29, defined as an HAI titer 
≥1:40 in serum against an H3N2 M2SR-like virus  
8. The number and percentage of participants in Cohorts 4 with putative seroprotection at 
baseline (Day 1) and on approximately Day 57, defined as an HAI titer ≥1:40 in serum 
against an H3N2 M2SR-like virus 
9. The number and percentage of participants in Cohorts 1, 2, 3 with neutralization titer 
≥1:40 in serum against an H3N2 M2SR-like virus on approximately Day 29 
10. The number and percentage of participants in Cohorts 4 with neutralization titer ≥1:40 in 
serum against an H3N2 M2SR-like virus at baseline (Day 1) and on approximately Day 
57 
11. Geometric Mean Titers (GMTs) of serum HAI against an H3N2 M2SR-like virus at 
baseline (Day 1) and on approximately Day 29 for participants in Cohorts 1, 2, and 3 
12. Geometric Mean Titers (GMTs) of serum HAI against an H3N2 M2SR-like virus at 
baseline (Day 1) and on approximately Day 57 for participants in Cohorts 4 
13. Geometric Mean Titers (GMTs) of serum neutralizing antibodies against an H3N2 
M2SR-like virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1) and on 
approximately Day 29 
14. Geometric Mean Titers (GMTs) in serum neutralizing antibodies against an H3N2 
M2SR-like virus for participants in Cohorts 4at baseline (Day 1) and on approximately 
Day 57  
15. Geometric Mean Fold Rise (GMFR) and proportions in serum with greater than or equal 
to 2- and 4-fold rises from baseline (Day 1) in HAI titers against an H3N2 M2SR-like 
virus for participants in Cohorts 1, 2, and 3 on approximately Day 29  
16. Geometric Mean Fold Rise (GMFR) and proportions in serum with greater than or equal 
to 2- and 4-fold rises from baseline (Day 1) in HAI titers against an H3N2 M2SR-like 
virus for participants in Cohorts 4 on approximately Day 57 

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17. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in serum neutralization titers against an H3N2 M2SR-
like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29  
18. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in serum neutralization titers against an H3N2 M2SR-
like virus for participants in Cohorts 4 on approximately Day 57  
19. Mean secretory IgA (sIgA) responses, as measured by the binding antibody multiplex 
assay (BAMA) in nasal lavage specimens, against an H3N2 M2SR-like virus for 
participants in Cohorts 1, 2, and 3 before vaccination and on approximately Day 29 
20. Mean secretory IgA (sIgA) responses, as measured by the binding antibody multiplex 
assay (BAMA) in nasal lavage specimens, against an H3N2 M2SR-like virus for 
participants in Cohorts 4 before vaccination and on approximately Day 57  
21. Mean change (difference) from baseline in secretory IgA (sIgA) responses, as measured 
by the binding antibody multiplex assay (BAMA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29  
22. Mean change (difference) from baseline in secretory IgA (sIgA) responses, as measured 
by the binding antibody multiplex assay (BAMA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohorts 4 on approximately Day 57 
 
Exploratory 
23. Antigen-specific T-cell responses to vaccine immunogens, at baseline (Day 1) and 
following one or two doses of vaccine 
24. Humoral antibody, as detected by one or more assays that measure responses to NA, at 
baseline (Day 1) and following one or two doses of vaccine or IIV4 
25. Serum antibody, as detected by HAI and neutralization, directed against homologous 
H3N2 strains  at baseline (Day 1) and after IIV4 administration 
26. Antigen-specific T-cell responses to vaccine immunogens, at baseline (Day 1) and 
measured after IIV4 administration 
27. Serum antibody to additional influenza antigens, as detected by multiplex ELISA assays 
and/or protein microarrays with subsequent evaluation of HAI and neutralization when 
multiplex ELISA screen is positive, directed against heterologous strains at baseline (Day 
1) and following one or two doses of Sing2016 M2SR H3N2 vaccine 
28. Humoral and/or mucosal antibody neutralizing and extra-neutralizing function against 
homologous and heterologous strains based on positive responses detected by methods 
described above.  
29. Associations between serum antibody responses and age, dose level, sex, prior receipt of 
seasonal influenza vaccine(s) and other variables over time. 
30. Associations between mucosal antibody responses and age, dose level, sex, prior receipt 
of seasonal influenza vaccine(s) and other variables over time.   
31. Geometric Mean Titers (GMTs) of plasma HAI against an H3N2 M2SR-like virus at 
baseline (Day 1) and on approximately Day 29 for participants in Cohorts 1, 2, and 3 
32. Geometric Mean Titers (GMTs) of plasma HAI against an H3N2 M2SR-like virus at 
baseline (Day 1) and on approximately Day 57 for participants in Cohort 4 
33. Geometric Mean Titers (GMTs) of plasma neutralizing antibodies against an H3N2 
M2SR-like virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1) and on 
approximately Day 29  

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34. Geometric Mean Titers (GMTs) of plasma neutralizing antibodies against an H3N2 
M2SR-like virus for participants in Cohort 4 at baseline (Day 1) and on approximately 
Day 57 
35. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma HAI titers against an H3N2 M2SR-like virus 
for participants in Cohorts 1, 2, and 3 on approximately Day 29 
36. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma HAI titers against an H3N2 M2SR-like virus 
for participants in Cohort 4 on approximately Day 57 
37. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma neutralization titers against an H3N2 M2SR-
like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29 
38. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma neutralization titers against an H3N2 M2SR-
like virus for participants in Cohort 4 on approximately Day 57  
39. The number and percentage of participants in Cohorts 1, 2, and 3 with putative 
seroprotection at baseline (Day 1) and on approximately Day 29, defined as a plasma 
HAI titer ≥1:40 against an H3N2 M2SR-like virus 
40. The number and percentage of participants in Cohort 4 with putative seroprotection at 
baseline (Day 1) and on approximately Day 57, defined as a plasma HAI titer ≥1:40 
against an H3N2 M2SR-like virus 
41. The number and percentage of participants in Cohorts 1, 2, 3 with plasma neutralization 
titer ≥1:40 against an H3N2 M2SR-like virus at baseline (Day 1) and on approximately 
Day 29 
42. The number and percentage of participants in Cohort 4 with plasma neutralization titer 
≥1:40 against an H3N2 M2SR-like virus at baseline (Day 1) and on approximately Day 
57 
43. Geometric mean titers (GMTs) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1, before 
vaccination) and on approximately Day 29 
44. Geometric mean titers (GMTs) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohort 4 at baseline (Day 1, before 
vaccination) and on approximately Day 57 
45. Geometric mean fold rise (GMFR) and proportions with greater than or equal to 2- and 4-
fold rises from baseline (Day 1) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29 
46. Geometric mean fold rise (GMFR) and proportions with greater than or equal to 2- and 4-
fold rises from baseline (Day 1) in secretory IgA (sIgA) endpoint titers, as measured by 
the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens , against an 
H3N2 M2SR-like virus for participants in Cohort 4 on approximately Day 57 
47. Mean total secretory IgA (sIgA), as measured by the enzyme-linked immunosorbent 
assay (ELISA) in nasal lavage specimens , against an H3N2 M2SR-like virus for 

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participants in Cohorts 1, 2, and 3 at baseline (Day 1, before vaccination) and on 
approximately Day 29 
48. Mean total secretory IgA (sIgA), as measured by the enzyme-linked immunosorbent 
assay (ELISA) in nasal lavage specimens, against an H3N2 M2SR-like virus for 
participants in Cohort 4 at baseline (Day 1, before vaccination) and on approximately 
Day 57 
49. Mean change (difference) from baseline in total secretory IgA (sIgA), as measured by the 
enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 29 
50. Mean change (difference) from baseline in total secretory IgA (sIgA), as measured by the 
enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against an 
H3N2 M2SR-like virus for participants in Cohort 4 on approximately Day 57 
51. Mean normalized secretory IgA (sIgA), as measured by the enzyme-linked 
immunosorbent assay (ELISA) in nasal lavage specimens , against an H3N2 M2SR-like 
virus for participants in Cohorts 1, 2, and 3 at baseline (Day 1, before vaccination) and on 
approximately Day 29 
52. Mean normalized secretory IgA (sIgA), as measured by the enzyme-linked 
immunosorbent assay (ELISA) in nasal lavage specimens , against an H3N2 M2SR-like 
virus for participants in Cohort 4 at baseline (Day 1, before vaccination) and on 
approximately Day 57 
53. Mean change (difference) from baseline in normalized secretory IgA (sIgA), as measured 
by the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against 
an H3N2 M2SR-like virus for participants in Cohorts 1, 2, and 3 on approximately Day 
29 
54. Mean change (difference) from baseline in normalized secretory IgA (sIgA), as measured 
by the enzyme-linked immunosorbent assay (ELISA) in nasal lavage specimens, against 
an H3N2 M2SR-like virus for participants in Cohort 4 on approximately Day 57 
55. Geometric Mean Titers (GMTs) of plasma or serum NA antibodies against an H3N2 NA 
for participants in Cohorts 1, 2, and 3 at baseline (Day 1) and on approximately Day 29 
56. Geometric Mean Titers (GMTs) of plasma or serum NA antibodies against an H3N2 NA 
for participants in Cohort 4 at baseline (Day 1) and on approximately Day 57 
57. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma or serum NA antibodies against an H3N2 
NA for participants in Cohorts 1, 2, and 3 on approximately Day 29 
58. Geometric Mean Fold Rise (GMFR) and proportions with greater than or equal to 2- and 
4-fold rises from baseline (Day 1) in plasma or serum NA antibodies against an H3N2 
NA for participants in Cohort 4 on approximately Day 57 
59. The number and percentage of participants in Cohorts 1, 2, 3 with NA antibody titer 
≥1:40 against an H3N2 NA at baseline (Day 1) and on approximately Day 29 
60. The number and percentage of participants in Cohort 4 with NA antibody titer  ≥1:40 
against an H3N2 NA-like virus at baseline (Day 1) and on approximately Day 57 
 
 
4. 
STUDY DESIGN 
 

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4.1 
Overall Design 
This is a Phase 1b, randomized, double-blind, dose-escalating, age de-escalating, placebo-
controlled study of 200 children 6 months through 17 years of age, inclusive, who are in good 
health and meet all eligibility criteria. To allow for simultaneous enrollment at the four research 
sites, the number of children in each cohort and the total number of children in the entire study 
may slightly deviate from the targeted numbers mentioned throughout the protocol. 
 
The study design includes 7 cohorts in Protocol Versions 1-4. Given a number of non-safety-
related contingencies (timelines, budgets, recruiting, and lack of additional vaccine lots),  
Cohorts 5, 6, and 7 will no longer be enrolled as of Protocol Version 5.0. Cohorts 5, 6, and 7 
included children 6 to 23 months old. The study-related objectives and endpoints have been 
updated to reflect that only Cohorts 1 through 4, were enrolled.   Descriptions of Cohorts 5, 6, 
and 7 have not been removed from the remainder of the protocol design. 
All participants in Cohorts 1, 2, 3, and 4, will be “non-naïve”, having been vaccinated against 
seasonal influenza in a prior season. Cohorts 5, 6, and 7 will be 6 to 23 month-olds who are 
influenza-naïve (no previous influenza vaccine and no previous documented influenza infection). 
 
A cohort of 45 healthy children 9-17 years old (Cohort 1) will receive a single dose of vaccine at 
the 109 TCID50 dose of the Sing2016 M2SR H3N2 vaccine or placebo. At the same time, the 
second cohort (Cohort 2) will begin enrollment.  
 
Cohort 2 will consist of 45 healthy children 2-8 years old who will also receive a single dose of 
the vaccine, but they will be given at 108 TCID50 or placebo. Once there is sufficient evidence of 
safety and tolerability in Cohorts 1 and 2 and enrollment has completed of all 45 in each of these 
cohorts, the third cohort (Cohort 3) will begin enrollment.  
 
Cohort 3 will consist of 25 healthy children 2-8 years old who will receive a single dose of the 
109 TCID50 dose of vaccine or placebo. Once there is sufficient evidence of safety and 
tolerability in Cohort 3 and enrollment has completed for this cohort, the fourth and fifth cohorts 
(Cohorts 4 and 5) will begin enrollment.  
 
Cohort 4 will consist of 25 healthy children 2-8 years old who will receive 2 doses of the 109 
TCID50 dose of vaccine or placebo. Participants in Cohort 4 will enroll concurrently with 
participants in Cohort 5. 
 
Cohort 5 (termed lead-ins) will consist of 8 children, 6-23 months old, who will be randomized 
to receive two doses of 107 TCID50 Sing2016 M2SR (n=6) or two doses of placebo (n=2).  
 
Once there is sufficient evidence of safety and tolerability in Cohort 5 and enrollment has 
completed for Cohort 5, Cohort 6 will open for enrollment. Once 6 of the 8 participants in 
Cohort 5 have completed Day 4 after the second dose (e.g., Study Day 32), the following process 
will be implemented prior to receipt of the second dose  for any participant in Cohort 6: 
 
1. An email will be sent from the site to the SDMCC attesting that all data entry through 
Day 4 after the second dose (e.g., Study Day 32) for at least 6 participants in Cohort 5 has 
been completed. 

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2. The SDMCC will perform data cleaning as it pertains to assessment of the halting rules. 
3. The SDMCC will assess if there were any halting rules met based on data entered in the 
data system and email DMID indicating what halting rules, if any, were met based on 
data entered in the data system. 
a. If any of the halting rules were met, the study will halt and a review by the SMC, 
sponsor, and investigators will convene as specified in Section 7.1.  
b. If none of the halting rules were met, DMID will confirm that the study may 
proceed as per-protocol.   
 
Cohorts 6 will consist of a lead-in group of 8 children, 6-23 months old followed by an 
additional 18. The first 8 will be randomly assigned to receive either two doses of 108 TCID50 of 
the Sing2016 M2SR (n=6) or two doses of placebo (n=2). Once the first 8 children have 
completed Day 8, the SRC will review the safety and determine if Cohort 7 may begin 
enrollment. Enrollment of the remaining 18 Cohort 6 participants may continue during SRC 
review and must be completed before enrollment into Cohort 7 can begin. 
 
Cohort 7 will consist of 26 healthy children 6 to 23 months old, 18 of whom will be randomly 
assigned to receive 2 doses of the 109 TCID50 dose of vaccine and 8 of whom will be randomly 
assigned to receive placebo.  
 
This study is designed to assess the safety and immunogenicity of one or two doses of the 
Sing2016 M2SR H3N2 influenza vaccine delivered intranasally. Eligible participants will be 
enrolled and randomly assigned to receive the Sing2016 M2SR H3N2 vaccine (one or two doses) 
or placebo. An overview of the planned cohorts is presented in Table 2. All participants will be 
administered the IIV4 seasonal influenza vaccine a minimum of 28 days after last IP dose or later 
once it is available (anticipated September – November). IIV4 may be administered outside of 
the study window described in the protocol, in case of public health emergency or as determined 
necessary by institutional policy, regulatory authorities, sponsor or principal investigator. 
Management of participants who meet this criterion will be as determined suitable by site-
specific procedures. COVID-19 vaccines which are fully approved or approved under EUA may 
be received during study participation. COVID-19 vaccines may not be received within the 10 
days before receipt of the study product nor planned to be received during a reactogenicity 
period (the day of and 7 days following each intranasal vaccination) of the study product. 
Participants who experience a COVID-19 exposure during study participation will be managed 
as per institutional protocol. 
 
Due to the limited availability of product, and funding to support additional years of enrollment 
into Cohorts 5, 6, and 7, the decision was made to stop enrollment after the final participant was 
enrolled into Cohort 4. 
 
Table 2. Cohorts  
Cohort Age  
 Experience 
Vaccine 
Dose 
Doses 
Participants 
1 
9-17 yrs. 
Non-naive 
Sing2016 
M2SR H3N2  
109 TCID50 
1 
30 
Placebo 
- 
1 
15 

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2 
2-8 yrs. 
Non-naive 
Sing2016 
M2SR H3N2  
108 TCID50 
1 
30 
Placebo  
- 
1 
15 
3 
2-8 yrs. 
Non-naive 
Sing2016 
M2SR H3N2  
109 TCID50 
1 
15 
Placebo  
- 
1 
10 
4 
2-8 yrs. 
Non-naive 
Sing2016 
M2SR H3N2  
109 TCID50 
2 
15 
Placebo  
- 
2 
10 
5 
6-23 mo. 
Naïve 
Sing2016 
M2SR H3N2  
107 TCID50 
2 
6 
 
 
Placebo  
 
2 
2 
6 
6-23 mo. 
Naïve 
Sing2016 
M2SR H3N2  
108 TCID50 
2 
18 
 
 
 
Placebo  
- 
2 
8  
 
7 
6-23 mo. 
Naïve 
Sing2016 
M2SR H3N2  
109 TCID50 
2 
18 
 
 
 
Placebo  
- 
2 
8 
 
Total Sample size  
200 
 
Schedule of assessments can be found in Section 1.2, Schedule of Activities. 
Protocol Schema can be found in Section 1.3, Protocol Schema. 
Dose escalation or dose-ranging details are found in Section 6.1.2, Dosing and Administration 
Full details of interim analysis are found in Section 9.4.6, Planned Interim Analyses 
 
4.2 
Scientific Rationale for Study Design 
Currently available inactivated and recombinant influenza vaccines induce neutralizing 
antibodies that recognize the virus envelope protein HA. These antibodies effectively induce 
immunity, but the efficacy of these vaccines depends on a close match between the vaccine 
immunogen and circulating viruses. Such vaccines are therefore relatively ineffective against 
newly emerging viruses or viruses that have drifted away from the original vaccine viral strain. 
About 90% of vaccines of the currently available inactivated vaccines have limited or no broadly 
cross-reactive (heterosubtypic) immunity to protect against divergent or drifted strains of the 
virus. Currently available trivalent (IIV3) or quadrivalent (IIV4) preparations provide the HA 
antigen and either none or limited and non-standardized amounts of the NA antigen. Moreover, 
at least one HA component of these vaccines must often be updated each influenza season to 
account for accumulated point mutations in the HA protein that allow the viruses to evade 
circulating human immune response. In addition, while a strong relationship has been established 
between pre-existing T-cell immunity and illness severity in persons seronegative for influenza 
virus-specific antibody, no or minimal cellular immune responses have been shown to be elicited 
following vaccination with these conventional preparations [3].  
 

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Although live influenza virus vaccines may offer immunologically superior responses because 
they induce diverse types of adaptive responses, only one such product, FluMist®, has been 
approved in the US, and is presently indicated only for certain persons 2-49 years of age [18]. 
Accumulating data with FluMist® also indicate that pre-existing, cross-reactive immunity, 
present in most adults, limits vaccine virus replication, which in turn mitigates a consistently 
effective immune response. 
 
In an effort to overcome the limitations of currently available influenza vaccines, FluGen, Inc. is 
developing a novel live influenza virus vaccine platform known as “M2SR”. This platform is 
based on a replication-defective recombinant influenza virus that does not express M2 protein 
(and hence, the derivation of the “M2SR” naming convention [M2 deficient Single Replication 
vaccine]). The M2SR vaccine virus is comprised of the following: (1) 5 of the 8 gene segments 
of the donor virus, Influenza A/Puerto Rico/8/34 (PR8), an attenuated strain that has been used 
for decades in traditional inactivated influenza vaccine manufacturing; (2) two genetic segments 
for influenza virus surface protein antigens, HA and NA, derived from any selected type A 
influenza strain; and (3) an M gene segment that cannot express the M2 protein. In the vaccine 
virus, the M2 protein is acquired from the cell substrate used to grow the vaccine, M2 Vero cells, 
which stably expresses and supplies M2 protein for vaccine virus growth. The resulting vaccine 
virus can infect normal cells in the respiratory epithelium of the vaccine recipient and then 
uncoat and initiate infection in a manner similar to a wild-type influenza virus, thereby evoking 
an immune response. However, because the M2SR genome does not encode for the M2 protein, 
no viral progeny are produced after the initial (single-round) infection, such that no cell-to-cell 
spread, or subsequent shedding of virus occurs.  
 
The HA and NA of an influenza A/Brisbane/10/2007-like H3N2 virus were chosen for inclusion 
in the prototype monovalent vaccine that was initially tested in humans, and this prototype 
vaccine is referred to here as “Bris10 M2SR H3N2 vaccine”. A second investigational (updated) 
H3N2 M2SR vaccine encodes the HA and NA of the A/Singapore/INFIMH-16-0019/2016 
reference virus, the H3N2 strain that was recommended for the 2018-2019 influenza season and 
is referred to here as “Sing2016 M2SR H3N2 vaccine”. Sing2016 M2SR H3N2 vaccine contains 
a similar PR8 backbone to Bris10 M2SR H3N2 vaccine but with nine amino acid changes 
distributed over five genes (PB1, PB2, PA, NP, and NS1) that facilitate growth in the production 
cell line, M2VeroA. The mechanism of attenuation (deletion of the M2 gene) is not affected. 
This updated vaccine of the M2SR platform was generated in a similar manner as Bris10 M2SR 
H3N2 vaccine. Sing2016 M2SR H3N2 vaccine is the vaccine that will be evaluated in this study.  
 
4.3 
Justification for Dose 
Previous clinical experience with the M2SR vaccine platform includes a First Time in Human 
(FTIH) Phase 1 dose escalation study in healthy adults, a Phase 2a challenge study in healthy 
adults, an on-going Phase 1b dose escalation study in healthy adults, and an on-going Phase 1 
safety and immunogenicity study in 9-17 years, sponsored by NIAID/DMID (DMID 17-0012, 
NCT03553940 – preliminary results posted). A total of 410 adults and 43 children, respectively, 
have been enrolled in these studies to date.  
 
In the FTIH study, 72 adults (24 participants per dose level) received a single dose of Bris10 
M2SR H3N2 vaccine at levels of 106, 107, or 108 TCID50. In the challenge study, 52 adults 

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received a single dose of Bris10 M2SR H3N2 vaccine, with 48 subsequently challenged with a 
drifted H3N2 strain. The M2SR influenza vaccines evaluated to date have not displayed any 
evidence of shedding in human participants (n=285) evaluated in the FTIH, Phase 2a and Phase 
1b studies. These results align with the biology of the M2SR virus; that is, the replication-
deficient phenotype is intrinsic to the virus (not based on host response) due to the deletion of the 
essential M2 gene. 
 
In a Phase 1 study of 9-17 year-olds, 43 participants (out of 50 planned) were enrolled and 
received one 108 TCID50 dose of Bris10 M2SR H3N2 vaccine or placebo, followed by one dose 
of licensed quadrivalent influenza vaccine (IIV4) administered 3 months later; data from this 
study remain blinded as of March 2021. Although the allocation to vaccine or placebo is not yet 
unblinded, among participants who received one dose of 108 TCID50 M2SR vaccine, there have 
been no reported SAEs and no halting rules met. 
 
For the Phase 1b dose escalation study, 206 healthy male and non-pregnant female participants 
ages 18-49 years old, having microneutralization (MN) titers of ≤ 20 to H3N2 
A/Singapore/INFIMH-16-0019/2016, were randomized 1:1:1:1:1 to receive two administrations 
28 days apart of Sing2016 M2SR H3N2 vaccine at three dose levels, Bris10 M2SR H3N2 
vaccine at one dose level or placebo. Treatment groups included Sing2016 M2SR H3N2 vaccine 
at 108 TCID50 (n=38), 108.5 TCID50 (n=35), and 109 TCID50 (n=30); Bris10 M2SR H3N2 vaccine 
at 108 TCID50 (n=39); and placebo (n=35). The first participant first dose was in September 2019 
and the last participant completed the Day 209 follow-up visit (180 days following the second 
dose) in June 2020. The active phase of this Phase 1b dose escalation study was completed with 
no SAEs or observations that would halt the study. 
  
Participants who received Bris10 M2SR H3N2 vaccine (108 TCID50 dose) in the Phase 1a and 
Phase 2a studies demonstrated broad-spectrum immune responses. In the FTIH study, vaccine-
induced serum and mucosal antibody responses were cross-reactive against multiple 
antigenically different H3N2 viruses including recent strains that belong to the same clade 
(3c.3b) as the challenge virus used in the Phase 2a study. In the Phase 2a study, data demonstrate 
that the vaccine was effective in prevention of infection after challenge with an antigenically 
drifted virus, but mainly among participants for whom a humoral immune response to the 
vaccine was detected. Participants who demonstrated a 2-fold increase from baseline in serum 
antibody response (~50% of the M2SR vaccine cohort) by MN to the intranasal M2SR vaccine 
had lower viral load and influenza-like symptoms than placebo recipients. These preliminary 
results suggest that M2SR could potentially provide higher efficacy levels if a greater proportion 
of M2SR vaccine recipients can demonstrate an appropriate serological response.   
 
In the subsequent Phase 1b study, a single 109 TCID50 dose of Sing2016 M2SR H3N2 vaccine 
generated significantly increased serum HAI responses compared to the 108 TCID50 of Bris10 
M2SR H3N2 vaccine that had provided protection against infection and illness in the earlier 
human influenza challenge study.  HAI titers ≥40 were achieved in 0%, 23% and 58% of 
participants after the first dose of placebo, 108, or 109 TCID50 M2SR vaccine, respectively 
(p<0.003).  Increases also were stimulated in serum MN titers to drifted strains of H3N2 and in 
serum NA inhibition (NAI) and mucosal sIgA titers. Further increases in serum and mucosal 
immune response were noted after a second IN vaccination.  

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The purpose of this proposed Phase 1b dose-finding clinical study is to assess the safety, 
tolerability/reactogenicity, and immunogenicity of the Sing2016 M2SR H3N2 vaccine, including 
doses from 108 TCID50 to 109 TCID50, in a pediatric population. The same Sing2016 M2SR 
H3N2 vaccine which will be used in this study is currently being investigated in adults, as 
described above. Demonstration of broadly reactive responses in adolescents and children, like 
those found to be protective against challenge in adults, would support further clinical 
development of the M2SR vaccine platform in this age group. 
 
5. 
STUDY POPULATION 
 
5.1 
Inclusion Criteria 
1. Participant is a male or female child aged 6 months to 17 years inclusive at time of 
enrollment (each cohort has its own age upper and lower limits2) 
1Cohort 1: 9-17 years (on or after the 9th birthday and before the 18th birthday at the time of the first dose); 
Cohorts 2, 3, and 4: 2-8 years (on or after the 2nd birthday and before the 9th birthday at the time of the first 
dose); Cohorts 5, 6, and 7: 6 months to 23 months (on or after the 6th month of life based on calendar day 
and before the second birthday at the time of the first dose)  
2. For Cohorts 1 to 4, receipt of at least 2 doses of seasonal influenza vaccine in the past.  
3. For Cohorts 5 to 7, receipt of no seasonal influenza vaccines in the past and no documented 
history of laboratory-confirmed influenza illness 
4. Parent/guardian of the participating child provides written informed permission and 
participating child provides assent2 prior to initiation of any study procedures   
2as appropriate by age or development and approved by the IRB 
5. Parent/guardian and participant, as appropriate, are able to understand and comply with 
planned study procedures and are available for all study visits 
6. Participant is in good health as assessed by the principal investigator or other designated 
study investigator3  
3based on medical history and physical examination (physical examination may be done as part of routine 
medical care or specifically for eligibility screening) 
7. Parent/guardian of the participating child agrees not to allow the participant to join another 
clinical trial that includes an investigational agent or device during the study period   
8. A female participant of child-bearing potential4 agrees to abstain from sexual intercourse or 
to correctly use an acceptable method of contraception5  
4A female of child-bearing potential is defined as a female who is post-menarchal and not sterilized via 
tubal ligation, bilateral oophorectomy, salpingectomy, hysterectomy, or successful Essure® placement 
(permanent, non-surgical, non-hormonal sterilization) with documented radiological confirmation test at 
least 90 days after the procedure. This applies only to participants in Cohort 1.  
5Acceptable methods of contraception must be used from 30 days prior to vaccination until 60 days after 
the last study vaccination (not IIV4) and include full abstinence from sexual intercourse with a male 
partner, monogamous relationship with vasectomized partner who has been vasectomized for 180 days or 
more or shown to be azoospermic prior to the participant receiving the study vaccination, barrier methods 
such as condoms or diaphragms/cervical cap, intrauterine devices, NuvaRing®, and licensed hormonal 
methods such as implants, injectables, or oral contraceptives (“the pill”).  
 
 
 
 

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9. A female participant of child-bearing potential4 must have a negative urine pregnancy test 
within 24 hours prior to each study product 
10. A male who is sexually active with a female of childbearing potential4 must agree to use an 
acceptable method of contraception6  
6From the time of the first dose of study vaccine until 60 days after receipt of the last dose study vaccine, 
only in Cohort 1. The only acceptable method of contraception for males who are sexually active with 
females of childbearing potential is condoms.  
 
5.2 
Exclusion Criteria 
1. Has a body temperature of 38.0°C/100.4°F (oral or axillary) or greater 
or another acute illness7 within the 72 hours prior to study vaccination   
7Potential participants who are recovering from an acute illness and have residual minimal symptoms, 
which, in the opinion of the site principal investigator or appropriate sub-investigator, will not likely 
affect the evaluation of outcome measures are not ineligible. Temperature evaluation will not be 
performed as a study procedure on participants prior to administration of seasonal influenza vaccine  
2. Has any medical or mental health disease or condition8 that would render study participation 
unsafe, or would interfere with the evaluation of the responses 
8in the opinion of the site principal investigator or appropriate sub-investigator 
3. Has a history of provider-diagnosed asthma requiring the use of medications at any age, or 
has had a wheezing episode or use of medications to treat asthma in the 12 months prior to 
screening. 
4. Has immunosuppression as a result of an underlying illness or treatment, a recent history 
or current use of immunosuppressive or immunomodulating disease therapy  
5. Has a diagnosis of or history of malignant neoplastic disease   
6. Has taken oral, parenteral (intramuscular or intravenous), inhaled, or nasal corticosteroids of 
any dose within 30 days prior to study vaccination   
7. Has known HIV, hepatitis B, or hepatitis C infection  
8. Has known hypersensitivity or allergy to any components of the study vaccine or material in 
the nasal delivery device9  
9Vaccine components: sucrose, sodium chloride, phosphate, glutamate; delivery device material: 
polycarbonate, polypropylene, synthetic rubber  
9. Has a history of severe reactions following previous immunization with licensed or 
unlicensed influenza vaccines   
10. Has a history of an anatomic disorder of the nares or nasopharynx   
11. Has a history of chronic sinus infections  
12. Has a history of or currently smokes or vapes  
13. Has a history of Guillain-Barré syndrome   
14. Use of aspirin- or salicylate-containing products in the 30 days prior to or intends to use these 
products in the 30 days following administration of the investigational vaccine  
15. Has a history of documented influenza or receipt of influenza antiviral treatment in the 4 
months prior to the first vaccination 
16. Receipt of any antiviral drug within the week prior to or following the investigational 
vaccine. 
17. Receipt of a licensed live vaccine within 30 days prior to the first study vaccination or plans 
to receive a licensed live vaccine within the 30 days after the last study vaccination.   
18. Receipt of licensed inactivated non-influenza vaccine10 within 14 days prior to the 1st study 
vaccination, or plans to receive licensed, inactivated vaccine within the 30 days after the 
last study vaccination 

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10 Participants will be asked to avoid receipt of any routine licensed vaccines or vaccines under emergency 
use authorization during the periods described.  
19. Receipt of an influenza vaccine within the 4 months prior to the first study vaccination or 
plans to receive an influenza vaccine following the last study vaccination. Seasonal IIV4 will 
be received by participants as part of this trial.   
20. Receipt of immunoglobulin or other blood products within the 6 months prior to the first 
study vaccination or plans to receive during the period of study participation. 
21. Receipt of an experimental11 agent or device within the 6 months prior to the first 
study vaccination or expects to receive an experimental agent or device during the study 
period   
11Products for treatment or prevention of COVID-19, when received under Emergency Use Authorization 
[EUA] or full FDA approval and not as part of a clinical trial, will not be deemed “experimental” for the 
purposes of this criterion and will not make an otherwise eligible prospective participant ineligible.  
22. Is a family member of study personnel or personnel directly involved in the conduct or 
monitoring of the study 
23. Receipt of an approved or experimental product for treatment or prevention of COVID-19 
within the 10 days12,13 prior to study enrollment 
12Participants may enroll if greater than 10 days after receipt of the COVID-19 treatment or prevention. 
13Participants who are receiving COVID-19 vaccines around the time of dosing of the investigational 
product will be asked to avoid COVID-19 vaccination within the 10 days before any vaccination in the 
study and within any reactogenicity period (the day of and 7 days following each intranasal vaccination).  
24. Inability of the study team to collect 5 mL of blood from the participant before the first 
vaccination (pre-vaccination blood).  
 
5.2.1 
Exclusion of Specific Populations 
This study is evaluating the immunogenicity of the Sing2016 M2SR H3N2 vaccine in pediatric 
participants. This evaluation is limited to children and hence the study will exclude adult and 
geriatric populations.  
 
5.3 
Inclusion of Vulnerable Participants 
This study will provide clinical research data to inform influenza vaccine safety and 
immunogenicity in children. The children who take part in this study are considered vulnerable 
participants per the US Code of Federal Regulations, and site IRBs/IBCs will consider the 
potential risks and benefits to child participants as described in 45 CFR 46 and 21 CFR 50 
Subparts D (for children).  
 
With respect to these regulations, IRBs/IBCs will determine the level of risk to children in the 
categories specified. Documentation of this determination is required, and the risk category 
assigned by the IRB/IBC further determines the parental informed consent requirements for the 
study at each site.  
 
5.4 
Lifestyle Considerations 
No lifestyle restrictions will be expected of participants.  
 
5.5 
Screen Failures 
After the screening evaluations have been completed, the investigator or designee will review the 
inclusion/exclusion criteria and determine the participant’s eligibility for study enrollment and 

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randomization. Screen failures will be recorded on the screen failure log together with the reason 
for failure.  
 
5.6 
Strategies for Recruitment and Retention 
 
5.6.1 
Recruitment 
Sites may use a variety of recruitment approaches, that may include but are not limited to direct 
recruitment at clinics, referrals from other providers, registries, electronic health record (EHR) 
messages, and use of online and social networking websites and applications. Recruitment 
materials will educate parents/guardians and, where age appropriate, participants, on the details 
of the clinical trial and the previously known safety information.  
 
5.6.2 
Retention 
Once a child has received study product, study staff will make every effort to retain him or her in 
follow-up for the protocol-specified duration of follow-up, i.e., through the influenza season as 
defined in the protocol, thereby minimizing potential biases associated with loss to follow-up. 
 
5.6.3 
Compensation Plan for Participants  
Compensation will be provided to the participant’s parent/guardian based on each site’s standard. 
The amount and means of disbursing will be reviewed and approved by each site's IRB, or the 
central IRB, if applicable.  
 
5.6.4 
Costs 
There are no costs for the research tests, procedures, and study product while taking part in this 
trial. The investigational product and the seasonal influenza vaccine will be provided free of 
charge. Procedures and treatment for clinical care may be billed to the participant, participant’s 
insurance, or third party.   
 
6. 
STUDY PRODUCT 
 
6.1 
Study Product(s) and Administration 
 
6.1.1 
Study Product Description 
 
6.1.1.1 
Product 1: Sing2016 M2SR H3N2 Intranasal Influenza Vaccine  
The Sing2016 M2SR vaccine is provided as a frozen liquid formulation in 2 mL cryovials.  Each 
vial contains ~0.6 mL of vaccine formulated to contain infectious viral particles of Sing2016 
M2SR encoding the HA and NA of influenza virus strain A/Singapore/INFIMH-16-0019/2016 
(H3N2). The virus is suspended in Sucrose Phosphate Glutamate with Sodium Chloride (SPG-
NaCl) buffer comprised of 10% sucrose, 5 mM glutamic acid, 136.9 mM sodium chloride, 2.67 
mM potassium chloride, 1.47 mM potassium dihydride phosphate and 8.1 mM disodium 
phosphate, at pH 7.2. The Sing2016 M2SR vaccine is clear to opalescent, colorless to slightly 
yellow suspension.  
 

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6.1.1.2 
Product 2: SPG buffer to be used as the diluent for Product 1 
The vaccine diluent, SPG-NaCl buffer (10% sucrose, 5 mM glutamic acid, 136.9 mM sodium 
chloride, 2.67 mM potassium chloride, 1.47 mM potassium dihydride phosphate and 8.1 mM 
disodium phosphate, at pH 7.2), is provided as a frozen, liquid formulation. It is a clear, colorless 
solution that is used to prepare dilutions of the investigational vaccine for the lower dose 
treatments.  
6.1.1.3 
Product 3: Placebo 
The placebo consists of a commercially prepared 0.9% sodium chloride for injection, USP.  
6.1.1.4 
Product 4: Seasonal Inactivated Influenza Vaccine (IIV4) 
All participants without contraindications will be given injectable IIV4 using a licensed product. 
All available IIV products licensed for children in the US are now quadrivalent.  
 
6.1.1.5 
Product 5: MAD300™ Sprayer Device 
The intranasal delivery device, i.e., nasal spray device, is comprised of a MAD300™ mucosal 
atomization device and a 1 mL Henke-Ject (low dead space) syringe. The MAD300™ is made 
from radiation-stable medical-grade polycarbonate material and is compliant with USP Class VI 
and ISO 10993 requirements. It is manufactured by Teleflex Medical and is a conical shaped 
component with a Luer-lock feature for attachment to the filled syringe. For intranasal 
administration of the Sing2016 M2SR vaccine and placebo, the MAD300™ conical shape forms 
a plug in the nostril and rapid depression of the syringe plunger atomizes the liquid as it passes 
through the device nozzle into a fine mist.  
 
6.1.2 
Dosing and Administration 
Participants will receive one of the following: the 109 TCID50 dose or the 108 TCID50 dose or the 
lowest dose of 107 TCID50, of the Sing2016 M2SR H3N2 vaccine or placebo. Due to the limited 
availability of product and funding to support additional years of enrollment into Cohorts 5, 6, 
and 7, the decision was made to stop enrollment after the final participant was enrolled into 
Cohort 4. This means no participant will receive the 107 TCID50  dose. 
 
The Sing2016 M2SR H3N2 vaccine will be provided to the site as a frozen, liquid formulation 
filled at a volume of 0.5 mL in 2.0 mL cryovials that are stored at ≤ -65°C. It is a clear to 
opalescent, colorless to yellow solution. Once thawed for use, the M2SR must not be refrozen for 
later use. 
 
A pharmacist or his/her designee will thaw the vial contents to room temperature soon before 
dose administration, as described in the Manual of Procedures (MOP). The contents will be 
diluted to the target dosing concentration with provided Sucrose-Phosphate-Glutamate-NaCl 
(SPG-NaCl) diluent for each participant. The SPG-NaCl buffer is provided as a frozen, liquid 
formulation and in single use vials that are stored at ≤ -65°C. It is a clear, colorless solution. 
Once thawed for use, the SPG-NaCl must not be refrozen for later use. 
 
The final diluted product will be drawn into two 1 mL disposable polypropylene syringes 
(Henke-Ject) and then each syringe will be fitted with a MAD300™ nasal device (MAD300; 
Teleflex, Morrisville, NC, USA) for intranasal delivery, using one intranasal delivery device (a 
syringe fitted with a nasal device) per naris. 
 

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Detailed instructions on preparation, concealing the syringe, timing from thaw to preparation to 
administration, and further study product details are found in the MOP. 
  
Placebo control will be given as the comparator to the Sing2016 M2SR H3N2 vaccine. The 
saline solution placebo will be administered on the same schedule as the Sing2016 M2SR H3N2 
vaccine product (Table 3) and will also be administered using the MAD300™ nasal device.  
 
Seasonal IIV4 is given intramuscularly at the dose and volume recommended by the 
manufacturer for the age of the participant. 
 
Table 3: Age and Dose Cohorts 
Cohort 
Age  
Experience 
Product Dose & Name 
Route 
Frequency 
1 
9-17y 
Non-naive 
109 TCID50 Sing2016 M2SR 
H3N2 vaccine or placebo 
IN 
Single dose, D1 
2 
2-8y 
Non-naive 
108 TCID50 Sing2016 M2SR 
H3N2 vaccine or placebo 
IN 
Single dose, D1 
3 
2-8y 
Non-naive 
109 TCID50 Sing2016 M2SR 
H3N2 vaccine or placebo 
IN 
Single dose, D1 
4 
2-8y 
Non-naive 
109 TCID50 Sing2016 M2SR 
H3N2 vaccine or placebo 
IN 
Two doses: D1, 
D29 
5 
6-23m 
Naive 
107 TCID50 Sing2016 M2SR 
H3N2 vaccine or placebo 
IN 
Two doses: D1, 
D29 
6 
6-23m 
Naive 
108 TCID50 Sing2016 M2SR 
H3N2 vaccine or placebo 
IN 
Two doses: D1, 
D29 
7 
6-23m 
Naive 
109 TCID50 Sing2016 M2SR 
H3N2 vaccine or placebo 
IN 
Two doses: D1, 
D29 
 
6.1.3 
Dose Escalation 
In this study, Cohort 1 will first be administered 109 TCID50 Sing2016 M2SR H3N2 or placebo, 
and Cohort 2 will be administered 108 TCID50 Sing2016 M2SR H3N2 vaccine or placebo 
intranasally. Subsequent cohorts will be permitted to begin enrollment when criteria, as 
described in the protocol, have been met.  
 
6.1.4 
Dose Modifications 
If adverse events are deemed to be occurring at frequencies or with severities that were 
unexpected, the sponsor, investigators, and SMC may take one of a number of actions. These 
include halting the study and adjusting the dose given to subsequent participants or cohorts. No 
changes will be implemented without IRB approval.  
 
6.2 
Preparation/Handling/Storage/Accountability 
 
6.2.1 
Acquisition and Accountability 
The study product (vaccine and diluent) will be provided by FluGen, Inc. The seasonal IIV4, 
MAD300™ Sprayer Device, and saline will be sourced as specified by the MOP or advised by 
the study sponsor, which maybe locally or by DMID Clinical Materials Services (CMS). The 

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product will be stored as instructed. Records will be maintained that document receipt, release 
for dosing, disposal, or return to the sponsor.  
 
Product 1: Sing2016 M2SR H3N2 Intranasal Influenza Vaccine 
Product 2: SPG-NaCl buffer to be used as the diluent for Product 1 
Product 3: Saline 
Product 4: Seasonal IIV4  
Product 5: MAD300™ Sprayer Device 
 
Upon request by DMID, study products 1, 2, 3, 4 and 5 will be transferred to the following 
address: 
DMID Clinical Materials Services (CMS) 
Fisher BioServices 
20439 Seneca Meadows Parkway 
Germantown, MD 20876 
Phone: 240-477-1350 
Fax: 240-477-1360 
Email: DMID.CMS@thermofisher.com 
 
Based on availability, DMID will advise on sourcing of additional specific products which may 
include CMS or site-specific procurement. This includes saline placebo, IIV4, cryovials, Henke-
Ject syringes and shipping supplies for this study. The study products will be provided through 
the DMID CMS to the participating clinical sites prior to the start of this trial upon request and 
with prior approval from DMID. Should the site PI require additional study products, further 
instructions will be provided in the protocol-specific MOP. 
 
Accountability: After receipt of the study products, the site principal investigator is responsible 
for study product distribution and disposition and has ultimate responsibility for study product 
accountability. The site principal investigator may delegate to the participating site’s research 
pharmacist responsibility for study product accountability. The participating site’s research 
pharmacist will be responsible for maintaining complete records and documentation of study 
product receipt, accountability, dispensation, storage conditions, and final disposition of the 
study product(s). All study product(s), whether administered or not, must be documented on the 
appropriate study product accountability record or dispensing log. The sponsor’s monitoring staff 
will verify the participating site’s study product accountability records and dispensing logs per 
the site monitoring plan. 
 
Used and unused of study products will be retained until study conclusion or until accountability 
via verification of inventory or monitoring has occurred and written notification stating retention 
is no longer required is received as applicable per the site’s SOPs.   
 
Accountability may be performed through verification of inventory and during monitoring.  
DMID does not require used containers of study product to be maintained at the research 
pharmacy until the clinical monitors have confirmed the disposition of all study products. 
Retention of used study product containers for monitoring is only required when the local 
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product containers, then the used CTM vials can be destroyed per the site’s SOPs with a second 
staff member’s observation and signed verification (two signatures) that the used CTM vials 
were destroyed. 
 
Used study products may be destroyed in accordance with site-specific SOPs following each 
monitoring visit where study product accountability is monitored, and resolution of any 
discrepancies. 
 
Final disposition of the unused study products will be determined by DMID and communicated 
to the participating sites by the DMID Clinical Project Manager. 
 
6.2.2 
Formulation, Appearance, Packaging, and Labeling 
 
6.2.2.1 
Product 1: Sing2016 M2SR H3N2 Intranasal Influenza Vaccine 
The vaccine is manufactured in M2VeroA cells that provide the internal protein M2 for vaccine 
virus production. Cells are infected with the master virus seed and the infected cell culture 
supernatant is harvested at the peak of infection. The resulting virus-infected medium is then 
processed during downstream events to purify live virus from cell debris and production 
reagents. Steps are taken during purification to remove Vero cell DNA and host cell proteins and 
contaminants. The purified virus is formulated into the SPG formulation buffer that helps to 
stabilize the live virus. Instructions on sterile filtration and preparation of the drug product can be 
found in the MOP. 
 
The Sing2016 M2SR H3N2 vaccine will be provided frozen and in cryovials. An unblinded 
pharmacist or their designee will thaw the vial contents to room temperature prior to dose 
administration, as detailed in the MOP. For all dose cohorts, the contents will be diluted if 
necessary, to the target dosing concentration with provided SPG-NaCl diluent for each 
participant.  
 
The investigational product (whether undiluted [neat] or diluted) will be drawn into two 
intranasal delivery devices per participant dose. The plunger of each delivery device will be 
compressed to spray atomized solution into each nostril at administration.  
 
6.2.2.2 
Product 2: SPG-NaCl Buffer   
The SPG-NaCl buffer is provided. It is clear, colorless solution. It is used as the diluent for the 
intranasal vaccine, Product 1. Complete instructions on vaccine preparation are found in the 
protocol-specific MOP. 
 
6.2.2.3 
Product 3: Saline placebo 
The placebo consists of 0.9% sodium chloride for injection, USP (containing no preservatives 
and suitable for intranasal administration) and is a clear, colorless solution. It will be sourced by 
DMID CMS in single-use containers. The unblinded pharmacist or designee will aseptically 
withdraw volumes of the placebo as needed to fill the intranasal delivery devices.  
 

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6.2.2.4 
Product 4: Seasonal IIV4 
The licensed seasonal influenza vaccine will be acquired by DMID CMS with storage and 
administration as per manufacturers recommendations.  
 
6.2.2.5 
Product 5: MAD300™ Nasal Device 
MAD300™ nasal devices are supplied in individual non-sterile pouches and are attached to 
sterile 1-mL single-use syringes before use (see Section 6.1.1.5). MAD300™ pouches will be 
stored as specified in the MOP.  
 
Refer also to the MAD300™ Instructions for Use (IFU) as well as to the protocol-specific MOP. 
 
Each of these study products, except the MAD300™ nasal device, will be labeled according to 
manufacturer specifications and include the statement “Caution: New Drug – Limited by Federal 
Law to Investigational Use.” 
 
6.2.3 
Product Storage and Stability 
 
6.2.3.1 
Product 1: Sing2016 M2SR H3N2 Intranasal Influenza Vaccine 
Long term storage is at ≤ -65°C. Stability studies performed by FluGen demonstrate that the 
thawed vial contents remain functionally stable when diluted, if necessary, with SPG buffer to all 
doses required for the protocol as defined in the MOP.  
 
6.2.3.2 
Product 2: SPG-NaCl Diluent   
SPG-NaCl buffer is stored frozen at ≤ -65°C. Stability studies indicate that the thawed vial 
contents remain stable for at least 48 hours when filled into the delivery device and held at 2-
8ºC. 
 
6.2.3.3 
Product 3: Saline placebo 
Stored at 20°C to 25°C (68°F to 77°F) (See USP Controlled Room Temperature). Placebo 
consists of a commercially prepared, physiological saline for injection, USP. Placebo, saline 
solution, is stable at room temperature. 
 
6.2.3.4 
Product 4: Licensed Seasonal IIV  
Licensed seasonal IIV4 will be stored as per manufacturers recommendations.  
 
The temperature of the storage units for study product will be recorded daily (excluding non-
business days and holidays, as applicable), continuously monitored, and recorded during the 
duration of this trial per the participating CIVIC site’s standard operating procedures, and 
documentation will be maintained. If the temperature fluctuates outside of the required range, the 
affected study product(s) must be quarantined at the correct storage temperature and labeled as 
‘Do Not Use’ (until further notice). The research pharmacist must alert the site principal 
investigator and study coordinator, if the temperature fluctuates outside of the required range.  
 
In the event the temperature fluctuates outside of the required range, the affected study 
product(s) must not be administered. The site principal investigator or responsible person should 
immediately contact the DMID Product Support Team at 

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DMIDProductSupportTeam@niaid.nih.gov for further instructions before any additional study 
vaccinations are administered. Based on the information collected, DMID and/or the 
manufacturers will determine whether the affected study product(s) can be used. If it cannot be 
used, the site will receive specific instructions on how to return the affected study product(s) to 
the DMID CMS or destroy it on site. Additional instructions for quarantine of product are 
provided in the protocol-specific MOP. 
 
6.2.4 
Preparation 
See the protocol-specific MOP appendices for detailed information on the preparation, labeling, 
storage and administration of study vaccine for each group. It is recommended that the study 
vaccine preparation be performed by the participating CIVIC site’s pharmacist on the same day 
of study vaccine administration. However, for logistical purposes, it is possible to prepare study 
vaccine prior to the day of dosing as long as dose administration occurs within 48 hours of 
vaccine stock removal from ultracold storage. 
 
For each participant, a vial or vials of the study product and SPG-NaCl diluent as needed will be 
thawed and doses prepared as per the MOP.  
 
The vaccine will be delivered intranasally (up to 0.3 mL per nostril as described in the MOP) 
using the intranasal delivery device. MAD nasal sprayer device employs a cone-shaped nozzle 
with an atomizer in the 4.3 mm tip and is attached to a 1 mL polypropylene syringe via a Luer 
lock feature.  A  MAD nasal sprayer device was previously successfully utilized for the 
intranasal delivery of live, wild-type A/H1N1 influenza virus in a human challenge study 
conducted as a Phase 1 study under US IND #BB-14969 by the National Institute of Allergy and 
Infectious Diseases, NIH (Memoli 2015; intranasal delivery of live, A/H1N1 influenza virus in a 
human challenge study under US IND 19213 (DMID protocol 18-0010); and a Phase 1 study 
under US IND # 18170 for intranasal delivery of H3N2 M2SR (DMID protocol 17-0012) [19]. 
The prepared dose will be drawn into two intranasal delivery devices that will be used to spray 
the contents into the participant’s nostrils. Each nostril will receive approximately half of the 
targeted dose.  
 
6.3 
Measures to Minimize Bias: Randomization and Blinding 
 
6.3.1 
Treatment Assignment Procedures 
Per ICH E6 GCP, screening records will be kept at the participating CIVIC site to document the 
reason why an individual was screened, but failed trial entry criteria. The reasons why 
individuals failed screening will be recorded in the Statistical and Data Management 
Coordinating Center’s (SDMCC) Advantage eClinicalSM (Electronic Data Capture System). 
Once consented and upon entry of demographic data and confirmation of eligibility for this trial, 
the participant will be enrolled in the electronic data capture system. The participant will be 
enrolled and randomly assigned to either treatment or placebo control.  
 
Enrollment of participants will be done online using the enrollment module of Advantage 
eClinicalSM. The randomization code will be prepared by statisticians at the SDMCC and 
included in the enrollment module for this trial. Advantage eClinicalSM will assign each 
participant to a treatment arm after the demographic and eligibility data have been entered into 

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the system. A designated individual at the CIVIC site will be provided with a code list for 
emergency unblinding purposes, which will be kept in a secure place. 
 
Instructions for use of the enrollment module are included in the Advantage eClinicalSM User’s 
Guide. Manual back-up procedures and instructions are provided for use in the event that the 
participating CIVIC site temporarily loses access to the internet or the online enrollment system 
is unavailable. 
 
6.3.2 
Randomization and Blinding 
This is a randomized double-blinded study.  
 
6.3.3 
Blinding and Masking Procedures 
This is a double-blind (masked) clinical trial. Participants, site investigators, and study personnel 
performing any study-related assessments following study product administration are blinded to 
product received. Laboratory personnel performing immunological assays will receive serum 
blinded to participant ID number, specimen visit number, and allocation group. 
 
The randomization scheme will be generated by the SDMCC and provided to unblinded study 
personnel (i.e., research pharmacists performing study vaccination preparations and unblinded 
study vaccine administrators) at the participating CIVIC sites. 
 
The unblinded or blinded study vaccine administrator is a study personnel member credentialed 
to administer vaccines. The unblinded vaccinator may also participate in dose preparation but 
will not be involved in study-related assessments or have participant contact for data collection 
following study vaccine administration. When participants receive IIV4, it is not necessary that 
vaccine is given by an unblinded member of the study team.  
 
The SRC and the SMC will receive data in aggregate and the SMC may be presented data by 
aliased treatment group. The actual treatment assignments will be provided to the SMC at their 
request, but the SRC, as part of their review, will remain blinded. The SMC may also be 
provided with expected and observed rates of the expected AEs in an unblinded fashion and may 
request the treatment assignment be unblinded for an individual participant if required for safety 
assessment. The SMC will review grouped and unblinded data in the closed session only. 
 
6.4 
Study Intervention Compliance 
Participants will be directly observed at the time of dosing by a member of the clinical research 
team who is licensed to administer the study product. Administration will be documented and 
entered the eCRF. 
 
6.5 
Concomitant Therapy 
For this protocol, a prescription medication is defined as a medication that can be prescribed only 
by a properly authorized/licensed clinician. Medications to be reported in the Case Report Form 
(CRF) are concomitant prescription medications, over-the-counter medications, and 
supplements. 
 

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Administration of any medications, therapies, or vaccines will be recorded on the appropriate 
eCRF. Concomitant medications will include all current medications and medications taken in 
the 30 days prior to enrollment. Study team will record, on the source document, medications 
taken starting from 30 days prior to the screening visit (or enrollment visit, if only one visit for 
screening and enrollment), but only enter into the eCRF, those medications taken up to 30 days 
prior to the enrollment. At subsequent visits, until Visit 4 (Day 29) for Cohorts 1, 2, and 3 (those 
receiving 1 dose of IN study product) and until Visit 7 (Day 57) for Cohorts 4, 5, 6, and 7 (those 
receiving 2 doses of IN study product) updates to previous concomitant medications and 
additional medications taken in the interim will be recorded. No concomitant medications will be 
recorded after Day 57. Prescription and over-the-counter drugs will be included as well as 
herbals, vitamins, and supplements. Use of a new medication should prompt evaluation for the 
occurrence of any AE, AESI, or SAE, if within the respective reporting period.  
 
Other than those that preclude enrollment, as found in the eligibility criteria, medications that 
might interfere with the evaluation of the investigational product(s) should not be used from Day 
1 until the time when, for the participant’s cohort, concomitant medications are no longer being 
recorded, unless clinically indicated as part of the participant’s health care. Use of antipyretics 
will not be encouraged. Parents may administer them to treat, but not to prevent, fever.  
 
Medications in this category include the prohibited medications per the Participant Exclusion 
Criteria (see Section 5.2 Exclusion Criteria). Site team members will discourage prophylactic 
antipyretics but will encourage antipyretics to treat fever or other symptoms that may be relieved 
with these medications. In addition, the site PI or appropriate sub-investigator may identify other 
medications, herbals, vitamins, or supplements that should not be used due to a risk to participant 
safety or assessment of reactogenicity and immunogenicity. 
 
6.5.1 
Rescue Medicine 
No rescue medications are applicable in this trial.  
   
7. 
STUDY INTERVENTION DISCONTINUATION AND 
PARTICIPANT DISCONTINUATION/WITHDRAWAL 
 
7.1 
Halting Criteria and Discontinuation of Study Intervention 
 
7.1.1 
Study Halting Criteria Study-Wide 
These halting criteria refer to all enrolled participants, study-wide, in aggregate and are 
cumulative. Should one occur, study enrollment will be paused for review by the SMC, sponsor, 
and investigators:   
 
1. Any participant experiences laryngospasm, bronchospasm, or anaphylaxis within 24 
hours after administration of study product if deemed related to the study product by the 
study investigator. These events must be reported to the medical monitor within 24 hrs of 
investigator awareness.   
2. Two or more participants experience generalized urticaria (defined as occurring at more 
than two body parts) within 72 hours after administration of study product that is 
considered related to study product   

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3. Any participant experiences an SAE after administration of study product that is 
considered related to study product    
4. Any participant experiences acute weakness of limbs and/or cranial nerve innervated 
muscles (description of potential signal of GBS) after administration of study product   
5. 10% of participants (minimum 3 participants) experience the same grade 3 local solicited 
adverse event or systemic solicited adverse event (reactogenicity events, as defined in the 
protocol) 
10% of participants (minimum 3 participants) experience the same grade 3 unsolicited 
AE, in the same HLT by MedDRA coding, considered related to study product 
 
7.1.2 
Study Halting Criteria for “Lead-In” Participants 
The 8 participants in Cohort 5 and the first 8 participants in Cohort 6 are termed “lead-ins” 
because data on safety and tolerability from Days 1 to 8 from these participants will allow for the 
following cohort to begin enrollment. Given that the lead-ins are young (6-23 months), 
influenza-naïve, and only 8 participants in Cohorts 5 and 6, the halting criteria are slightly more 
stringent. If any of the following events occurs, study enrollment will be paused for review by 
the SMC, sponsor, and investigators:   
 
Any participant experiences laryngospasm, bronchospasm, or anaphylaxis within 24 hours after 
administration of study product if deemed related to the study product by study investigator. 
These events must be reported to the medical monitor within 24 hrs of investigator awareness.  
 
1. Two or more participants experience generalized urticaria (defined as occurring at more than 
two body parts) within 72 hours after administration of study product that is considered 
related to study product   
2. Any participant experiences an SAE after administration of study product that is considered 
related to study product    
3. Any participant experiences acute weakness of limbs and/or cranial nerve innervated muscles 
(description of potential signal of GBS) after administration of study product   
4. Two or more participants experience the same grade 3 local solicited adverse event or 
systemic solicited adverse event (reactogenicity events, as defined in the protocol) 
5. Two or more participants experience the same grade 3 unsolicited AE, in the same HLT by 
MedDRA coding, considered related to study product. 
 
7.1.3 
Individual Halting Criteria 
The study intervention will be discontinued in a participant if any of the following occurs: 
 
1. Laryngospasm, bronchospasm, or anaphylaxis within 24 hours after administration of study 
product if deemed related to the study product by the study investigator. These events must 
be reported to the medical monitor within 24hrs of investigator awareness. 
2. Generalized urticaria (defined as occurring at more than two body parts) within 72 hours 
after administration of study product that is considered related to study product   
3. SAE after administration of study product that is considered related to study product    
4. Acute weakness of limbs and/or cranial nerve innervated muscles (description of potential 
signal of GBS) after administration of study product   
 

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7.1.4 
Criteria for Redosing 
Cohorts 1, 2, and 3 will receive a single dose of Sing2016 M2SR H3N2 vaccine or placebo. 
Cohorts 4, 5, 6, and 7 will receive 2 doses, the first on Day 1 and the second on approximately 
Day 29. Participants in Cohorts 4, 5, 6, and 7 may receive the second dose if no individual or 
study halting criteria have been met and the following redosing criteria are met:  
 
Eligibility for Second Dose 
Inclusion Criteria 
1. Parent/guardian of the participating child continues to agree to permit participation 
2. Participant remains in good health as assessed by the principal investigator or other 
designated study investigator 
 
Exclusion criteria 
1. Has a body temperature 38.0°C/100.4°F (oral or axillary) or greater 
or another acute illness1 within the 72 hours prior to study vaccination   
1A potential participant who is recovering from an acute illness and has residual minimal symptoms, which, 
in the opinion of the site principal investigator or appropriate sub-investigator, will not likely affect the 
evaluation of outcome measures is not ineligible. 
2. Has any new medical or mental health disease or condition that, in the opinion of the site 
principal investigator or appropriate sub-investigator, would render study participation 
unsafe, or would interfere with the evaluation of the responses   
3. Has immunosuppression as a result of an underlying illness or treatment, a recent history 
or current use of immunosuppressive or immunomodulating disease therapy  
4. Has a new diagnosis of neoplastic disease   
5. Has taken oral, parenteral, or nasal corticosteroids of any dose since the first vaccination   
6. Has taken inhaled corticosteroids since the first vaccination   
7. Has known HIV, hepatitis B, or hepatitis C infection  
8. Has known hypersensitivity or allergy to any components of the study vaccine (sucrose, 
sodium chloride, phosphate, glutamate), or material in nasal delivery device 
(polycarbonate, polypropylene, synthetic rubber)   
9. Use of aspirin or salicylate containing products since the first vaccination   
10. Had documented influenza or received specific influenza antiviral treatment since first 
vaccination   
11. Received any antiviral drug since first vaccination   
12. Received a licensed live vaccine since first study vaccination, or plans to receive a 
licensed live vaccine within the 30 days after the last study vaccination unless provided 
as part of this study  
13. Received a licensed inactivated vaccine since the first study vaccination, or plans to 
receive a licensed, inactivated vaccine within the 30 days after the last study vaccination    
14. Plans to receive an influenza vaccine, other than the one offered as part of participation in 
this study, within the 30 days following the last study vaccination    
15. Received immunoglobulin or other blood products since first study vaccination  
16. Received an experimental agent or device since first study vaccination or expects 
to receive an experimental agent or device during the trial-reporting period 
 
7.1.5 
Follow up for Participants who Discontinued Study Intervention 

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Discontinuation from receipt of Sing2016 M2SR H3N2 vaccine or placebo does not mean 
discontinuation from the study, and remaining study procedures may be completed as indicated 
by the study protocol.  Participants who receive a single dose of vaccine or placebo, but joined 
the study in Cohorts 4, 5, 6, or 7, and were thereby assigned to receive 2 doses of vaccine or 
placebo, will still be offered the seasonal licensed influenza vaccine (IIV4) at the time indicated 
in the protocol. Such participants will be followed on the schedule of activities in the same 
manner as participants in Cohorts 1, 2, and 3 (who were assigned to receive only a single dose of 
vaccine or placebo). If a clinically significant finding is identified (including, but not limited to 
changes from baseline) after enrollment, the investigator or qualified designee will determine if 
any change in participant management is needed. Any new clinically relevant finding after study 
product administration will be reported as an adverse event (AE). 
 
7.2 
Participant Withdrawal from the Study and Replacement 
Parents/guardians of participants are free to withdraw their child from participation in the study 
at any time upon request. An investigator may discontinue or withdraw a participant from the 
study for the following reasons: 
 
1. Study non-compliance that in the opinion of the investigator poses an increased risk or 
compromises the validity of the data  
2. If any clinical adverse event (AE), laboratory abnormality, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the participant, in the opinion of the investigator 
3. Medical disease or condition, or new clinical finding(s) for which continued participation, in 
the opinion of the investigator, might compromise the safety of the participant, interfere with 
the participant’s successful completion of this study, or interfere with the evaluation of 
responses 
4. Participant lost to follow-up 
 
If the parent/guardian of the participant agrees, every attempt will be made to follow all AEs 
through resolution. 
 
Participants whose parents withdraw them, or who are withdrawn from this study, or are lost to 
follow-up after signing the informed consent form (ICF) but prior to administration of study 
product may be replaced, at the discretion of the sponsor.   
 
Given it is crucial to have an adequate pre-vaccination blood specimen from all children who 
enroll, participants from whom pre-vaccination blood is not successfully collected are not 
eligible to participate in the study.  
 
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
appropriate Case Report Form (CRF).  
 
7.3 
Lost to Follow-Up 
A participant will be considered lost to follow-up if he or she fails to return for scheduled visits 
and is unable to be contacted by the study site staff.  
 

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8. 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
Screening Procedures (Day -28 to -1, or Day 1): 
Screening may occur on Days -28 to -1 or may occur on the day of vaccination, Day 1. During 
the screening visit, after the study is described to the parent/guardian and child, when 
appropriate, the parent/guardian will provide written permission and the participant, when 
appropriate, will provide assent, prior to any study-related procedure. Eligibility will be 
determined. 
 
Study staff will collect demographic information, medical history, concomitant medications, and 
either perform or review the physical examination (that is, the physical exam may be performed 
by the participant’s clinical provider, who is not a member of the study team or by a qualified 
study team member). No safety laboratory tests will be collected. Blood for immune response 
and nasal washes will be collected at the vaccination visit, prior to vaccination, to establish a 
baseline. Female participants of childbearing potential will have a urine pregnancy test 
performed and recorded as negative within the 24 hours prior to vaccination. This test will not be 
done at a screening visit if the visit is not within 24 hours prior to vaccination.  Blood will be 
collected for immunological assays and a nasal wash will be collected for immunological assays 
on Day 1, prior to vaccination.  
 
8.1.1 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
If a physiologic parameter, e.g., body temperature, is outside of the protocol-specified range, 
then the measurement may be repeated once if, in the judgment of the investigator, the 
abnormality is unlikely to represent the true body temperature. A physiologic parameter may also 
be repeated if there is a technical problem with the measurement caused by a malfunctioning or 
inappropriate measuring device, or for any other reason that the investigator believes has led to a 
spurious result. 
  
A participant may be re-screened if there is a transient disease status (e.g., participant 
complained of a “cold or fever” and met a temporary delaying enrollment criterion of acute 
illness or fever)  or if a protocol eligibility criterion that is not met at the initial time of screening, 
will be met by rescreening at a later date (e.g., a medication taken within exclusionary window at 
the time of first screening that would not be within exclusionary window at a later rescreen). 
  
No participant may be screened more than twice due to a screening failure result as defined 
above.  
  
Parents of participants will be provided the results of any abnormal clinical laboratory results 
(such as a respiratory viral panel result) and will be referred to their primary healthcare provider. 
Research laboratory results will not routinely be provided to the parent of the participant.  
 
8.2 
Efficacy / Immunogenicity Assessments 
There are no efficacy evaluations in this study. Immunological evaluations include humoral 
immune responses, cellular immune responses, and mucosal immune responses following one or 
two doses of Sing2016 M2SR H3N2 vaccine or placebo. These evaluations also include 

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immunological evaluations following seasonal influenza vaccine provided to participants after 
having received 1 or 2 doses of Sing2016 M2SR H3N2 vaccine or placebo. 
 
8.2.1 
Humoral Immune Evaluations After Sing2016 M2SR H3N2 Vaccine 
Humoral immune evaluations will include HAI, neutralization, anti-HA, and anti-NA antibodies 
against the homologous vaccine (Sing2016M2SR H3N2) antigens. Responses to heterologous 
antigens may also be assessed. Analysis of extra-neutralizing function of antibodies to 
homologous and heterologous strains may also be performed on an exploratory basis according 
to positive responses obtained for assays described above and specimen availability. 
 
8.2.2 
Cellular Immune Evaluations After Sing2016 M2SR H3N2 Vaccine  
Cellular immune evaluations will include T-cell responses and antigen-binding B cells to 
homologous antigen epitopes and may include assessment of responses to heterologous antigen 
epitopes. Also, these evaluations might include B memory assessment of responses by ELISpot 
if enough cells are available. 
 
8.2.3 
Mucosal Immune Evaluations After Sing2016 M2SR H3N2 Vaccine 
Mucosal immune evaluations will include measurement of secretory IgA from nasal wash 
specimens (sIgA) against homologous vaccine antigens. Additional measurements may include 
HAI, neutralization, as well as anti-HA and anti-NA IgG and/or IgM antibodies and analysis of 
extra neutralizing antibody function on an exploratory basis according to specimen availability.  
 
8.2.4 
Immune Evaluations After Seasonal Influenza Vaccine (IIV4)  
Humoral immune evaluations after seasonal influenza vaccine will include serum HAI and 
neutralization as well as mucosal secretory IgA to vaccine-containing strains. Additional 
measurements may include anti-HA, anti-NA antibodies and extra-neutralizing antibody function 
according to responses obtained in assays described above and specimen availability.  
Cellular response analysis after seasonal influenza vaccine will include T-cell responses and 
antigen-binding B cells and may include responses to heterologous strains; analysis of B memory 
responses by ELISpot may be included according to cell availability.  
 
8.3 
Safety and Other Assessments 
Safety will be assessed by evaluations of the frequency and severity of solicited AEs 
(reactogenicity), unsolicited non-serious AEs, AESIs, NOCMCs, and SAEs.  
 
8.3.1 
Solicited AEs (Reactogenicity)  
Solicited AEs can be divided into immediate post-vaccination events, local reactogenicity events, 
and systemic reactogenicity events. The immediate events will be recorded by the study staff in 
the 20 minutes after each vaccination. All solicited events with corresponding grades as 
described in Section 8.4.3 will be evaluated. These severity grades include mild, moderate, or 
severe.  
 
Local and systemic reactogenicity events will then be recorded by the parent/guardian daily from 
Day 1 to Day 8 after the first vaccine or placebo and the 7 days following the second vaccination 
for participants in Cohorts 4, 5, 6, and 7 with the use of a memory aid. The local events are those 

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in and around the nares, where the vaccine is administered. Local and systemic events are 
described in Table 4, below. 
 
8.3.2 
Unsolicited Non-Serious AEs 
We will record unsolicited non-serious AEs, both medically attended and not, from the time of 
vaccination on Day 1 until 28 days after the last vaccination (Day 29 for Cohorts 1, 2, and 3 and 
approximately Day 57 for Cohorts 4, 5, 6, and 7). 
 
8.3.2.1 
Suspected Unexpected Serious Adverse Reactions (SUSAR) 
A SUSAR is any SAE where a causal relationship with the study product is at least a reasonable 
possibility but is not listed in the Investigator Brochure, Package Insert, and/or Summary of 
Product Characteristics. 
 
8.3.3 
Adverse Events of Special Interest (AESIs).  
Given that there is a theoretical possibility that intranasal influenza vaccines will lead to 
increased rates of acute onset wheeze and that these events may not always be captured by the 
other safety assessments, these events will be deemed AESIs. AESIs will be collected from the 
time of the first study vaccination on Day 1 until individual participant study completion, but 
with special emphasis paid to the 28 days following each intranasal vaccination. Details on the 
evaluation of this AESI are found in Appendix B – Evaluation Criteria for Acute Wheezing.  
 
8.3.4 
New Onset Chronic Medical Conditions (NOCMCs).  
NOCMCs are defined as any new ICD-10 diagnosis that is applied to the participant during the 
duration of the study, after receipt of the study agent, that is expected to continue for at least 3 
months and requires continued health care intervention. These events will be collected from the 
time of the first study vaccination on Day 1 until the end of the study period (the final visit done 
in April of the calendar year following enrollment). 
 
8.3.5 
Serious Adverse Events (SAEs).  
SAEs will also be collected from the time of the first study vaccination on Day 1 until the end of 
the study period (the final visit done in April of the calendar year following enrollment). 
 
8.3.6 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
The site PI or appropriate sub-investigator is responsible for recording all AE/SAEs that are 
observed or reported during this trial, when they occur in the timeframes described, regardless of 
relationship to study product. AE/SAEs, abnormal laboratory test values, or abnormal clinical 
findings will be collected, assessed, documented, reported, and followed appropriately.  
 
8.4 
Adverse Events and Serious Adverse Events 
 
8.4.1 
Definition of Adverse Events (AE) 
Adverse event means any untoward medical occurrence associated with the use of an 
intervention in humans, whether considered intervention-related (21 CFR 312.32 (a)). An AE 
can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of medicinal (investigational) 

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product. Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE.  However, if the severity or frequency of any 
pre-existing medical condition increases, it will be recorded as an AE. 
 
Adverse events can be further divided into solicited adverse events and unsolicited adverse 
events. Solicited adverse events are those for which the study team will specifically query the 
participant or parent/guardian whether they occurred. Unsolicited adverse events are those events 
that the participant or parent/guardian reports occurring without being queried about the specific 
event.  
 
All AEs will be assessed for severity and relationship to study intervention (see section 8.3.3). 
All AEs, solicited and unsolicited, will be captured on the appropriate data collection form.  
Information to be collected for AEs includes event description, date of onset, assessment of 
severity, relationship to study product and alternate etiology (assessed only by those with the 
training and authority to make a diagnosis and listed on the Form FDA 1572 as an investigator), 
date of resolution, seriousness, and outcome. AEs occurring during the trial collection and 
reporting period will be documented appropriately regardless of relationship. 
   
AEs will be followed through resolution. 
 
8.4.2 
Solicited Adverse Events 
Solicited adverse events are anticipated local and systemic adverse events for which consistent 
collection of information is desired. For this study, these include local respiratory tract 
reactogenicity events and systemic reactogenicity events (Section 8.3.1).  
 
8.4.3 
Local Reactogenicity  
The following Toxicity Grading Scales will be used to grade solicited local (respiratory) and 
systemic (constitutional) reactions:  
 
Table 4: Grades of Local & Systemic Reactogenicity Assessments, By Age Group 
Ages 9-17 Years 
Symptoms 
Grade 1 
Grade 2 
Grade 3 
Local (Including Respiratory) Reactogenicity 
Rhinorrhea 
(Runny Nose) 
Noticeable but does not 
interfere with daily activity  
 
Moderate 
discomfort/interferes 
with daily activity 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 
Stuffy 
nose/congestion  
Noticeable but does not 
interfere with daily activity  
Moderate 
discomfort/interferes 
with breathing 
through the nose  
Unable to breathe 
through the nose, or 
prevents daily 
activity or seeks 
medical encounter 

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Sneezing  
Noticeable but does not 
interfere with daily activity 
Moderate 
discomfort. 
Interferes with daily 
activity 
Significant 
discomfort/prevents 
daily activity 
Nasal 
pain/irritation  
Noticeable but does not 
interfere with daily activity 
Moderate 
discomfort/interferes 
with daily activity 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 
Nasal bleeding/ 
epistaxis  
Total duration of all 
episodes in a 24-hour period 
<30 minutes 
Total duration of all 
episodes in a 24-
hour period > 30 
minutes, but did not 
require a visit for a 
medical encounter 
Any bleeding that 
required visit for 
medical encounter 
Sinus 
pressure/pain  
Noticeable but does not 
interfere with daily activity  
Moderate 
discomfort. 
Interferes with daily 
activity 
Significant 
discomfort/prevents 
daily activity 
Sore throat (may 
include scratchy 
or painful throat) 
Noticeable but does not 
interfere with eating and/or 
drinking 
Moderate 
discomfort. 
Interferes with 
eating and/or 
drinking  
Significant 
discomfort/prevents 
eating and/or 
drinking or seeks 
medical encounter 
 
 
Cough  
Noticeable but does not 
interfere with daily activity 
or sleeping 
Moderate 
discomfort/interferes 
with daily activity or 
sleeping 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 
Trouble 
breathing or 
shortness of 
breath  
Noticeable but does not 
interfere with daily activity 
or not troubled by 
breathlessness except on 
vigorous exercise 
Moderate 
discomfort/interferes 
with daily activity or 
short of breath with 
regular movement 
activities such as 
when hurrying on a 
level surface or 
walking up a slight 
incline and need to 
stop 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter or too 
breathless to leave 
the house, or 
breathless when 
undressing, 
preventing normal 
activities 
Systemic Reactogenicity 
Feverishness 
(may include 
chills or 
shivering)  
No interference with  
daily activity  
Some interference 
with daily activity 
Significant  
interference, 
prevents daily 
activity  

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Fatigue 
(tiredness)  
No interference with daily 
activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents daily 
activity 
Malaise (general 
unwell feeling)  
No interference with daily 
activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents  
daily activity  
Myalgia (general 
body aches, 
general muscular 
pain)  
No interference with daily 
activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents  
daily activity  
Arthralgia 
(general joint 
pains)  
No interference with daily 
activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents  
daily activity  
Headache  
No interference with daily 
activity 
Some interference 
with daily activity 
Significant 
interference, 
prevents daily 
activity and/or seeks 
medical encounter 
Flushing  
Asymptomatic flushing  
Symptoms, some 
interference with 
daily activity  
Symptomatic, 
significant 
interference, 
prevents daily 
activity  
Decreased 
activity  
No interference with daily 
activity 
Some interference 
with daily activity 
Significant 
interference, 
prevents daily 
activity  
Decreased 
appetite  
Loss of appetite without 
decrease in oral intake 
Loss of appetite 
associated with 
decreased oral intake 
Loss of appetite 
without oral intake, 
seek medical care 
Abdominal pain  
No interference with daily 
activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents daily 
activity 
Nausea  
Transient (<24 hours) or 
intermittent and no or 
minimal interference with 
oral intake  
Persistent nausea 
resulting in 
decreased oral intake 
for 24 to 48 hours  
Persistent nausea 
resulting in minimal 
oral intake for > 48 
hours, or rehydration 
 indicated (e.g., IV 
fluids)  

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Vomiting  
Transient or intermittent, 
and no interference with 
daily activity; minimal 
interference with oral intake  
Frequent episodes 
with no dehydration 
and interferes with 
some daily activity  
Persistent vomiting, 
resulting in 
orthostatic 
hypotension or 
aggressive 
rehydration (e.g., IV 
fluids) or prevents 
normal daily  
activity  
Diarrhea  
Transient or intermittent, 
and no interference with 
daily activity; minimal 
interference with oral  
intake 
Frequent episodes 
with no dehydration 
and interferes with 
some daily activity  
Persistent diarrhea, 
resulting in 
orthostatic 
hypotension or 
aggressive 
rehydration (e.g., IV 
fluids) or  
prevents normal 
daily activity  
Eye pruritus  
Transient of intermittent or 
minimal interference  
and no intervention  
Persistent or 
frequent episodes, 
some interference 
with daily activity  
Significant 
symptoms, prevents 
daily activity, or 
seeks medical  
attention  
Eye redness 
Asymptomatic eye redness  
Symptomatic eye 
redness, some 
interference with 
daily activity  
Eye redness prevents 
daily activity or 
seeks medical 
encounter  
Allergic Skin 
Reaction 
Pruritus with or without 
rash, no medical 
intervention  
Localized urticaria, 
with intervention  
Generalized 
urticaria, 
anaphylaxis, or  
angioedema  
Fever* - oral or 
axillary† 
38.0°C – 38.4°C (100.4°F – 
101.1°F)  
38.5°C – 38.9°C 
(101.2°F – 102.0°F)  
>38.9°C  
(>102.0°F) 
• * A fever can be considered not related to the study product if an alternative etiology can 
be documented.  
† Participants must not eat or drink anything hot or cold prior to taking oral temperature 
 
Ages 4-8 Years 
Symptoms 
Grade 1 
Grade 2 
Grade 3 
Local (Including Respiratory) Reactogenicity 
Rhinorrhea (Runny Nose) 
Noticeable but 
does not interfere 
with daily activity  
 
Moderate 
discomfort/interferes 
with daily activity 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 

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Stuffy nose/congestion  
Noticeable but 
does not interfere 
with daily activity  
Moderate 
discomfort/interferes 
with breathing 
through nose  
Unable to breathe 
through nose, or 
prevents daily 
activity or seeks 
medical encounter 
Sneezing  
Noticeable but 
does not interfere 
with daily activity 
Moderate 
discomfort. 
Interferes with daily 
activity 
Significant 
discomfort/prevents 
daily activity 
Nasal pain/irritation  
Noticeable but 
does not interfere 
with daily activity 
Moderate 
discomfort/interferes 
with daily activity 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 
Nasal bleeding/ epistaxis  
Total duration of 
all episodes in a 
24-hour period 
<30 minutes 
Total duration of all 
episodes in a 24-
hour period > 30 
minutes 
Any bleeding that 
required visit for 
medical encounter 
Facial Pain  
Noticeable but 
does not interfere 
with daily activity  
Moderate 
discomfort. 
Interferes with daily 
activity 
Significant 
discomfort/prevents 
daily activity 
Sore throat (may include 
scratchy or painful throat) 
 
Noticeable but 
does not interfere 
with eating and/or 
drinking 
Moderate 
discomfort. 
Interferes with 
eating and/or 
drinking  
Significant 
discomfort/prevents 
eating and/or 
drinking or seeks 
medical encounter 
 
 
Cough  
Noticeable but 
does not interfere 
with daily activity 
or sleeping 
Moderate 
discomfort/interferes 
with daily activity or 
sleeping 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 
Trouble breathing, 
shortness of breath  
Noticeable but 
does not interfere 
with daily activity 
or not troubled by 
breathlessness 
except on vigorous 
exercise 
Moderate 
discomfort/interferes 
with daily activity or 
short of breath with 
regular movement 
activities such as 
when hurrying on 
the level or walking 
up a slight incline 
and need to stop 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter or too 
breathless to leave 
the house, or 
breathless when 
undressing, 
preventing normal 
activities 
Systemic Reactogenicity 

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Feverishness (may include 
chills, shivering, 
sweating)  
No interference 
with  
daily activity  
Some interference 
with daily activity 
Significant  
interference, 
prevents daily 
activity  
Fatigue (tiredness)  
No interference 
with daily activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents daily 
activity 
Malaise (general unwell 
feeling)  
No interference 
with daily activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents  
daily activity  
Myalgia (general body 
aches/muscular pain)  
No interference 
with daily activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents  
daily activity  
Arthralgia (general joint 
pains)  
No interference 
with daily activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents  
daily activity  
Headache  
No interference 
with daily activity 
Some interference 
with daily activity 
Significant 
interference, 
prevents daily 
activity and/or seeks 
medical encounter 
Decreased activity  
No interference 
with daily activity 
Some interference 
with daily activity 
Significant 
interference, 
prevents daily 
activity  
Decreased appetite  
Loss of appetite 
without decrease 
in oral intake 
Loss of appetite 
associated with 
decreased oral 
intake 
Loss of appetite 
without oral intake, 
seek medical care 
Abdominal pain  
No interference 
with daily activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents daily 
activity 
Nausea  
Transient (<24 
hours) or 
intermittent, and 
with no or 
minimal 
interference with 
oral  
intake  
Persistent nausea 
resulting in 
decreased oral 
intake for 24 to 48 
hours  
Persistent nausea 
resulting in minimal 
oral intake for > 48 
hours, or 
rehydration 
 indicated (e.g., IV 
fluids)  

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Vomiting  
Transient or 
intermittent AND 
no interference 
with daily activity; 
minimal 
interference with 
oral intake  
Frequent episodes 
with no dehydration 
and interferes with 
some daily activity  
Persistent vomiting, 
resulting in 
orthostatic 
hypotension or 
aggressive 
rehydration (e.g., IV 
fluids) or prevents 
normal daily  
activity  
Diarrhea  
Transient or 
intermittent AND 
no interference 
with daily activity; 
minimal 
interference with 
oral  
intake  
Frequent episodes 
with no dehydration 
and interferes with 
some daily activity  
Persistent diarrhea, 
resulting in 
orthostatic 
hypotension or 
aggressive 
rehydration (e.g., IV 
fluids) or  
prevents normal 
daily activity  
Eye pruritus  
Transient of 
intermittent or 
minimal 
interference  
and no 
intervention  
Persistent or 
frequent episodes, 
some interference 
with daily 
 activity  
Significant 
symptoms, prevents 
daily activity, or 
seeks medical  
attention  
Eye redness  
Asymptomatic eye 
redness  
Symptomatic eye 
redness, some 
interference with 
daily activity  
Eye redness 
prevents daily 
activity or seeks 
medical encounter  
Allergic Skin Reactions 
Pruritus with or 
without rash, no  
medical 
intervention  
Localized urticaria, 
with intervention  
Generalized 
urticaria, 
anaphylaxis, or  
angioedema  
Fever* - oral or axillary† 
38.0°C – 38.4°C  
100.4°F – 101.1°F  
38.5°C – 38.9°C  
101.2°F – 102.0°F  
>38.9°C  
>102.0°F  
• * A fever can be considered not related to the study product if an alternative etiology can 
be documented.  
† Participants must not eat or drink anything hot or cold prior to taking oral temperature 
 
Ages 6-47 Months 
Symptoms 
Grade 1 
Grade 2 
Grade 3 
Local (Including Respiratory) Reactogenicity 
Rhinorrhea (Runny 
Nose) 
Noticeable but does 
not interfere with 
daily activity  
 
Moderate 
discomfort/interferes 
with daily activity 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 

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Stuffy nose/congestion  
Noticeable but does 
not interfere with 
daily activity  
Moderate 
discomfort/interferes 
with breathing 
through nose  
Unable to breathe 
through nose, or 
prevents daily 
activity or seeks 
medical encounter 
Sneezing  
Noticeable but does 
not interfere with 
daily activity 
Moderate 
discomfort. 
Interferes with daily 
activity 
Significant 
discomfort/prevents 
daily activity 
Nasal bleeding/ 
epistaxis  
Total duration of all 
episodes in a 24-
hour period <30 
minutes 
Total duration of all 
episodes in a 24-
hour period > 30 
minutes 
Any bleeding that 
required visit for 
medical encounter 
Cough  
Noticeable but does 
not interfere with 
daily activity or 
sleeping 
Moderate 
discomfort/interferes 
with daily activity or 
sleeping 
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter 
Trouble 
breathing/shortness of 
breath  
Noticeable but does 
not interfere with 
daily activity  
Moderate 
discomfort/interferes 
with daily activity  
Significant 
discomfort/prevents 
daily activity or 
seeks medical 
encounter  
Systemic Reactogenicity 
Sleepiness (may include 
increased napping)  
No interference with 
daily activity  
Some interference 
with daily activity  
Significant 
interference, 
prevents daily 
activity 
Irritability/Fussiness  
No interference with 
daily activity 
Some interference 
with daily activity 
Significant 
interference, 
prevents daily 
activity and/or seeks 
medical encounter 
Decreased activity  
No interference with 
daily activity 
Some interference 
with daily activity 
Significant 
interference, 
prevents daily 
activity  
Decreased appetite  
Loss of appetite 
without decrease in 
oral intake 
Loss of appetite 
associated with 
decreased oral intake 
Loss of appetite 
without oral intake, 
seek medical care 

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Vomiting  
Transient or 
intermittent, and no 
interference with 
daily activity; 
minimal interference 
with oral intake  
Frequent episodes 
with no dehydration 
and interferes with 
some daily activity  
Persistent vomiting, 
resulting in 
dehydration 
requiring aggressive 
rehydration (e.g., IV 
fluids) or prevents 
normal daily  
activity  
Diarrhea  
Transient or 
intermittent, and no 
interference with 
daily activity; 
minimal interference 
with oral  
intake  
Frequent episodes 
with no dehydration 
and interferes with 
some daily activity  
Persistent diarrhea, 
resulting in 
dehydration or 
aggressive 
rehydration (e.g., IV 
fluids) or  
prevents normal 
daily activity  
Eye pruritus  
Transient of 
intermittent or 
minimal interference  
and no intervention  
Persistent or 
frequent episodes, 
some interference 
with daily activity  
Significant 
symptoms, prevents 
daily activity, or 
seeks medical  
attention  
Eye redness 
Asymptomatic eye 
redness  
Symptomatic eye 
redness, some 
interference with 
daily activity  
Eye redness prevents 
daily activity or 
seeks medical 
encounter  
Allergic Skin Reactions  
Pruritus with or 
without rash, no  
medical intervention  
Localized urticaria, 
with intervention  
Generalized 
urticaria, 
anaphylaxis, or  
angioedema  
Fever* - oral or 
axillary† 
38.0°C – 38.4°C 
100.4°F – 101.1°F  
38.5°C – 38.9°C 
101.2°F – 102.0°F  
>38.9°C  
>102.0°F  
• * A fever can be considered not related to the study product if an alternative etiology can 
be documented.  
† Participants must not eat or drink anything hot or cold prior to taking oral temperature 
 
8.4.4 
Unsolicited Events 
All AEs spontaneously reported by the parent/guardian or participant and/or in response to an 
open question from study personnel or revealed by observation, physical examination, or other 
diagnostic procedures will be recorded on the appropriate case report form. All reported 
unsolicited AEs are graded in accordance with the protocol toxicity tables. 
 
8.4.5 
Special Reporting of Adverse Events 
Following notification from the site PI or appropriate sub-investigator, DMID, the 
Investigational New Drug (IND) sponsor, will report any suspected adverse reaction that is both 
serious and unexpected. DMID will report an AE as a suspected adverse reaction only if there is 
evidence to suggest a causal relationship between the drug and the AE. DMID will notify the 

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FDA and all investigators (i.e., the participating site PI to whom the sponsor is providing drug 
under its IND(s) or under any PI’s IND(s)) in an IND safety report of potential serious risks from 
clinical trials or any other source, as soon as possible, but in no case later than 15 calendar days 
after the sponsor determines that the information qualifies for reporting as specified in 21 CFR 
Part 312.32. DMID will also notify the FDA of any unexpected fatal or life-threatening 
suspected adverse reaction as soon as possible, but in no case later than 7 calendar days after the 
sponsor’s initial receipt of the information. Relevant follow up information to an IND safety 
report will be submitted as soon as the information is available. Upon request from the FDA, 
DMID will submit to the FDA any additional data or information that the agency deems 
necessary, as soon as possible, but in no case later than 15 calendar days after receiving the 
request. 
 
All serious adverse events designated as “not related” to study product(s) will be reported to the 
FDA at least annually in a summary format. 
 
8.4.6 
Definition of Serious Adverse Events  
An adverse event or suspected adverse reaction is considered "serious" if, in the view of either 
the investigator or sponsor, it results in any of the following outcomes (21 CFR 312.32 (a)):  
• death 
• a life-threatening adverse event 
• inpatient hospitalization or prolongation of existing hospitalization 
• a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or  
• a congenital anomaly/birth defect.  
 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the patient or participant and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition. Examples of such medical events include 
allergic bronchospasm requiring intensive treatment in an emergency room or at home, 
convulsions that do not result in inpatient hospitalization, etc. 
 
All SAEs, as with any AE, will be assessed for severity and relationship to study intervention 
(see section 8.3). All SAEs will be recorded on the appropriate SAE data collection form and 
eCRF. All SAEs will be followed through resolution by a licensed study physician (for IND 
studies, a physician listed on the Form FDA 1572 as the site Principal Investigator or Sub-
Investigator). All SAEs will be reviewed and evaluated by DMID and will be sent to the SMC 
(periodic review unless related) and the IRB/IEC when required. 
 
8.4.7 
Classification of an Adverse Event 
The determination of seriousness, severity, and causality will be made by an on-site investigator 
who is qualified (licensed) to diagnose AE information, provide a medical evaluation of AEs, 
and classify AEs based upon medical judgment. This includes but is not limited to physicians, 
physician assistants, and nurse practitioners.  
 

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8.4.7.1 
Severity of Event 
All AEs will be assessed by the study clinician using the following grading system for AE 
severity.  
 
For adverse events (AEs) not included in the protocol defined grading system, the following 
guidelines will be used to describe severity.  
• Mild (Grade 1):  Events that are usually transient and may require only minimal or no 
treatment or therapeutic intervention and generally do not interfere with the participant’s 
usual activities of daily living. 
• Moderate (Grade 2):  Events that are usually alleviated with additional specific 
therapeutic intervention.  The event interferes with usual activities of daily living, causing 
discomfort but poses no significant or permanent risk of harm to the research participant. 
• Severe (Grade 3):  Events interrupt usual activities of daily living, or significantly affects 
clinical status, or may require intensive therapeutic intervention.  Severe events are 
usually incapacitating. 
AEs characterized as intermittent require documentation of onset and duration of each episode.  
The start and stop date of each reported AE will be recorded on the appropriate data collection 
form and eCRF. Changes in the severity of an AE will be documented to allow an assessment of 
the duration of the event at each level of intensity. 
 
8.4.7.2 
Relationship to Study Intervention 
For each reported adverse reaction, the Principal Investigator or designated clinical investigators 
will make the determination of relation as defined by:  
• Related - The AE is known to occur with the study intervention, there is a reasonable 
possibility that the study intervention caused the AE, or there is a temporal relationship 
between the study intervention and event. Reasonable possibility means that there is 
evidence to suggest a causal relationship between the study intervention and the AE. 
• Not Related – There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study 
intervention and event onset, or an alternate etiology has been established. 
 
8.4.8 
Time Period and Frequency for Event Assessment and Follow-Up 
For this study, the following time periods for reporting of AEs will be followed: 
1. Solicited AEs (reactogenicity events): From the time of vaccination on Day 1 until Day 8, 
for the first vaccination; and from the time of the second vaccination until 7 days later, 
for those participants who receive 2 vaccinations (Cohorts 4, 5, 6 and 7).  
2. Unsolicited non-serious AEs (medically attended and not medically attended): From the 
time of vaccination on Day 1 until Day 29, for the first vaccination; and from the time of 
the second vaccination until 28 days later, for those participants who receive 2 
vaccinations (Cohorts 4, 5, 6, and 7).  
3. AESIs (wheezing episodes): From the time of each vaccination until 28 days later, 
parents of participating children will be asked at every interaction with study team about 
wheezing episodes and all episodes will be evaluated as described in Appendix B – 
Evaluation Criteria for Acute Wheezing. For events occurring beyond 28 days after each 
vaccination, the parents will have been counseled to notify the study team, and all 

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relevant data on the wheezing episode will be collected, but there will be no other active 
attempts at surveillance.  
4. NOCMCs, and SAEs: From the time of vaccination on Day 1 until the final study visit (in 
the month of April of the calendar year following enrollment) for all cohorts.  
 
8.4.9 
Adverse Event Reporting 
 
8.4.9.1 
Investigators Reporting of AEs 
Information on all AEs should be recorded on the eCRF. All clearly related signs, symptoms, 
and results of diagnostic procedures performed because of an AE will be grouped together and 
recorded as a single diagnosis. If the AE is a laboratory abnormality that is part of a clinical 
condition or syndrome, it will be recorded as the syndrome or diagnosis rather than the 
individual laboratory abnormality. Each AE will also be described in terms of duration (start 
and stop date), severity, association with the study product, action(s) taken, and outcome. 
 
8.4.10 
Serious Adverse Event Reporting 
 
8.4.10.1 
Investigators Reporting of SAEs 
Any AE that meets a protocol-defined serious criterion must be submitted immediately (within 
24 hours of site awareness) on an SAE form to the DMID Pharmacovigilance Group, at the 
following address:  
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX Number: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
 
In addition to the SAE form, select SAE data fields must also be entered into the DCC system. 
Please see the protocol-specific MOP for details regarding this procedure.  
 
Other supporting documentation of the event may be requested by the DMID Pharmacovigilance 
Group and should be provided as soon as possible. The DMID Medical Monitor will review and 
assess the SAE for regulatory reporting and potential impact on study participant safety and 
protocol conduct. 
 
At any time after completion of the study, if the site principal investigator or appropriate sub-
investigator becomes aware of an SAE that is suspected to be related to study product, the site 
principal investigator or appropriate sub-investigator will report the event to the DMID 
Pharmacovigilance Group. 
 
8.4.10.2 
Regulatory Reporting of SAEs 
Following notification from the site Principal Investigator or appropriate sub-investigator, 
DMID, as the IND sponsor, will report any suspected unexpected serious adverse event 
(SUSAR) as an IND safety report to the FDA and will notify all participating site Principal 

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Investigators (i.e., all Principal Investigators to whom the sponsor is providing drug under its 
IND(s) or under any Principal Investigator’s IND(s)) of potential serious risks from clinical 
studies or any other source, as soon as possible. DMID will report to the FDA any unexpected 
fatal or life-threatening suspected adverse reaction as soon as possible, but in no case later than 7 
calendar days after the sponsor’s initial receipt of the information. If the event is not fatal or life-
threatening, the IND safety report will be submitted within 15 calendar days after the sponsor 
determines that the information qualifies for reporting as specified in 21 CFR Part 312.32.  
Relevant follow up information to an IND safety report will be submitted as soon as the 
information is available. Upon request from FDA, DMID will submit to the FDA any additional 
data or information that the agency deems necessary, as soon as possible, but in no case later 
than 15 calendar days after receiving the request.   
 
SAEs that are not SUSARs will be reported to the FDA at least annually in a summary format 
which includes all SAEs.   
 
8.4.11 
Reporting Events to Participants 
Not applicable  
 
8.4.12 
Adverse Events of Special Interest 
Early clinical data indicates Sing2016 M2SR H3N2 vaccine is well tolerated in study 
participants. However, we will collect information about one AESI: wheezing.  This AESI will 
be collected from the time of each study vaccination until the last visit for each individual 
participant. Details about wheezing and uniform collection of data are found in Appendix B – 
Evaluation Criteria for Acute Wheezing.   
 
All AESIs will be assessed, recorded, and followed as described above under adverse events.  In 
addition, AESIs will be reported on an SAE form to the DMID Pharmacovigilance Group, at the 
following address:  
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX Number: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
 
8.4.13 
Reporting of Pregnancy 
All pregnancies that occur among study participants while enrolled in the study will be reported. 
If a female becomes pregnant before receiving study product, no product will be given, and her 
participation will end. Since the only cohort that includes females of childbearing potential is 
Cohort 1, and participants in this cohort are scheduled to receive a single dose of study product, 
it is not expected that a female scheduled for 2 doses of study product will become pregnant after 
one dose. For a female who becomes pregnant, we will request permission to continue to follow 
her, per protocol, for tolerability, safety, and immunogenicity. She may decline any or all 
subsequent study follow-up. We will also request to follow her pregnancy and record the 
outcome. This follow-up will include pregnancy outcome (termination, pre-term birth, term 

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birth) and newborn outcome (live birth, fetal demise, stillbirth; presence of any congenital 
anomalies). No in-person visits will be required for pregnancy outcome determination.  
 
8.5 
Unanticipated Problems 
 
8.5.1 
Definition of Unanticipated Problems (UP) 
The Department of Health and Human Services Office for Human Research Protections (OHRP) 
considers unanticipated problems involving risks to participants or others to include, in general, 
any incident, experience, or outcome that meets all the following criteria: 
• Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the Institutional Review 
Board (IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied; 
• Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and 
• Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
 
8.5.2 
Unanticipated Problem Reporting 
The investigator will report unanticipated problems (UPs) to the reviewing Institutional Review 
Board (IRB) and to the Statistical and Data Management Coordinating Center (SDMCC)/study 
sponsor and the lead principal investigator (PI). The UP report will include the following 
information: 
 
• Protocol identifying information: protocol title and number, PI’s name, and the IRB 
project number; 
• A detailed description of the event, incident, experience, or outcome;  
• An explanation of the basis for determining that the event, incident, experience, or 
outcome represents an UP;  
• A description of any changes to the protocol or other corrective actions that have been 
taken or are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following 
timeline:   
 
• UPs that are serious adverse events (SAEs) will be reported to the IRB and as per Section 
8.4.7 Serious Adverse Event Reporting within 24 hours of the investigator becoming 
aware of the event.  
• Any other UP will be reported to the IRB as per IRB guidelines and to the DCC/study 
sponsor within three days of the investigator becoming aware of the problem.  
 
8.5.3 
Reporting Unanticipated Problems to Participants 
Not applicable  
 

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9. 
STATISTICAL CONSIDERATIONS 
 
9.1 
Statistical Hypotheses 
This study is not designed to achieve pre-determined levels of power or precision to address the 
primary, secondary, or exploratory objectives. The power and precision allowed by the study’s 
sample size are discussed in Section 9.2, and the general methodology planned to address study 
objectives is described in Section 9.4. Specific statistical hypotheses and details about planned 
tables, figures, and data listings will be presented in a separate statistical analysis plan. 
 
9.2 
Sample Size Considerations 
Table 5 gives the probability of observing one or more safety events, such as a reactogenicity 
event or an adverse event of a particular classification, for varying sample sizes, given 
underlying true event probabilities. 
 
Table 5: Probability (%) of observing at least one safety event, given varying underlying event 
probabilities and sample sizes 
Group 
Group Size 
Underlying 
Event 
Probability 
 
 
 
 
 
0.01%  
(Very Rare) 
0.1% (Rare) 
1% 
(Uncommon) 
10% 
(Common) 
Cohorts 5-6 
lead-in safety 
review, 
Cohorts 6-7 
placebo 
N=8 
0.08 
0.797 
7.73 
57 
Cohort 3-4 
placebo 
N=10 
0.1 
1 
9.6 
65.1 
Individual 
placebo 
group or 
Cohort 3-4 
vaccine 
group 
N=15 
0.15 
1.5 
14 
79.4 
Individual 
vaccine 
group in 
Cohorts 6-7 
N=18 
0.18 
1.78 
16.5 
85 
Cohorts 1-2 
safety review 
N=25 
0.25 
2.5 
22.2 
92.8 
Individual 
vaccine 
group in 
Cohorts 1-2; 
N=30 
0.3 
3 
26 
95.8 

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Cohorts 3-4 
aggregate 
vaccine 
groups, post 
first-dose 
Any 
Sing2016 
M2SR H3N2 
Vaccine, 
Cohorts 1-4 
N=90 
0.9 
8.6 
59.5 
>99.9 
Any 
Sing2016 
M2SR H3N2 
Vaccine 
N=132 
1.31 
12.4 
73.5 
>99.9 
 
 
Among the 25 participants in each of Cohorts 1-2 considered for the safety reviews prior to 
enrolling the subsequent cohorts, there will be a 22.2% chance of observing at least one 
uncommon event and a 92.8% chance of observing at least one common event (bolded and 
underlined in Table 5). 
 
The primary objective of this study is to determine the safety and tolerability of the Sing2016 
M2SR H3N2 influenza vaccine, which will be assessed via the incidence of adverse events of 
different types.  While the study was not designed to achieve a pre-determined level of precision, 
confidence intervals will be estimated for some of the binary endpoints. Table 6 presents the 
exact 95% Clopper-Pearson confidence intervals that would result from observing varying 
numbers of events in groups of possible interest. 
 
 
 

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Table 6: Estimated event probabilities (%) and associated exact 95% Clopper-Pearson 
confidence intervals resulting from varying numbers of events observed in groups of different 
sizes. 
Group Size 
 
Number of Events Observed 
 
0 
4 
5 
7 
9 
12 
15 
45 
66 
Cohorts 5-6 
lead-in safety 
review, 
Cohorts 6-7 
placebo 
(N=8) 
0 
(0, 
36.
9) 
50 
(15.7, 
84.3) 
62.5 
(24.5, 
91.5) 
87.5 
(47.3, 
99.7) 
- 
- 
- 
 
 
 
 
 
- 
- 
Cohort 3-4 
placebo 
(N=10) 
0 
(0, 
30.
8) 
40 
(12.2, 
73.8) 
50 
(18.7, 
81.3) 
70 
(34.8, 
93.3) 
90 
(55.5, 
99.7) 
- 
- 
 
 
 
- 
- 
Individual 
placebo 
group or 
Cohort 3-4 
vaccine group 
(N=15) 
0 
(0, 
21.
8) 
26.7 
(7.8, 
55.1) 
33.3 
(11.8, 
61.6) 
46.7 
(21.3, 
73.4) 
60 
(32.3, 
83.7) 
80 
(51.9, 
95.7) 
100 
(78.2, 
100) 
 
 
 
 
 
- 
- 
Individual 
vaccine group 
in Cohorts 6-
7 (N=18) 
0 
(0, 
18.
5) 
22.2 
(6.4, 
47.6) 
27.8 
(9.7, 
53.5) 
38.9 
(17.3, 
64.3) 
50 (26, 
74) 
66.7 
(41, 
86.7) 
83.3 
(58.6, 
96.4) 
 
 
 
- 
- 
Cohorts 1-
2safety 
review 
(N=25) 
0 
(0, 
13.
7) 
16 
(4.5, 
36.1) 
20 
(6.8, 
40.7) 
28 
(12.1, 
49.4) 
36 (18, 
57.5) 
48 
(27.8, 
68.7) 
60 
(38.7, 
78.9) 
 
 
 
- 
- 
Individual 
vaccine group 
in Cohorts 1-
2; Cohorts 3-
4 aggregate 
vaccine 
groups, post 
first-dose 
(N=30) 
0 
(0, 
11.
6) 
13.3 
(3.8, 
30.7) 
16.7 
(5.6, 
34.7) 
23.3 
(9.9, 
42.3) 
30 
(14.7, 
49.4) 
40 
(22.7, 
59.4) 
50 
(31.3, 
68.7) 
 
 
 
 
 
 
 
 
- 
- 
Any 
Sing2016 
M2SR H3N2 
Vaccine, 
Cohorts 1-4 
(N=90) 
0 
(0, 
4) 
 
 
 
4.4 
(1.2, 
11) 
5.6 
(1.8, 
12.5) 
7.8 
(3.2, 
15.4) 
10 
(4.7, 
18.1) 
13.3 
(7.1, 
22.1) 
16.7 
(9.6, 
26) 
 
 
 
50 
(39.3, 
60.7) 
73.3 
(63, 
82.1) 

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Any 
Sing2016 
M2SR H3N2 
Vaccine 
(N=132) 
0 
(0, 
2.8
) 
3.0 
(0.8, 
7.6) 
3.8 
(1.2, 
8.6) 
5.3 
(2.2, 
10.6) 
6.8 
(3.2, 
12.5) 
9.1 
(4.8, 
15.3) 
11.4 
(6.5, 
18) 
 
 
 
 
- 
50.0 
(41.2, 
58.8) 
For illustration, if zero events of a certain type were observed in an individual vaccine group or 
in Cohorts 3-4 aggregate vaccine groups after one dose with N=30 participants, true event 
probabilities above 11.6% could be ruled out at the α=0.025 level, compared to 2.8% in the 
combined vaccine groups (N=132).  Binomial confidence intervals are widest at 50% events 
observed; the estimates associated with approximately (or exactly) 50% observed events are 
bolded in Table 6.  Within an individual vaccine group or in Cohorts 3-4 aggregate vaccine 
groups after one dose with N=30 participants, the maximum half-width for a 95% CI would be 
18.7%, compared to 10.7% across active vaccine groups in Cohorts 1-4. In the case of substantial 
loss to follow-up or missed visits, these estimates give an idea of the loss of precision due to 
decreasing the effective sample size. 
 
While no formal hypothesis testing is planned for this study, some limited statistical testing may 
be conducted to aid interpretation of the strength of the results.  Comparisons would be primarily 
focused on vaccine to placebo within a given cohort, due to the likelihood of confounding in 
most results due to age, dose level, or the number of doses (see Section 9.4.1 for details on 
analysis groupings).   
 
Table 7 presents the probability of a binary event of interest (e.g., adverse event, reactogenicity, 
seroconversion, etc.) in a vaccine group of size N=30 (Cohorts 1 or 2) that would be detectable 
with 80% or 90% power given varying event probabilities in a placebo group of size N=15, using 
two-sided Barnard’s exact tests at the α=0.05-level. 
  
Table 7: Detectable probabilities (%) with 80% or 90% power when comparing binary event 
probabilities between groups with two-sided Barnard’s exact tests with α=0.05. 
Probability in Placebo Group 
(N=15) 
Detectable Probability in Vaccine Group  
(N=30) 
 
80% power 
90% power 
0.1% (Rare) 
29.0 
32.9 
1% (Uncommon) 
31.5 
36.5 
10% (Common) 
48.4 
54.4 
50% (Very Common) 
88.2 
92.4 
 
There would be 80% power to detect a difference of 29% between vaccine and placebo groups 
for a rare event (0.1% probability) in the placebo group, compared to a difference of at least 48% 
for a common or very common event in the placebo group. 
 
9.3 
Populations for Analyses 
• The enrolled population will include all participants enrolled and randomized 

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• The Safety population will include all participants who receive at least one study 
influenza vaccination. 
• The Modified Intent-to-Treat (mITT) population will consist of all participants who 
received study influenza vaccination and reported baseline and at least one post-baseline 
immunogenicity result. 
• The Per-Protocol (PP) population will include participants in the mITT population with 
the following exclusions of data: 
o Data from participants determined to be ineligible at baseline (determined 
thereafter) 
o Data from participants in Cohorts 4, 5, 6, and 7 subsequent to missed second 
study vaccinations 
o Data from any visit that occurs substantially out of window as defined in the SAP 
o Data from all visits subsequent to major protocol deviations, as defined in the 
SAP, that could impact the validity of later data, such as: 
§ Receipt of immunosuppression or any medications that may be associated 
with impaired responsiveness 
§ Receipt of any non-study investigational drug/investigational 
vaccine/licensed vaccine 
 
The enrolled population will be used for summaries of participant disposition as well as 
participant demographics and baseline characteristics. The Safety population will be used for all 
analyses of adverse events and reactogenicity.  Immunogenicity analyses will be done in the 
mITT population, and if sufficient data (>10% of secondary analysis observations) would be 
excluded by the PP exclusions, the PP population will be used for sensitivity analyses. Certain 
analyses may be done on subsets of these populations, such as by-dose analysis, where 
applicable, and these would be specified in the analysis plan. 
 
9.4 
Statistical Analyses 
The primary clinical database for this study will consist of safety (adverse events and 
reactogenicity) and baseline/demographic data. Once the last participant has completed the final 
visit, the primary clinical database will be cleaned, monitored, and locked. After clinical 
database lock and receipt of secondary immunogenicity data, a set of topline tables will be 
generated by the Statistical and Data Management Coordinating Center (SDMCC), including 
summaries of clinical safety and secondary immunogenicity data. The topline report will be 
made available to the study team for planning subsequent trials and may be presented in a public 
forum or used for publication in collaboration with lead PI. These analyses will be considered 
final and will be included in the Clinical Study Report (CSR) as well. The CSR, comprised of the 
final analyses of safety and available immunological data, will be subsequently completed. Any 
available data from the exploratory endpoints may also be included.  Additional exploratory 
endpoint data not available at the time of CSR preparation may be included in one or more 
addenda to the CSR, manuscript(s), or other report. A formal Statistical Analysis Plan (SAP) that 
specifies all planned analyses will be finalized prior to generating the topline report and CSR.   
 

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In addition to the descriptive analyses of study data for SRC and SMC review, an interim 
analysis is planned for while the study is ongoing to allow for an early assessment of the humoral 
immunogenicity of the study vaccine and to aid in decision-making regarding future studies.  
These analyses would not affect the conduct of this trial via early stopping, and participant-level 
unblinding will not occur until the clinical database is locked.  Additional details on interim 
analyses are given in Section 9.4.6. 
 
The formal SAP will elaborate on the analyses described here. 
 
9.4.1 
General Approach 
For descriptive analyses, continuous variables will be summarized by the non-missing sample 
size, mean, standard deviation, and the minimum, first quartile, median, third quartile, and 
maximum.  Categorical variables will be summarized by frequencies and percentages of 
observed levels, based on the non-missing sample size. Titers will be summarized using the 
geometric mean, first across replicates to compute one value for each individual sample and then 
applied again across participants within the same group. 
 
Generally, summaries will be presented by analysis group including cohort and vaccination 
group (i.e., Cohort 1 – active, Cohort 1 – placebo, Cohort 2 – active, etc., called “group” 
throughout this section). Post-first dose safety and immunogenicity results for active Cohorts 
3+4 will be combined as both cohorts receive the same dose and belong to the same age group. 
Additional analysis groupings may be specified in the analysis plan. 
 
For safety endpoints, summaries may also include a column for all participants receiving active 
study vaccination and all participants receiving placebo. Data listings will be presented sorted by 
clinical site or other grouping variable, participant, and then by visit number within participant, 
where applicable. All tables will be annotated with the total population size relevant to that table, 
including any missing observations. The analysis population for each exhibit will be clearly 
indicated. 
 
Prior receipt of IIV4, season of prior receipt of IIV4, and time between prior receipt of IIV4 and 
study vaccination, as well as receipt of the COVID-19 vaccine during the study period and the 
timing of receipt of the COVID-19 in relation to study vaccination may be considered in 
exploratory analyses to adjust for potential confounding.  
 
The study was not designed for any formal group comparisons, but hypothesis testing may be 
performed for some endpoints and would be considered exploratory.  Adjustments for 
multiplicity or more stringent thresholds for significance than the typical α=0.05 type-I error may 
be used for immunogenicity analyses involving numerous endpoints. Details on any hypothesis 
testing will be described in the standalone SAP. 
 
9.4.2 
Analysis of the Primary Endpoint(s) 
The primary objective of the study is to assess the safety and tolerability of 1 or 2 doses of the 
study vaccination, as determined by the incidence of solicited reactogenicity events, unsolicited 

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serious and non-serious adverse events, adverse events of special interest, and new-onset chronic 
medical conditions. 
 
Safety objectives will be assessed in the Safety population.  The vaccine and placebo groups 
from Cohorts 3 and 4 will be combined for post-first dose analyses and presented separately after 
the second dose in Cohort 4.  Solicited local and systemic reactogenicity events will be 
summarized via the number and percentage of participants reporting each event, and any 
solicited reactogenicity event, within the 7 days following each dose in each group.  These will 
be tabulated by severity, by dose, and post-any dose.  Exact 95% Clopper-Pearson confidence 
intervals for the probability of each event post-each and any dose will be computed for each 
group.  The frequency of each solicited event will be summarized as well.  Severity will be 
determined as described in Section 8.4.3. 
 
Non-serious unsolicited adverse events and adverse events of special interest occurring within 
the 28 days post-each dose and post-either dose will be summarized by MedDRA system organ 
class (SOC) and preferred term (PT), severity, and dose, as well as post-any dose.  Serious 
adverse events and new-onset chronic medical conditions reported through the end of the study 
period will be summarized similarly.  Abnormal clinical laboratory values will be considered 
unsolicited adverse events and graded as in Section 8.4.2, and these will be summarized 
separately.  For unsolicited events, summaries will also be made by relationship to study vaccine, 
as described in Section 8.4.4. 
 
Adverse events and clinical laboratory results will be presented in data listings as well. 
 
9.4.3 
Baseline Descriptive Statistics 
Baseline and demographic characteristics will be summarized by group, and separately by 
clinical site, with summary statistics as described in Section 9.4.1.  These characteristics will 
include age, sex, ethnicity, race, and prior receipt of seasonal vaccination. 
 
9.4.4 
Analysis of the Secondary Endpoint(s) 
Humoral immunogenicity of the study vaccination will be assessed via hemagglutination 
inhibition (HAI) in serum, neutralization (Neut) in serum; and secretory IgA (sIgA) as measured 
by the binding antibody multiplex assay (BAMA), assays against the homologous strain of H3N2 
M2SR-like virus.  These assays will be analyzed separately by analysis groups defined in Section 
9.4.4.   
 
HAI and Neut titers will be summarized by analysis group at baseline and  Day 29or Day 57 
using the geometric mean (GMT) and the number and proportion of participants reporting a 
titer ≥ 1:40 (putative seroprotection for HAI).  For post-baseline time points, the geometric 
mean fold-rise (GMFR) and the number and proportion of participants reporting at least 2-fold 
and at least 4-fold rises from baseline will also be presented.  Associated 95% confidence 
intervals will be computed for each of these, using the t-distribution for GMTs and GMFRs and 
exact Clopper-Pearson binomial CIs for the binary endpoints. Bootstrap confidence intervals 
may be calculated for continuous endpoints instead if the t-distribution is determined to be 

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unsuitable for continuous endpoints. Additional details of analyses of secondary endpoints will 
be provided in the SAP.  
 
9.4.5 
Analysis of the Exploratory Immunogenicity Endpoints 
The exploratory objectives of the study involve further characterization of the immunogenicity of 
the study vaccine and the seasonal IIV4 vaccine given to each participant after the study 
vaccination(s). These will include additional assessments of humoral responses as well as 
cellular immunogenicity (T cell responses). 
 
For analysis of exploratory immunological endpoints, immune responses will be described by 
time point and analysis group, similarly to the summaries of secondary immunological outcomes.  
For titer data, geometric means will be computed for each group at each time point (GMTs), and 
geometric mean fold-rise (GMFR) at post-baseline visits will be calculated as well, where 
applicable. Confidence intervals corresponding to these statistics will be computed for each 
group, but formal hypothesis testing is not planned for descriptive summaries of exploratory 
endpoints. Other immunological endpoints will include frequencies of T cell subsets, which will 
be described by mean or median frequencies within each group and the associated 95% CIs 
depending on distributional properties. Additional details will be provided in the SAP.  
 
Responses in serum will be compared with analogous responses in plasma. These analyses will 
be described in the SAP. 
 
Exploratory analyses of humoral immunogenicity assays will include statistical modeling of 
seroprotection and/or appropriately transformed titers over time, while accounting for age, dose 
level, sex, and prior seasonal vaccination status. These analyses will be described in the SAP, 
along with further details for analyses of exploratory immunogenicity endpoints and exploratory 
analyses of secondary immunogenicity endpoints. 
 
9.4.6 
Planned Interim Analyses 
 
9.4.6.1 
Interim Safety Analyses 
There are two safety committees that will be convened for this study: The Safety Review 
Committee (SRC) and the Safety Monitoring Committee (SMC).  
 
The SRC will review blinded safety data to allow for progression through the cohorts as 
described in the study design section, Section 4.1. The SMC will review safety data if criteria for 
progression to enrollment of a subsequent cohort is not met, a halting rule (as defined in Section 
1.1) occurs, or as regularly scheduled per the separate SMC charter. 
 
Interim safety reviews may include enrollment and demographic information, clinical laboratory 
tests, and AE/SAEs. During SMC reviews, additional data may be requested by the SMC.  The 
reviews will be blinded to study assignment, with unblinded study assignments available upon 
SMC request. As an outcome of each SMC review, the SMC will make a recommendation to 
continue, modify, or terminate this study. 

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9.4.6.2 
Interim Analyses 
As described in Section 9.4, an interim analysis focused primarily on immunogenicity will be 
conducted while the study is ongoing. These analyses, including primary, secondary and 
exploratory endpoints, may be made available to the pharmaceutical partner for planning for 
future studies, with no public dissemination of results, and will not be used to make any 
decisions concerning the conduct of this trial.  Analyses from specimens (serum, plasma, nasal 
washes) collected through the Day 29 visit (Visit 4) for Cohorts 1 to 3 and through D57 visit 
(Visit 4C) for others will be presented in aggregate by cohort and vaccine/placebo. Interim safety 
analyses through the Day 29 visit (Visit 4) for Cohorts 1 to 3 and through D57 visit (Visit 4C) 
for others will only be presented aggregated by cohort, to avoid the possibility of unblinding due 
to rare adverse events occurring only in active vaccine or placebo groups.  
 
9.4.7 
Sub-Group Analyses 
Most analyses will be conducted by age group (i.e., by Cohort). Other exploratory analyses may 
be conducted by sex or by previous seasonal vaccination status, to determine potential 
confounders other than age that could impact safety, reactogenicity, or immunogenicity of the 
study vaccination. 
 
9.4.8 
Tabulation of Individual Participant Data 
Safety data, reactogenicity data, and immunogenicity data will be presented in listings, as 
described in Section 9.4.1. 
 
10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
 
10.1 
Regulatory, Ethical, and Study Oversight Considerations 
Each site principal investigator will obtain IRB approval for this protocol and any amendments 
to be conducted at his/her research site(s) followed by DMID must receive the documentation 
that verifies IRB/IEC-approval for this protocol, informed consent, and associated documents 
prior to the recruitment, screening, and enrollment of participants. Study activities may not begin 
until all approvals including both DMID as well as IRB are attained. 
 
10.1.1 
Informed Consent Process 
Before any study procedures are performed, parental permission and participant assent, where 
appropriate, will be obtained and documented for all study participants. All participants undergo 
a consent process that includes both a verbal discussion of study information as well as written 
documentation. The key information provided will include the purpose of the study, the 
procedures and experimental aspects of the study, study interventions/products, probability for 
random assignment to treatment groups, risks and discomforts, the expected duration of the 
participant’s participation in the trial, any expected benefits to the participant, and alternative 
treatments and procedures that may be available to the participant. The explanation will be 
organized and presented in lay terminology and using language that facilitates understanding 

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why one might or might not want to allow one’s child to participate or, in the case of assenting 
minors, why they themselves might or might not want to participate. 
 
The parents/guardians of participants will be informed that records identifying the participant 
will be kept confidential, and, to the extent permitted by the applicable laws and/or regulations, 
will not be made publicly available and, if the results of the trial are published, the participant’s 
identity will remain confidential. The parents/guardians of participants will be informed whether 
private information collected from this research and/or specimens will be used for additional 
research, even if identifiers are removed.  
 
The parents/guardians of participants will be informed that the monitor(s), auditors(s), IRB, 
NIAID, and regulatory authority(ies) will be granted direct access to the participant’s original 
medical records for verification of clinical trial procedures and/or data without violating the 
confidentiality of the participant, to the extent permitted by the applicable laws and regulations, 
and that, by signing a written parental permission form, the parent/guardian of the participant is 
authorizing such access.  
 
The parents/guardians of participants, and participants themselves when assenting, will be 
allowed sufficient time to consider participation in this research trial and have the opportunity to 
discuss this trial with their family, friends, or legally authorized representative, or think about it 
prior to agreeing to participate. 
 
Parental permission forms and assent forms for children will be IRB-approved, and participants 
will be asked to read and review the written forms. Parents/guardians of participants will sign the 
parental permission form, and participants will sign the written assent form, as required, prior to 
starting any study procedures being done specifically for this trial. Once signed, a copy of the 
forms will be given to the parent/guardian of the participant(s) for their records. 
   
New information will be communicated by the site principal investigator to parents/guardians of 
participants who provide permission for their children to participate in this trial in accordance 
with IRB requirements.  The parental permission document and assent forms will be updated, 
and parents/guardians of participants will be asked to provide updated permission per IRB 
requirements, if necessary.  Participants may be asked to re-assent, if required by the IRB, 
following updated information.  
 
The participant’s parent or guardian will be asked to consent for future use of specimens for 
secondary research; genetic testing on these samples may be performed. Please refer to Section 
10.1.4.1. 
 
10.1.1.1 
Requirements for Permission by Parents/Guardians and Assent by Children (in 
case of a minor)  
The study proposes to enroll participants 6 months to 17 years of age. Investigators or their 
designee will conduct the consent process with the parent(s)/legal guardian, who will be given an 
IRB/IEC-approved permission form, which may be referred to as a consent form, to read, review, 
and sign prior to any study procedures. In addition, assent will be obtained in accordance with 
the approval by the site or central IRB. 

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The participant who reaches the age of majority or becomes emancipated during the study will 
be consented at the next visit prior to study procedures being performed.  When no further visits 
are planned but the participant’s participation is ongoing, the consent will be obtained via 
IRB/IEC-approved processes. 
 
10.1.1.2 
Other Informed Consent Procedures 
The rights and privacy of human participants who participate in genomic or phenotypic research 
studies will be protected at all times. The consent process, including relevant language in the ICF 
will provide an explanation of the potential risks to the parents of the individual study 
participants. The consent will include whether individual participant data will be shared through 
a NIAID-designated controlled access data repository. Clinical metadata, genomic, or other 
datasets or a subset of the clinical and other metadata that may potentially identify human 
participants will not be released in unrestricted databases. Parents of participants will be 
informed that the evolution of genomic technology and analytical methods raises the risk of re-
identification, even when specimens are de-identified. The parents of the participants will be 
asked to consent specifically to genetic testing that may be performed under secondary human 
participant research. 
 
10.1.2 
Study Termination and Closure 
Section 7, Study Intervention Discontinuation and Participant Discontinuation/Withdrawal 
describes the temporary halting of the study. 
 
This study may be prematurely terminated if there is sufficient reasonable cause, including but 
not limited to: 
• Determination of unexpected, significant, or unacceptable risk to participants 
• Insufficient compliance to protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Regulatory authorities determine it will be terminated 
 
If the study is prematurely terminated, the Principal Investigator (PI) will promptly inform study 
participants and the Institutional Review Board (IRB) and regulatory authorities as applicable. 
Study participants will be contacted, as applicable, and be informed of changes to study visit 
schedule. The PI will ensure appropriate follow-up for the participants, as necessary. 
 
The sponsor will notify regulatory authorities as applicable.   
  
10.1.3 
Confidentiality and Privacy 
Participant confidentiality is strictly held in trust by the participating investigators, their staff, 
and the sponsor(s) and their agents. This confidentiality is extended to cover clinical information 
relating to participants, test results of biological samples, and all other information generated 
during participation in the study. No identifiable information concerning participants in the study 
will be released to any unauthorized third party. Participant confidentiality will be maintained 
when study results are published or discussed in conferences.   
 

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The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the participants in this study.  The 
clinical study site will permit access to such records. 
 
All source records including electronic data will be stored in secured systems in accordance with 
institutional policies and federal regulations.   
 
All study data and research specimens that leave the site (including electronic transmission of 
data) will be identified by a coded number that is linked to a participant through a code key 
maintained at the clinical site.  The code key, including names or readily identifiable information 
is kept confidential and will not be transmitted off site. 
 
As this research is funded by the NIH, it is covered by NIH policy which effectively issues the 
research a Certificate of Confidentiality. By this policy, researchers cannot be forced to disclose 
or provide, in any Federal, State, or local civil, criminal, administrative, legislative, or other 
proceeding, the name of such individual or any such information, document, or biospecimen that 
contains identifiable, sensitive information about the individual and that was created or compiled 
for purposes of the research, unless such disclosure or use is made with the consent of the 
individual to whom the information, document, or biospecimen pertains. 
 
The Certificate cannot be used to resist a demand for information from personnel of the United 
States Government that is used for auditing or evaluation of federally funded projects, like this 
study, or for information that must be released to meet the requirements of the Federal Food and 
Drug Administration (FDA). 
 
A Certificate of Confidentiality does not prevent the participant or the participant’s parent or 
guardian from voluntarily releasing information about themselves or their child or their or their 
child’s involvement in this research. If any person or agency obtains a written consent to receive 
research information, then the researchers may not use the Certificate to withhold that 
information.   
 
The Certificate of Confidentiality does not prevent the researchers from reporting without the 
participant’s or participant’s parent or guardian’s consent, information that would identify the 
participant as a participant in the research project regarding matters that must be legally reported 
including child and elder abuse, sexual abuse, or wanting to harm themselves or others.   
 
Participant study information will not be released without the written permission of their 
parent/guardian, except as necessary for oversight by the protocol Principal Investigator or 
designee, the study funders, and applicable regulatory bodies. 
 
All research laboratory specimens, evaluation forms, reports, and other records that leave the site 
will be identified by coded number only to maintain participant confidentiality. 
 
10.1.4 
Future Use/Secondary Use of Stored Specimens and Data  

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Secondary Human Participant Research is the re-use of identifiable data or identifiable 
biospecimens that were collected from some other ‘‘primary’’ or ‘‘initial’’ activity, such as the 
data and samples collected in this protocol. This section will detail the samples and data 
available for secondary research. Any use of the sample or data, however, will be presented in a 
separate protocol and require separate IRB approval.   
 
10.1.4.1 
Samples for Secondary Research 
Leftover research samples not required for the laboratory testing specified in this protocol will be 
stored for potential future studies with the subject’s consent. Specimens will be coded. Any 
future testing laboratory will not have access to the code, and therefore will not be able to 
identify study participants. Genetic testing on these samples may be performed with 
corresponding permissions as part of the Biorepository consent (DMID 19-0025).  
 
The following types of samples will be stored and used for secondary research: 
• Residual biological specimens- Any leftover primary research sample after laboratory 
testing is completed per protocol and which the protocol explicitly states is allowed to be 
stored and used for secondary research, and for which the research participant or the 
participant’s parent or legal guardian gave specific consent. 
• Repository research sample- Samples will be collected with the participant’s or the 
parent/guardian’s consent in this protocol with the intent to store for additional research 
(i.e., samples collected beyond those needed for primary research) and will be used in 
future studies. 
 
In addition to this protocol, participants and their parent/guardian will be asked to consent to a 
separate biorepository protocol (DMID 19-0025) for the use of residual samples and associated 
data in future research. 
 
Participants and their parent/guardian will be asked for consent for storage of samples for 
secondary use. Samples will be stored indefinitely at a DMID-designated storage facility. Each 
sample will be encoded (labeled) only with a barcode and a unique tracking number to protect 
subject confidentiality. Secondary research with coded samples and data may occur, however, 
subject confidentiality will be maintained as described for this protocol. An IRB review of the 
secondary research using coded specimens is required. 
 
Residual/Repository Research Samples, upon written request and approval from DMID and any 
approvals required by the site, may be shared for secondary research with investigators at the 
participating site, with researchers at other CIVICs sites or other institutions, or company-
designated research laboratories. The samples will not be sold or used directly for production of 
any commercial product. DMID will authorize shipment from the repository. 
 
Reports from secondary research will not be kept in the participants’ health records or shared 
with participants or their parents/guardians, unless required by law. Reports will not be sent to 
the specimen repository. The participant’s or the parent/guardian’s decision for secondary 
research can be changed at any time by notifying the study doctors or nurses in writing. If the 

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participant changes his/her decision, the samples will be destroyed if the samples have not been 
used for research or released for a specific research project. 
 
10.1.4.2 
Data Sharing for Secondary Research 
Data from this study may be used for secondary research. All of the individual participant data 
collected during the trial will be identified by a coded number that is linked to a participant 
through a code key maintained at the clinical site. The code key is kept confidential and will not 
be transmitted off site. All of the individual participant data collected during the trial will be 
made available after deidentification. The Protocol, Statistical Analysis Plan, and Analytic Code 
will also be made available. These data will be available immediately following publication, with 
no end date.   
 
The investigator may request removal of data on individual study participants from NIH data 
repositories in the event that a research subject withdraws or changes his or her consent. 
However, some data that have been distributed for approved research use cannot be retrieved. 
 
10.1.5 
Key Roles and Study Governance 
This study is sponsored by DMID.  The Principal Investigator, DMID Clinical Project Manager, 
DMID Medical Officer, and DMID Scientific Lead are listed on the cover page. Other study 
team members and roles are listed in the Manual of Procedures (MOP). 
 
10.1.6 
Safety Oversight 
The SRC Safety Review Committee consists of the PI at each participating site (or their 
delegate), representatives of the sponsor (at least the Medical Monitor and the Medical Officer), 
representatives of the manufacturer (at least the Medical Lead or delegate), and representatives 
of the SDMCC. The SRC will review the data required to allow progression from cohort to 
cohort. These reviews occur as described in the study design section, Section 4.1. Ad hoc 
reviews may also be called for by the investigators or sponsor. Decisions are dependent on the 
failure to meet halting criteria when the required follow-up data are available. The SRC 
deliberations may occur by teleconference, videoconference, or email. If the SRC determines 
that the study data have met the criteria necessary to begin a subsequent cohort, the sponsor will 
notify the sites of the determination. If the SRC is unable to determine that the criteria necessary 
to begin a subsequent cohort have been met, or if halting rules have been met, the SMC will 
convene. 
 
The SMC is an independent group of at least 3 experts that monitors participant safety and 
advises DMID. SMC members will be separate and independent of study personnel participating 
in this trial and should not have scientific, financial, or other conflicts of interest related to this 
trial. The SMC will consist of members with appropriate expertise to contribute to the 
interpretation of data from this trial. A quorum will consist of a simple majority. 
 
The SMC will hold an organizational meeting prior to enrollment. At this meeting, the SMC will 
review the charter, protocol, ICF, and safety report template. 
 
The SMC will conduct the following reviews: 
1. Review of safety if a halting rule is met 

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2. Review of safety if the SRC requests a review 
3. Review of safety, tolerability, and trial progress at the following times: after the first 
season of vaccinations, after the second season of vaccinations, at the conclusion of the 
trial 
4. Ad hoc review as needed when requested by the sponsor, investigators, or SMC 
 
Procedures for SMC reviews/meetings will be defined in the SMC charter. The SMC will review 
applicable data including, but not limited to, enrollment, demographics, dosing, data, laboratory 
data, and safety data at scheduled time points during this trial as defined in the SMC charter. The 
SMC will review blinded aggregate data in the open session of the SMC meetings.  Unblinded 
data (grouped by treatment) may be reviewed in the closed session only. When possible, these 
data will be “aliased” in that the SMC will review the data by group, but the identity of the group 
will not be divulged. The SMC may, if necessary, review grouped data in which the identities of 
the groups are divulged.  
 
Additional data may be requested by the SMC, and interim statistical reports may be generated 
as deemed necessary and appropriate by DMID. As an outcome of each review/meeting, the 
SMC will make a recommendation as to the advisability of proceeding with study drug 
administration, and to continue, modify, or terminate this trial. 
 
 
10.1.7 
Clinical Monitoring 
Site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct 
of the trial is in compliance with the currently IRB/IEC-approved protocol/amendment(s), ICH 
GCP, and applicable regulatory requirement(s). Clinical monitoring also verifies any critical 
study procedures are completed following specific instructions in ancillary documents referenced 
in the protocol such as a Manual of Procedures.  
  
Monitoring for this study will be performed by DMID. Details of clinical site monitoring are 
documented in a Clinical Monitoring Plan (CMP). The CMP describes in detail who will conduct 
the monitoring, at what frequency monitoring will be done, at what level of detail monitoring 
will be performed, and the distribution of monitoring reports. Monitoring visits will include, but 
are not limited to, review of regulatory files, accountability records, eCRFs, ICFs, medical and 
laboratory reports, site study intervention storage records, training records, and protocol and 
GCP compliance. Site monitors will have access to each participating site, study staff and all 
study documentation according to the DMID-approved CMP. Study monitors will meet with all 
participating site PIs to discuss any problems and outstanding issues and will document site visit 
findings and discussions.  
 
10.1.8 
Quality Assurance and Quality Control 
To ensure the reliability of study data, the site will develop a Clinical Quality Management Plan 
(CQMP).  The CQMP will describe routine internal quality control (QC) and quality assurance 
(QA) activities for the purposes of measuring, documenting, and reporting study conduct, 
protocol adherence, human participant protections, and reliability of the protocol-driven data 
collected. A process for addressing in a timely manner any data quality issues (for example, 

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collecting, recording, and reporting data), systemic issues (for example, protocol conduct, non-
compliance, human participant protections), and implementation of Corrective and Preventative 
Action (CAPA) procedures. 
 
The SDMCC will implement quality control procedures beginning with the data entry system 
and generate data quality control checks that will be run on the database. Any missing data or 
data anomalies will be communicated to the site(s) for prompt clarification and resolution. 
 
10.1.9 
Data Handling and Record Keeping 
 
10.1.9.1 
Data Collection and Management Responsibilities 
Clinical research data (including, but not limited to, AE/SAEs, concomitant medications, 
medical history, physical assessments), and clinical laboratory data will be abstracted from the 
source documentation and/or collected on source document worksheet by study personnel then 
entered into eCRFs via a 21 CFR Part 11-compliant internet data entry system provided by the 
study data coordinating center. Alternatively, direct data entry may be employed. The data 
system includes password protection and internal quality checks, such as automatic range checks, 
to identify data that appear inconsistent, incomplete, or inaccurate.   
 
Data collection is the responsibility of the study personnel at the participating clinical study site 
under the supervision of the site principal investigator. During the study, the site principal 
investigator must maintain complete and accurate documentation for the study. 
 
The data coordinating center for this study will be responsible for data management, quality 
review, analysis, and reporting of the study data. The IND sponsor is responsible for review of 
data collection tools and processes, and review of data and reports. Adverse events will be coded 
according to the MedDRA dictionary version 23.0 or higher. At the end of the study, a copy of 
all datasets including annotated case report forms and data dictionary will be provided to DMID.  
 
A separate SDSP will be developed which describes the technical recommendations for the 
submission of human study data and related information in a standardized electronic format 
throughout product development. 
 
10.1.9.2 
Study Record Retention 
Study-related records, including the regulatory file, study product accountability records, consent 
forms, participant source documents and electronic records should be maintained for a period of 
2 years following the date a marketing application is approved for the investigational product for 
the indication for which it is being investigated; or, if no application is to be filed or if the 
application is not approved for such indication, until 2 years after the investigation is 
discontinued and FDA is notified. These documents should be retained for a longer period, 
however, if required by local policies or regulations. No records will be destroyed without the 
written consent of DMID. Consent forms with specimen retention linked to identifiable 
specimens will be maintained for as long as the specimens remain in identifiable format, and a 
minimum of three years after use of the identifiable specimens in nonexempt human participant 
research. 

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10.1.9.3 
Source Records 
Source data are all information, original records of clinical findings, observations, or other 
activities documented in a clinical trial necessary for the reconstruction and evaluation of the 
trial.  Each participating site will maintain appropriate medical and research records for this trial, 
in compliance with ICH GCP, regulatory, and institutional requirements.  Data recorded in the 
electronic case report form (eCRF) derived from source documents should be consistent with the 
data recorded on the source documents. All source documents should be completed in a neat, 
legible manner to ensure accurate interpretation of data.   
 
For this trial, investigators may, when necessary, use the electronic medical record as source 
data, but most of the information required will be collected from the parents/guardians and as 
part of the conduct of the study.  
 
10.1.10 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, any process that is 
noted in the protocol and refers to details in the MOP, or GCP requirements.  
Noncompliance may be either on the part of the participant, the investigator, or the study site 
staff. Following a deviation(s), corrective actions should be developed by the site and 
implemented promptly. All individual protocol deviations will be addressed in participant study 
records.   
 
It is the responsibility of the site principal investigator and personnel to use continuous vigilance 
to identify and report deviations within five working days of identification of the protocol 
deviation, or within five working days of the scheduled protocol-required activity. All deviations 
must be promptly reported to DMID per the protocol deviation reporting procedures. Protocol 
deviations must be sent to the local IRB/IEC per their guidelines.  The site principal investigator 
and personnel are responsible for knowing and adhering to their IRB requirements. A completed 
copy of the DMID Protocol Deviation Form must be maintained in the Regulatory File, as well 
as in the participant’s chart if the deviation is participant specific.   
 
10.1.11 
Publication and Data Sharing Policy 
 
10.1.12 
Human Data Sharing Plan 
This study will be conducted in accordance with the following publication and data sharing 
policies and regulations: National Institutes of Health (NIH) Public Access Policy. This policy 
which ensures that the public has access to the published results of NIH-funded research. It 
requires scientists to submit final peer-reviewed journal manuscripts that arise from NIH funds to 
the digital archive PubMed Central upon acceptance for publication. 
 
10.1.13 
Genomic Data Sharing Plan 
Not applicable  
 
10.1.14 
Publication 
Following completion of the study, the Lead Principal Investigator is expected to publish the 
results of this research in a scientific journal. Data from the clinical trial may also be presented in 
CIVICs program seminars and the CIVICs annual meeting, subject to approval by DMID, or 

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used for publication as determined by the PI in collaboration with the study team and in 
accordance with DMID data sharing policy and the Clinical Trial Agreement. This study will 
adhere to the publication and data sharing policies and regulations of the sponsor: NIH Public 
Access Policy. This policy ensures that the public has access to the published results of NIH-
funded research. As such, the final peer-reviewed journal manuscripts will be accessible to the 
public on PubMed Central no later than 12 months after publication. 
 
10.1.15 
Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed 
and managed. Furthermore, persons who have a perceived conflict of interest will be required to 
have such conflicts managed in a way that is appropriate to their participation in the design and 
conduct of this trial. DMID has established policies and procedures for all study group members 
to disclose all conflicts of interest and will establish a mechanism for the management of all 
reported dualities of interest. 
 
10.2 
Additional Considerations 
 
10.2.1 
Research Related Injuries 
If it is determined by the participating site PI that an injury occurred to a participant as a direct 
result of the tests or treatments that are performed for this trial, then referrals to appropriate 
health care facilities will be provided to the participant and his or her parent/guardian. Study 
personnel will try to reduce, control, and treat any complications from this trial. Immediate 
medical treatment may be provided by the participating site, such as giving emergency 
medications to stop immediate allergic reactions due to the study vaccine. No financial 
compensation will be provided to the participant by NIAID, NIH, or the participating site for any 
injury suffered due to participation in this trial. 
 
10.3 
Abbreviations 
 
AE 
Adverse Event 
CAPA 
Corrective and Preventative Action Plan 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
CMP 
Clinical Monitoring Plan 
CMS 
Clinical Material Services 
CRF 
Case Report Form 
CROMS 
Clinical Research Operations & Management Support 
CSR  
Clinical Study Report 
DCC 
Data Coordinating Center 
DMID 
Division of Microbiology and Infectious Diseases 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HA 
Hemagglutinin 

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HAI 
Hemagglutination inhibition 
ICF 
Informed Consent Form 
ICH 
International Council for Harmonization 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
 
 
MITT 
Modified Intention-To-Treat 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
N 
Number (typically refers to participants) 
NA 
Neuraminidase 
NAI 
Neuraminidase Inhibition 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
OHRP 
Office for Human Research Protections 
PHI 
Protected Health Information 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
RVP 
Respiratory Virus Panel 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SRC 
Safety Review Committee 
SOA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
UP 
Unanticipated Problem 
US 
United States 
 
10.4 
Protocol Amendment History 
Version 
Date 
Description of Change  
Brief Rationale 
2.0 
02/11/2022 Minor changes incorporated 
including: 
Clarification on date when the 
study will start, acceptable 
mechanisms of contraception and 
co-enrollment of Cohort 4 & 5. 
The protocol was also further 
updated for consistency and 
additional clarifications provided.  
The protocol builds in 
additional conditions that 
may impact study conduct for 
further clarity/guidance for 
clinical sites 
3.0 
09/07/2022 Minor changes incorporated 
including: Additional clarification 
to avoid enrolling individual 
participants with a history of 
The protocol builds in 
additional clarification to 
study procedures as well as a 
decreased sample size given 

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wheezing episodes and including 
a narrower window for study visit 
2 related to evaluation of 
wheezing reported after receipt of 
study vaccine. Decrease in sample 
size for Cohort 4 from 45 to 25 
with corresponding changes in 
study schema and Statistical 
Considerations section. 
Incorporated study halting criteria 
if influenza activity is greater than 
10% locally or regional ILI 
activity is high. Other minor edits 
incorporated including correction 
of window for receipt of seasonal 
influenza vaccine (in Schedule of 
Activities), wording related to 
dose preparation of IP and 
evaluation of wheezing.  
it does not impact probability 
of identifying unexpected 
adverse reactions to study 
vaccine.  
4.0 
05/26/2023 Change the timing of and required 
reviews by the SRC after Cohort 
5. 
 
 
 
 
 
 
 
 
Descriptions of the antibody test 
for neuraminidase antibodies has 
been changed from NAI, a 
specific type of assay, to anti-NA, 
to allow for anti-neuraminidase 
assays other than NAI 
Allow administration of first 
dose in Cohort 6 after safety 
data of first dose in Cohort 5 
and allow for second doses in 
Cohort 6 to begin after at 
least 6 of 8 in Cohort 5 have 
completed through the third 
day after their second dose. 
 
 
To accommodate the optimal 
assay/s to measure antibodies 
against neuraminidase  
5.0 
12/7/2023 
Minor edits in the study design 
section indicating Cohorts 5-7 
will not be enrolled, clarification 
of endpoints and removal of ISM 
language 
Changes are made to 
accommodate FDA and other 
regulatory bodies 
requirements given non-
enrollment in Cohort 5-7 due 
to a number of non-safety 
related contingencies 
(timelines, budgets, 
recruiting, and lack of 
additional vaccine lots). 

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Other changes include more 
clarity on proposed 
secondary study objectives 
and associated secondary and 
exploratory endpoints. Other 
minor changes include 
clarification on anticipated 
interim analysis and 
publication plans. 
Additionally, given the 
sponsor no longer uses an 
ISM, ISM language has been 
removed from the protocol. 
 
 
 
 
11. 
APPENDIX 
 
11.1 
Appendix A – Blood and Fluid Collection Volumes by age group  
 
 

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Table 8: Blood and Fluid Collection volumes by Age 
 
 
11.2 
Appendix B – Evaluation Criteria for Acute Wheezing 
The Brighton Collaboration has set forth their recommendations for the evaluation of wheezing 
following vaccination[20]. This study will, in general, conform with those recommendations.  
 
Definition. 
Acute wheeze is the sudden onset of adventitious breath sounds that are high pitched or 
whistling. They are generated by turbulence of air in the intrathoracic airways due to airflow 
limitation. Acute wheeze has the following characteristics: 
Visit number 
1 
4 OR 4C 
6 
Target Volume by 
Day for Serum OR 
Whole Blood 
Max Volume by 
Day for Serum 
OR Whole Blood 
Target day 
1 
29 OR V4+28d 
V5+28d 
First day of window 
1 
26 OR V4+25d 
V5+25d 
Final day of window 
1 
32 OR V4+31d 
V5+31d 
Visit Type 
Vaccine Vaccine OR 
Follow 
Follow 
Visit venue 
Clinic 
Clinic 
Clinic 
Procedures 
per visit 
Serum Vol. 
(required for all ages) 
5ml 
Serum 
 
 
6mo-4yo: 5ml 
5yo-8yo: 5ml 
9yo-17yo: 10ml 
Serum 
 
 
6mo-4yo: 5ml 
5yo-8yo: 10ml 
9yo-17yo:10ml 
Additional Serum Vol. 
      6mo – 4yo 
      5yo - 8yo 
      9yo – 17yo  
 
None 
5 ml (if possible) 
5 ml 
Whole Blood Vol. 
      6mo – 4yo 
      5yo – 8yo 
      9yo – 17yo 
 
8.5ml (if possible) 
8.5 ml 
8.5 ml 
Whole Blood 
6mo-4yo: 0ml 
5-8yo: 8.5ml 
9-17yo: 17ml 
Whole Blood 
6mo-4yo: 8.5ml 
5-8yo: 8.5ml 
9-17yo: 17ml 
Additional Whole Blood 
Vol. 
      6mo – 4yo 
      5yo – 8yo 
      9yo – 17yo 
 
None 
None 
8.5 ml 
Target Volume by Day 
      6mo – 4yo 
      5yo – 8yo 
      9yo – 17yo 
 
5.0 ml 
13.5 ml 
27.0 ml 
Total Target by Age for study 
6mo – 4yo -15ml 
5yo – 8yo – 40.5ml  
9yo – 17yo – 81ml 
Max Volume by Day 
      6mo – 4yo 
      5yo – 8yo 
      9yo – 17yo 
 
13.5 ml 
18.5 ml 
27.0 ml 
Total Max by Age for study 
6mo – 4yo – 40.5ml 
5yo – 8yo – 55.5ml 
9yo – 17yo – 81ml 
Non-Blood Sample 
Nasal Lavage 
5-10 ml 
15-30 ml across all ages for study 

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1. The sounds must be of sudden onset (unexpectedly and without warning, leading to a 
marked change in a participant’s previously stable condition).  
2. The sounds must be “adventitious” which means extra or additional sounds heard over 
the participant’s normal breath sounds.  
3. The sounds must involve high-pitched breath sounds, which may sometimes be 
“whistling” in nature.  
4. The sounds must be audible on auscultation with a stethoscope by a healthcare provider 
and the sounds must be audible during expiration. (There may also be inspiratory sounds, 
but there must not be only inspiratory sounds.)  
 
Although parents and caregivers of children, or children themselves, may provide the study team 
or their healthcare providers with complaints they term “wheezing” or other complaints that lead 
to a healthcare provider diagnosing “acute wheeze”, the symptom of “wheezing” or any other 
symptom that a parent or caregiver of a child, or a child him- or herself, reports or endorses, will 
not be deemed as adequate to meet the case definition (although it is often the impetus for 
evaluation by a healthcare provider or the research team).  
 
When all the above criteria have been met, the child will be termed to have definite acute 
wheeze. When the study team is provided with information after the time when confirmation of 
acute wheeze is possible, such as at a future research follow-up visit, and the documentation of 
the episode cannot confirm or deny that it met the criteria, then the investigator, using his/her 
judgment, may record the event as “possible acute wheeze”. For example, a parent states that the 
child has a respiratory event that led to a health care visit and at the visit the child was given a 
bronchodilator, but the documentation required to meet the criteria were not recorded or are not 
available, such an even would be deemed “possible acute wheeze.”  
 
Timing. 
Enhanced surveillance of episodes of acute wheeze will occur in the 28 days after intranasal 
administration of study product. During the 28-day period post-vaccination, families will be 
given instructions to notify the research team immediately for all illnesses, including 
“wheezing”, respiratory symptoms such as cough, sneezing, rhinorrhea, tachypnea, chest 
indrawing, etc. at each encounter with the study team and via a weekly eDiary check conducted 
within +/- 3-day window.  If symptoms trigger an evaluation, a research clinician will conduct 
the evaluation, if possible. If an external physician completes the evaluation, data will be 
retrieved from the medical records of the participant.  The evaluation will include a standard set 
of clinical variables and a respiratory viral panel. Following this period of enhanced surveillance 
for acute wheeze, the study team will continue to collect information on all reports of wheezing 
until the end of each child’s participation, which spans until the last clinical evaluation of the 
participant in the following year (April of the following year). During this period, families will 
be instructed to let the study team know about any wheezing episodes but not all respiratory 
events, such as rhinorrhea and sneezing. Information on the wheezing events, including 
respiratory virus panel (RVP) results, if conducted as part of routine care, will be collected via 
chart abstraction.  
 
Data collection.  

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For each of these episodes, the participant will have collected, preferably by the research team, 
but if not possible then by an outside provider, a standard set of variables. If not all the variables 
are collected by an outside provider, the study team will record the available data. When 
possible, the research team will evaluate the acute episode. If not all the variables are available, 
to determine if the event met the criteria for definite acute wheeze, the investigator may report it 
as possible acute wheeze. If a complete RVP is not available but another microbiologic test was 
performed and provides an alternate explanation then this will be considered acceptable in lieu of 
investigator initiated RVP.  
 
Determining whether the episode meets or does not meet the definition. 
Episode evaluations will first determine if the definition of definite acute wheeze has been met, 
as defined above. Variables necessary to make that determination are the following: 1. Acute 
onset episode (y/n), 2. Auscultation by a healthcare provider using a stethoscope reveals breath 
sounds that are high-pitched/whistling, adventitious (y/n), and 3. The sounds are heard during 
expiration (y/n).  
 
Standard set of variables to be collected.  
If the definition of acute wheeze is not met, routine collection of AE variables, when in the time 
frame stated in the protocol for AEs, will be done.  
 
If the definition of acute wheeze is met, the following information will be collected, in order to 
characterize the acute wheeze episode: 
1. Date of onset of symptoms that led to evaluation. 
2. Date of meeting the case definition of acute wheeze 
3. Date when the AE resolves – this will be determined when symptoms resolve and does 
not require repeat physical exams. 
4. Relationship of person reporting the symptoms that prompted evaluation 
5. Role (investigator, outside healthcare provider, etc.) of person performing auscultation 
and respiratory evaluation. 
6. Accompanying respiratory findings: 
a. Respiratory Rate in breaths per minute (graded into normal or tachpneic, by age1) 
1The upper limit by age is as follows: 6–11mo - 38 breaths/min; 1–3 yo - 30  
breaths/min; 4–9yo - 24 breaths/min; 10–13yo - 22 breaths/min; 14–17yo –  
20 breaths/min [21] 
b. Inspiratory: Expiratory ratio (less than or equal to 1:3 [normal] or 1:3 or greater 
[prolonged]) 
c. Lower chest wall indrawing (yes or no) 
d. Central cyanosis (yes or no) 
e. Oxygen saturation by pulse oximetry (actual value and graded as <92% 
[significant hypoxia] or 92% or more [normal or mildly abnormal]) 
f. Inability or significant difficulty in breastfeeding, drinking, talking, based on age- 
(inability, difficulty, neither) 
g. Altered mental status/confusion as determined by the healthcare professional (yes 
or no) 
7. Accompanying non-respiratory findings: 
a. Temperature (including type of thermometer) 

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b. Blood pressure (if available) 
8. Treatment: 
a. Type 
b. Dose 
c. Duration 
9. Outcome 
10. Is this an SAE? 
11. Exposures other than the investigational product that could potentially be the etiology: 
infection, environmental, food 
12. Results of RVP, including COVID-19 test, or other infectious disease diagnostics 
performed.  
 
Severity Grades  
All episodes of acute wheeze will be graded based on the most severe time point within the 
single episode. Clinical discretion may be employed to determine when an event is deemed a 
new event, since signs and symptoms may come and go during a single wheezing event. The 
grading of the event should be as follows.  
 
Mild = Grade 1 = “Wheeze without respiratory distress or danger signs”. Definition: 
wheeze only- that is, without any of the additional findings in Grades 2 or 3. 
 
Moderate = Grade 2 = “Wheeze with respiratory distress but without danger signs”. 
Definition: wheeze plus one or both of the following: lower chest indrawing, I:E > 1:3, 
but none of the additional findings in Grade 3.   
 
Severe = Grade 3 = “Wheeze with danger signs”. Definition: wheeze plus one or more of 
the following: oxygen saturation < 92%, central cyanosis, altered mental 
status/confusion, inability to breastfeed, drink, or speak (depending on age) 
 
12. 
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