A Multicenter, Blinded, Randomized, Placebo-Controlled, Dose-Ranging Influenza Challenge Study in Healthy Adult Volunteers to Determine the Optimal Infection Dose and Safety of a Recombinant H3N2 (A/Texas/71/2017 (H3N2), Clade 3C3a) Influenza Challenge Virus
Phase: PHASE1
Conditions: Influenza
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Protocol 20-0005 
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A Multicenter, Blinded, Randomized, Placebo-Controlled, Dose-Ranging Influenza 
Challenge Study in Healthy Adult Volunteers to Determine the Optimal Infection Dose and 
Safety of a Recombinant H3N2 (A/Texas/71/2017 (H3N2), clade 3C3a) Influenza Challenge 
Virus 
 
Short Title: A/Texas/71/2017 (H3N2) Dose-ranging Challenge Study 
 
DMID Protocol Number: DMID 20-0005 
 
IND Sponsor: Division of Microbiology and Infectious Diseases (DMID) 
National Institutes of Allergy and Infectious Diseases, 
National Institutes of Health 
 
Version Number:  8.0 
 
04 May 2023 
 
 
 
 
 

Protocol 20-0005 
Version 8.0 
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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 Harmonisation 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 .......................................................................................................................... 6 
LIST OF FIGURES ........................................................................................................................ 6 
1. 
PROTOCOL SUMMARY ...................................................................................................... 7 
1.1 
Synopsis .......................................................................................................................... 7 
1.2 
Schedule of Activities (SoA) ........................................................................................ 15 
1.3 
Study Schema................................................................................................................ 20 
2. 
INTRODUCTION ................................................................................................................ 21 
2.1 
Study Rationale ............................................................................................................. 21 
2.2 
Background ................................................................................................................... 21 
2.2.1 
Purpose of Study ................................................................................................... 21 
2.3 
Risk/Benefit Assessment .............................................................................................. 22 
2.3.1 
Known Potential Risks .......................................................................................... 22 
2.3.2 
Known Potential Benefits ..................................................................................... 26 
2.3.3 
Assessment of Potential Risks and Benefits ......................................................... 27 
3. 
OBJECTIVES AND ENDPOINTS ...................................................................................... 28 
4. 
STUDY DESIGN.................................................................................................................. 32 
4.1 
Overall Design .............................................................................................................. 32 
4.2 
Scientific Rationale for Study Design ........................................................................... 36 
4.3 
Justification for Dose .................................................................................................... 36 
5. 
STUDY POPULATION ....................................................................................................... 38 
5.1 
Inclusion Criteria .......................................................................................................... 38 
5.2 
Exclusion Criteria ......................................................................................................... 40 
5.2.1 
Exclusion of Specific Populations ........................................................................ 42 
5.3 
Inclusion of Vulnerable Participants ............................................................................. 42 
5.4 
Lifestyle Considerations ............................................................................................... 42 
5.5 
Screen Failures .............................................................................................................. 43 
5.6 
Strategies for Recruitment and Retention ..................................................................... 43 
5.6.1 
Recruitment ........................................................................................................... 43 
5.6.2 
Retention ............................................................................................................... 43 
5.6.3 
Compensation Plan for Subjects ........................................................................... 44 
5.6.4 
Costs ...................................................................................................................... 44 
6. 
STUDY PRODUCT.............................................................................................................. 45 
6.1 
Clinical Challenge Strain .............................................................................................. 45 
6.1.1 
Dosing and Administration ................................................................................... 45 
6.1.2 
Dose Escalation ..................................................................................................... 45 

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6.1.3 
Dose Modifications ............................................................................................... 45 
6.2 
Preparation/Handling/Storage/Accountability .............................................................. 46 
6.2.1 
Acquisition and Accountability ............................................................................ 46 
6.2.2 
Formulation, Packaging, and Labeling ................................................................. 47 
6.2.3 
Product Storage and Stability................................................................................ 47 
6.2.4 
Preparation and Administration ............................................................................ 47 
6.3 
Measures to Minimize Bias: Randomization and Blinding .......................................... 48 
6.3.1 
Study Product Assignment Procedures ................................................................. 48 
6.3.2 
Randomization and Blinding ................................................................................ 48 
6.3.3 
Blinding and Masking Procedures ........................................................................ 48 
6.4 
Study Intervention Compliance .................................................................................... 49 
6.5 
Concomitant Therapy.................................................................................................... 49 
6.5.1 
Non-Research Standard of Care ............................................................................ 50 
7. 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL ..................................................................................... 51 
7.1 
Halting Criteria and Discontinuation of Study Intervention ......................................... 51 
7.1.1 
Study Halting Criteria ........................................................................................... 51 
7.2 
Participant Withdrawal from the Study and Replacement ............................................ 51 
7.3 
Lost to Follow-Up ......................................................................................................... 53 
8. 
STUDY ASSESSMENTS AND PROCEDURES ................................................................ 54 
8.1 
Screening and Outcome Procedures ............................................................................. 54 
8.1.1 
Screening Procedures ............................................................................................ 54 
8.1.2 
Discharge from Quarantine ................................................................................... 55 
8.1.3 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings .................................................................................................. 55 
8.1.4 
Outcome / Immunogenicity / Genetic Assessments ............................................. 56 
8.1.4.1 
Outcome Evaluations ........................................................................................ 56 
8.1.4.2 
Immunogenicity Assessments ........................................................................... 57 
8.1.4.3 
Genetic/Genomic Analyses ............................................................................... 57 
8.2 
Safety and Other Assessments ...................................................................................... 58 
8.2.1 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings .................................................................................................. 59 
8.3 
Adverse Events and Serious Adverse Events ............................................................... 60 
8.3.1 
Definition of Adverse Events (AE) ....................................................................... 60 
8.3.1.1 
Classification of an Adverse Event ................................................................... 61 
8.3.2 
Definition of Serious Adverse Events ................................................................... 61 
8.3.2.1 
Suspected Unexpected Serious Adverse Reactions (SUSAR) .......................... 62 
8.3.2.2 
Severity of Event ............................................................................................... 62 
8.3.2.3 
Relationship to Study Intervention ................................................................... 62 
8.3.3 
Time Period and Frequency for Event Assessment and Follow-Up ..................... 63 
8.3.4 
Adverse Event Reporting ...................................................................................... 63 
8.3.4.1 
Investigators Reporting of AEs ......................................................................... 63 
8.3.5 
Serious Adverse Event Reporting ......................................................................... 63 

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8.3.5.1 
Investigators Reporting of SAEs ...................................................................... 63 
8.3.5.2 
Regulatory Reporting of SAEs ......................................................................... 64 
8.3.6 
Reporting of Pregnancy ........................................................................................ 64 
8.4 
Unanticipated Problems ................................................................................................ 64 
8.4.1 
Definition of Unanticipated Problems (UP) ......................................................... 64 
8.4.2 
Unanticipated Problem Reporting ......................................................................... 65 
9. 
STATISTICAL CONSIDERATIONS.................................................................................. 66 
9.1 
Introduction ................................................................................................................... 66 
9.2 
Statistical Hypotheses ................................................................................................... 66 
9.3 
Sample Size Determination ........................................................................................... 66 
9.3.1 
Study Design ......................................................................................................... 66 
9.3.2 
Dose Escalation and Sample Size Allocation ....................................................... 66 
9.4 
Populations for Analyses .............................................................................................. 69 
9.5 
Statistical Analyses ....................................................................................................... 70 
9.5.1 
General Approach ................................................................................................. 70 
9.5.2 
Analysis of the Primary Endpoints ....................................................................... 71 
9.5.3 
Analysis of the Secondary Endpoints ................................................................... 72 
9.5.3.1 
Analysis of Secondary Endpoints ..................................................................... 73 
9.5.3.2 
Analysis of Secondary Immunological Endpoints ............................................ 73 
9.5.4 
Analysis of Exploratory Endpoints ....................................................................... 73 
9.5.4.1 
Exploratory Immunological Endpoints ............................................................. 74 
9.5.4.2 
Exploratory Genomic Endpoint ........................................................................ 74 
9.5.4.3 
Exploratory Transcriptomic Endpoint .............................................................. 74 
9.5.4.4 
Exploratory Physiological and Additional Clinical Endpoints ......................... 75 
9.5.5 
Baseline Descriptive Statistics .............................................................................. 75 
9.5.6 
Planned Interim and Early Analyses ..................................................................... 75 
9.5.6.1 
Interim Safety Analyses .................................................................................... 76 
9.5.6.2 
Interim Review to Determine the Dose for the Subsequent Cohort ................. 76 
9.5.6.3 
Early Analyses .................................................................................................. 76 
9.5.7 
Sub-Group Analyses ............................................................................................. 76 
9.5.8 
Tabulation of Individual Participant Data ............................................................. 77 
10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS ... 78 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ............................................ 78 
10.1.1 
Informed Consent Process .................................................................................... 78 
10.1.1.1 
Other Informed Consent Procedures ............................................................. 80 
10.1.2 
Study Termination and Closure ............................................................................ 80 
10.1.3 
Confidentiality and Privacy .................................................................................. 80 
10.1.4 
Future Use of Stored Specimens and Data ........................................................... 81 
10.1.4.1 
Samples for Secondary Research .................................................................. 82 
10.1.4.2 
Data Sharing for Secondary Research .......................................................... 82 
10.1.5 
Key Roles and Study Governance ........................................................................ 83 
10.1.6 
Safety Oversight.................................................................................................... 83 
10.1.7 
Clinical Monitoring ............................................................................................... 84 

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10.1.8 
Quality Assurance and Quality Control ................................................................ 84 
10.1.9 
Data Handling and Record Keeping ..................................................................... 84 
10.1.9.1 
Data Collection and Management Responsibilities ...................................... 85 
10.1.9.2 
Study Record Retention ................................................................................ 85 
10.1.9.3 
Source Records ............................................................................................. 86 
10.1.10 Protocol Deviations ............................................................................................... 86 
10.1.11 Publication and Data Sharing Policy .................................................................... 86 
10.1.12 Human Data Sharing Plan ..................................................................................... 87 
10.1.13 Genomic Data Sharing Policy ............................................................................... 87 
10.1.14 Publications ........................................................................................................... 87 
10.1.15 Conflict of Interest Policy ..................................................................................... 87 
10.2 
Additional Considerations ............................................................................................ 87 
10.2.1 
Research Related Injuries ..................................................................................... 87 
10.3 
Abbreviations ................................................................................................................ 88 
10.4 
Protocol Amendment History ....................................................................................... 91 
11. 
REFERENCES ................................................................................................................. 94 
12. 
Appendix A. Severity Grading for Vital Signs ................................................................. 95 
13. 
Appendix B. Severity Grading for Safety Laboratories.................................................... 96 
14. 
Appendix C: Venipuncture Volumes ................................................................................ 97 
15. 
Appendix D: FLU-PRO – Participant Reported Tool ....................................................... 99 
 
LIST OF TABLES 
Table 1. Schedule of Activities (SoA) .......................................................................................... 15 
Table 2. Dosing and Administration ............................................................................................. 45 
Table 3. Probability (%) of observing at least one safety event, given varying underlying event 
probabilities and dose group sizes ................................................................................................ 69 
Table 4. Commonly Used Abbreviations...................................................................................... 88 
 
 
LIST OF FIGURES 
Figure 1. Adaptive Dosing Schedule ............................................................................................ 20 
Figure 2. Probabilities of further enrollment at the current dose, given varying true infection 
probabilities, numbers of subjects enrolled at the current dose, and decision thresholds ............ 68 
Figure 3. Estimated symptomatic influenza probabilities and associated exact 95% CIs, given 
varying numbers of symptomatic influenza events observed and varying sample sizes .............. 69 
 
 
 
 

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1. 
PROTOCOL SUMMARY 
1.1 
Synopsis 
 
Rationale for Proposed Clinical Study 
Influenza is a seasonal respiratory disease that is a leading cause of hospitalizations, missed 
work, missed school and economic disruption. While there are currently a number of seasonal 
influenza vaccines available across numerous vaccine platforms, efforts are still underway to 
develop either an improved seasonal vaccine with greater efficacy, or a universal vaccine. The 
human challenge model has proven essential to determine the effectiveness of a vaccine in a 
controlled setting. This study is designed to determine the optimal infectious dose of the H3N2 
(A/Texas/71/2017 (H3N2), clade 3C3a) influenza virus challenge strain for use in future Phase I 
clinical trials evaluating vaccine candidates. 
Study Design 
The study will enroll and challenge up to 106 (plus approximately 8 shams) healthy adult 
volunteers with the H3N2 (A/Texas/71/2017 (H3N2), clade 3C3a) influenza virus challenge 
strain.  Subjects will be pre-screened for study inclusion to have serological HAI antibody titers 
of ≤1:40 against the clinical challenge strain.  Eligible participants will be enrolled sequentially 
into dosing cohorts and will be randomly assigned to receive a single dose of either placebo 
(sham inoculum) or a virus dose between 104 to 106 TCID50 (in an allocation of 1:12 to 1:17).  
Dose titration will be conducted under an adaptive escalation schedule (Figure 1) whereby 
dosing will start at the lowest dose (104 TCID50) and only escalate to the next dose if a pre-
determined infection and symptomatic attack rate are not met and the dose is determined to be 
safe and no pre-defined halting rule is met. The attack rate (AR), defined as the percentage of 
subjects meeting shedding and symptom criteria for symptomatic influenza virus infection (see 
clinical case definition below), will be determined for each challenge dose group in order to 
identify the optimal infectious dose (55%-80% AR).  This adaptive, dose-ranging approach 
allows adjustments to challenge dose group size and escalation dose schedule to be informed by 
the AR and safety results. 
Study Objectives  
Primary Objectives: 
1. To determine the optimal infectious dose1 of a recombinant influenza virus 
(A/Texas/71/2017 (H3N2), clade 3C3a) to be used as a clinical challenge strain in future 
vaccine efficacy or intervention studies as assessed by viral shedding and clinical 
symptoms 
2. To describe viral detection by quantitative and qualitative RT-PCR from study subjects 
at baseline and post-challenge 
3. To document clinical symptoms from self-reported surveys and standardized symptom 
scales at baseline and post-challenge 

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Secondary Objectives: 
1. To assess the safety profile of a live recombinant influenza strain (A/Texas/71/2017 
(H3N2), clade 3C3a) following challenge in healthy adult volunteers 
 
2. To describe the host serum hemagglutination inhibition and microneutralization 
antibody responses at baseline and post-challenge  
 
3. To describe anti-HA-stalk antibody titer at baseline and post-challenge 
 
Exploratory Objectives: 
1. To describe the host serum neuraminidase inhibiting antibody responses at baseline 
and post-challenge 
2. To describe the host immune responses (innate, serum antibody, nasal IgA) at baseline 
and post-challenge in at least a subset of subjects. 
3. To describe physiological responses from an influenza infection as assessed by 
wearable devices at baseline and post-challenge 
4. To describe systemic transcriptional responses at baseline and post-challenge in at least 
a subset of subjects 
 
5. To compare the clinical features of symptomatic RT-PCR-positive and RT-PCR-
negative illness post-challenge 
 
6. To explore alternative case definitions (e.g. MMID) for symptomatic RT-PCR-positive 
influenza virus infection 
 
7.  To determine the symptomatic background within uninfected participants and between 
sites 
 
8. To evaluate the plasmablast response post-challenge in a subset of study subjects 
 
9. To describe mucosal and systemic human immune cells in blood and NLF and/or nasal 
curettage at baseline and post-challenge in at least a subset of study subjects 
 
10. To describe T cell activation in cells from nasal curettage in a subset of study subjects 
 
11. To assess the association of human leukocyte antigen (HLA) class I and II alleles with 
clinical, immune and viral responses in at least a subset of subjects 
12. To evaluate cellular immune response at baseline and post-challenge in at least a 
subset of subjects 

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13. To describe viral detection by quantitative culture from study subjects at baseline and 
post-challenge 
1The optimal infectious dose is the minimal infectious dose that is considered safe and maximizes 
the AR and symptoms among the doses assessed 
Inclusion Criteria 
1. Provide written informed consent prior to initiation of any study procedure 
2. Are able to understand and comply with planned study procedures and be available for 
all study visits 
3. Agree to remain an inpatient for at least 7 days after challenge AND until they have no 
viral shedding1, determined by qualitative RT-PCR beginning on Study Day 6  
 
1No viral shedding is defined as two negative RT-PCR tests 12 or more hours apart 
4. Healthy2 males and non-pregnant, non-breastfeeding females aged ≥ 18 and < 46 years 
of age at enrollment 
2Healthy is defined in inclusion criteria #11. 
NOTE:  Female subjects of childbearing potential must have a negative serum 
pregnancy test at screening, a negative urine pregnancy test upon admission to the 
confinement unit AND a negative pregnancy test before any CXR (if ≥ 7 days have 
passed since a serum pregnancy test). 
5. Women of childbearing potential3 must agree to use or have practiced true abstinence4 or 
use at least one acceptable primary form of contraception5 for at least 30 days prior to 
challenge 
3Not of childbearing potential - post-menopausal females (defined as having a history of 
amenorrhea for at least one year) or a documented status as being surgically sterile 
(hysterectomy, bilateral oophorectomy, tubal ligation/salpingectomy, or Essure® 
placement with history of documented radiological confirmation test at least 90 days 
after the procedure). 
4True abstinence is 100% of time no sexual intercourse (male’s penis enters the female’s 
vagina). (Periodic abstinence [e.g. calendar, ovulation, symptothermal, post-ovulation 
methods] and withdrawal are not acceptable methods of contraception). 
5Acceptable forms of primary contraception include monogamous relationship with a 
vasectomized partner who has been vasectomized for180 days or more prior to the 
subject receiving the influenza challenge virus, intrauterine devices, birth control pills, 
and injectable/implantable/insertable hormonal birth control products. Must use at least 
one acceptable primary form of contraception for at least 30 days prior to challenge and 
at least one acceptable primary form of contraception during the remainder of the study 
or approximately 57 days after confinement. 
NOTE:  These criteria are applicable to female subjects in a heterosexual relationship 

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AND of child-bearing potential.  These criteria do not apply to subjects in a same sex 
relationship. 
6. Non-habitual smoker6 of tobacco, e-cigarettes or marijuana 
6Non-habitual smokers are those who smoke no more than four cigarettes, other tobacco 
products, e-cigarettes (to include vaping and Juuling products) or marijuana in a week 
and agree not to smoke cigarettes, other tobacco products, e-cigarettes and/or 
marijuana products during participation in the study. 
7. No self-reported or known history of alcoholism within the last 2 years and agrees to 
abstain from alcohol for at least one week before admission and throughout the 
confinement period 
8. No self-reported or known history of restricted drug use for at least 30 days prior to 
challenge and agrees to abstain from restricted drugs throughout the confinement period 
9. Negative drug urine toxicology result on screening (i.e., amphetamines, cocaine, and 
opiates) and on admission to the confinement unit (i.e., amphetamines, cocaine, and 
opiates)8 
8Select drug use may be allowed at Investigator’s discretion (e.g., prescribed 
amphetamines for ADHD) 
10. Agree not to use the listed prescription or over the counter medications9 within 7 days 
prior to and through confinement period, unless approved by the investigator 
9Oseltamivir, zanamivir, peramivir, baloxavir marboxil, amantadine (generic) and 
rimantadine (Flumadine and generic), aspirin, intranasal steroids, decongestants, 
antihistamines, and other non-steroidal anti-inflammatory drugs (NSAIDs) 
11. In good health10, and do not have clinically significant medical, psychiatric, chronic or 
intermittent health conditions including those listed in Exclusion Criteria (Section 5.2) 
10Good health, as determined by medical history, medication use and physical 
examination to evaluate ongoing chronic medical or psychiatric diagnoses or conditions, 
defined as those that have been present for at least 90 days, which would not affect the 
assessment of the safety of subjects or the immunogenicity of challenge. These medical 
diagnoses or conditions should be stable for the last 90 days (no hospitalizations, 
emergency room (ER) or urgent care for condition (excluding musculoskeletal 
conditions) and not listed in Exclusion Criteria (Section 5.2).  
Subjects may be on medications only if the condition or disease is stable and not 
deteriorating, if the medical intervention (such as device or medication) was not 
available during the maximal inpatient period of time, medications are not listed in the 
Exclusion Criteria (Section 5.2) and pose no additional risk to subject safety or 
assessment of adverse events. This also includes no change in prescription medication, 
dose or frequency as a result of new symptoms or deterioration of the medical diagnosis 
or condition in the 90 days prior to enrollment. Any prescription change that is due to 
change of health care provider, insurance company, etc., or that is done for financial 
reasons, as long as in the same class of medication, will not be considered a deviation of 
this inclusion criterion.  Any change in prescription medication due to improvement of a 

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disease outcome (e.g., lowering of the dosage or frequency), as determined by the site 
principal investigator (PI) or designated clinician licensed to make medical diagnoses 
and listed on Form FDA 1572, will not be considered a deviation of this inclusion 
criterion. 
12. Vital signs as follows:  
• Pulse is 47 to 99 beats per minute, inclusive 
• Systolic blood pressure is 85 to 139 mmHg, inclusive 
• Diastolic blood pressure is 55 to 89 mmHg, inclusive 
• SpO2 > 95%; RR < 18 
• Oral temperature is less than 100.6°F 
13. Eligibility laboratory values (WBC, Absolute Lymphocyte Count, Hgb, PLTs, ALT and 
Cr) are within acceptable parameters11  
11Labs within normal range or grade 1 abnormalities deemed not clinically significant 
by a study investigator are considered acceptable 
14. Body mass index (BMI) >18.5 and <40 kg/m2 at screening 
15. Other screening tests (ECG and CXR) are within normal reference range or not deemed 
clinically significant by the PI or appropriate sub-investigator12 
12Designated clinician licensed to make medical diagnoses and listed on the Form FDA 
1572 
16. Negative test for HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) at screening 
17. Negative respiratory virus panel by BIOFIRE® FILMARRAY® respiratory panel by 
bioMérieux or by Luminex xTAG® on Day -2, and Day -1 
18. Negative RT-PCR test for SARS-CoV 2 on screening and Day -2 
19. HAI antibody titer ≤1:40 against influenza A/Texas/71/2017 (H3N2) at screening 
20. Receipt of the recommended number of doses of an EUA authorized or licensed COVID-
19 vaccine product ≥ two weeks prior to confinement  
Exclusion Criteria 
1. Presence of self-reported or medically documented significant medical or psychiatric 
condition(s)1 
1Significant medical or psychiatric conditions include but are not limited to: 
a. Respiratory disease (e.g., chronic obstructive pulmonary disease [COPD], 
asthma) requiring daily medications [Inhaled, oral or intravenous (IV) 
corticosteroids, leukotriene modifiers, long and short acting beta agonists, 
theophylline, ipratropium, biologics] or any treatment for respiratory disease 
exacerbations (e.g., asthma exacerbation) within the last 5 years 
b. Presence of any febrile illness or symptoms suggestive of a respiratory infection 
within two weeks prior to challenge 
c. Significant cardiovascular disease (e.g., congestive heart failure, 

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cardiomyopathy, ischemic heart disease) or history of myocarditis or pericarditis 
as an adult. 
d. Neurological or neurodevelopmental conditions (e.g., epilepsy, stroke, seizures, 
encephalopathy, focal neurologic deficits, Guillain-Barre syndrome, 
encephalomyelitis or transverse myelitis). 
e. Ongoing malignancy or recent diagnosis of malignancy in the last five years 
(excluding basal cell carcinoma of the skin) 
f. 
Presence of an autoimmune disease. 
g. Immunodeficiency of any cause. 
h. History of diabetes. 
2. Presence of immunosuppression or any medications that may be associated with impaired 
immune responsiveness2 
2Including, but not limited to, corticosteroids exceeding 10 mg/day of prednisone 
equivalent, allergy injections, immunoglobulin, interferon, immunomodulators, cytotoxic 
drugs, or systemic corticosteroids or other similar or toxic drugs during the preceding 
12-month period prior to screening. Low dose topical and intranasal steroid 
preparations used for a discrete period of time are permitted (Section 6.5). 
3. Known allergy or intolerance to treatments for influenza (including any neuraminidase 
inhibitors or baloxavir marboxil). 
4. Known allergy to two or more classes of antibiotics (e.g., penicillins, cephalosporins, 
fluoroquinolones, or glycopeptides). 
5. Known allergy to excipients3 in the challenge virus inoculum. 
 
3 Sucrose, KH2PO4, K2HPO4, L-glutamic acid (in SPG diluent) 
6. Receipt or planned receipt of any investigational drug/investigational vaccine/licensed 
vaccine, except for a licensed or emergency use authorized COVID-19 vaccine product, 
within 30 days prior to the date of challenge. 
7. Prior enrollment in an influenza virus challenge study with an influenza virus of the same 
subtype within the past 2 years. 
8. Currently enrolled in any investigational study or intends to enroll in such a study within 
the ensuing study period.4 
4Co-enrollment in an observational study, an investigational study in a follow-up/post-
vaccination stage, or a study involving a licensed drug or biologic may be allowed at the 
investigator’s discretion. 
9. Receipt of any influenza vaccine four months prior to challenge or plans to receive 
influenza vaccine during the study (approximately 57 days after challenge). 
10. History of a previous severe allergic reaction to any drug or biologic with generalized 
urticaria, angioedema, or anaphylaxis. 
11. Receipt of blood or blood products during the six months prior to the planned date of 
challenge. 

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12. Plans to donate blood or blood products during the study (approximately 102 days). 
13. Any condition (including medical and psychiatric conditions) that, in the opinion of the 
Investigator, might interfere with the safety of the subject and/or study objectives. 
14. An ongoing symptomatic condition for which subject has had or has ongoing medical 
investigations but has not yet received a diagnosis or treatment plan e.g., ongoing fatigue 
without a diagnosis. 
15. Known close contact with anyone known to have or suspected to have a respiratory viral 
illness within 7 days prior to challenge. 
16. Significant abnormality altering anatomy of nose/nasopharynx (including significant 
nasal polyps), clinically significant nasal deviation, or nasal/sinus surgery within 180 
days prior to challenge. 
17. History in the last five years of chronic or frequent intermittent sinusitis. 
18. Recent history (180 days) of epistaxis or anatomic or neurologic abnormality impairing 
the gag reflex or contributing to aspiration. 
19. Currently using an internal cardiac device such as a pacemaker or other implanted 
electronic medical devices. 
Study Phase 
• Phase 1 
Study Population 
Healthy males and non-pregnant, non-breastfeeding females aged ≥ 18 and < 46 years of age 
with a serum HAI antibody titer of  1:40 against influenza A/Texas/71/2017 (H3N2), clade 
3C3a. 
Study Sites 
There will be two US sites. 
Study Intervention     
The study is designed to assess the influenza AR following challenge with recombinant H3N2 
(A/Texas/71/2017 (H3N2), clade 3C3a) influenza virus at sequentially increasing doses from 
104, 105, and up to 106 TCID50.   Initially, the trial will start with a challenge cohort receiving a 
single dose of 104 TCID50 of recombinant H3N2 (A/Texas/71/2017 (H3N2), clade 3C3a) 
influenza virus administered intranasally.  Subsequent cohorts will receive either the same dose 
or the dose will escalate to the next highest dose in a stepwise manner depending upon the AR 
observed.  Within each challenge cohort, subjects will be randomized to receive either challenge 
virus or an intranasally administered sham placebo.  For challenge cohorts 1A, 1B, 1C, 2A, 2B, 
and 2C the study will randomize subjects 12:1 targeted to challenge virus or sham placebo.  For 
challenge cohorts 3A and 3B the study will randomize subjects 17:1 targeted to challenge virus 
or sham placebo.  

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Study Duration 
This study will last for 7.5 months - one year depending upon the number of challenge cohorts 
enrolled given the adaptive dose escalation design. 
Participant Duration 
An individual subject will complete the study in about 102 days, from screening at Day -45 to -3 
to follow-up on Day 57 ± 7. 
Safety 
Between each challenge cohort, an Internal Safety Review Committee (ISRC) will review 
blinded subject data from Days 1 through Day 8 to assess if any of the halting criteria (Section 
7.1.1), including the occurrence of serious and severe influenza illness (Section 2.3.1), is met.   If 
none of the halting criteria is met, as assessed by the ISRC, the study can proceed to enroll the 
next challenge cohort according to the adaptive trial design.  If any of the halting criteria are met, 
an SMC will review the subject safety data in an ad hoc meeting.

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1.2 
Schedule of Activities (SoA) 
Table 1. Schedule of Activities (SoA) 
 
 
Study 
Screening 
Pre- 
Challenge 
Viral 
Challenge 
Post-Challenge 
Confinement period 
Follow-up29 
 
 
Study Days 
D -45 to -
3 
D -2 
D -1 
D1 
D
2 
D
3 
D
4 
D
5 
D6 
D
7 
D8 
D9+ 
D15 
± 3 
D29 
± 5 
D57 
± 7 
E1 
U2 
Visit Number 
00A 
00B 
00C 
1 
2 
3 
4 
5 
6 
7 
8 
 
9 
10 
11 
 
 
Type of Visit3 
C 
I 
I 
I 
I 
I 
I 
I 
I 
I 
I 
I 
C 
C 
C 
C 
C 
Clinical Procedures 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review/confirm eligibility criteria4 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review/confirm informed consent5 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confinement begins 
 
X6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confinement period 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Discharge from confinement unit 
 
 
 
 
 
 
 
 
 
 
X7 
X 
 
 
 
 
 
Initiate treatment course with antiviral 
 
 
 
 
 
 
 
 
 
 
X8 
 
 
 
 
X8 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height9 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight9 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant medications10 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Targeted physical exam11 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X24 
X 
X 
X 
X24 
X24 
Vital signs12 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X24 
X 
X 
X 
X24 
X24 
SpO213 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X24 
X 
X 
X 
 
 
Influenza challenge 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
FLU-PRO survey instrument PM14 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Modified Jackson Score AM and PM15 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Adverse event review16 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X24 
X 
X 
 
X24 
X24 

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Study 
Screening 
Pre- 
Challenge 
Viral 
Challenge 
Post-Challenge 
Confinement period 
Follow-up29 
 
 
Study Days 
D -45 to -
3 
D -2 
D -1 
D1 
D
2 
D
3 
D
4 
D
5 
D6 
D
7 
D8 
D9+ 
D15 
± 3 
D29 
± 5 
D57 
± 7 
E1 
U2 
Visit Number 
00A 
00B 
00C 
1 
2 
3 
4 
5 
6 
7 
8 
 
9 
10 
11 
 
 
Type of Visit3 
C 
I 
I 
I 
I 
I 
I 
I 
I 
I 
I 
I 
C 
C 
C 
C 
C 
SAE review 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECG (12 lead)  
X17 
 
 
 
 
 
 
 
X17 
 
 
 
 
 
 
 
 
Chest X-Ray18 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for HIV, HBV, and HCV 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum HCG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine HCG18 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine toxicology19 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety screening labs20 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HAI Serology screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety follow-up labs21   
 
 
 
X 
  
X 
 
 
 
X 
X24 
 
 
 
X25 
X24 
Research Laboratory Procedures 
Blood - for plasma and PBMC for T- 
and B- cell immunology   
 
 
X 
 
 
 
 
 
 
 
X24 
 
X 
X 
X 
X26 
X26 
Blood - Serum for antibody and cytokine 
assays22   
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X26 
 
Plasmablasts 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
Blood – PBMC for 
immunophenotyping31 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
 
 
 
Blood - RNA transcriptomics  
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X24 
X 
 
 
X26 
X26 
Blood – DNA – collected for genetic 
testing 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nasopharyngeal swab(s) for viral culture 
and/or qualitative23 and quantitative RT-
PCR 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Nasal absorptive matrices and nasal 
lavage for antibody and other assays 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X26 
X26 

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Study 
Screening 
Pre- 
Challenge 
Viral 
Challenge 
Post-Challenge 
Confinement period 
Follow-up29 
 
 
Study Days 
D -45 to -
3 
D -2 
D -1 
D1 
D
2 
D
3 
D
4 
D
5 
D6 
D
7 
D8 
D9+ 
D15 
± 3 
D29 
± 5 
D57 
± 7 
E1 
U2 
Visit Number 
00A 
00B 
00C 
1 
2 
3 
4 
5 
6 
7 
8 
 
9 
10 
11 
 
 
Type of Visit3 
C 
I 
I 
I 
I 
I 
I 
I 
I 
I 
I 
I 
C 
C 
C 
C 
C 
Curettage – nasal mucosa for t cell 
activation30 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X26 
X26 
Wearable Devices 
Garmin and/or Oura Ring27  
X28 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Faros device27 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
1. E=Early termination visit 
2. U=Unscheduled visit  
3. Visit type: C = Clinic, I = Inpatient 
4. Eligibility criteria may change from the day of screening to Study Day -3. To avoid admitting subjects to confinement with new 
exclusion criteria, the eligibility criteria will be reviewed and confirmed before the confinement stay. 
5. Subjects will have approximately a 48-hour window to decide if they would like to drop out of the study and leave the confinement 
unit before challenge virus is administered. 
6. Eligible subjects will be confined under respiratory isolation beginning on Study Day -2 (which is two days prior to the planned 
challenge). 
7. Subjects will remain in the confinement unit for a minimum of seven days after the date of challenge. Subjects will leave the unit 
after they meet the following discharge criteria: two consecutive negative NP swabs collected 12 hours apart (that are performed on 
Study Day 6 or thereafter) for influenza A by qualitative RT-PCR performed by the local clinical laboratory, are afebrile (< 
100.6°F/38.1°C), have SpO2 ≥ 95% on room air, show no moderate or severe influenza signs or symptoms by clinical evaluation, and 
are clinically and hemodynamically stable for 48 hours. Subjects who do not meet discharge criteria on Study Day 8 will remain in the 
confinement unit until the criteria are met. Study procedures for the confinement unit will be the same as on Study Day 8, except for 
no NLF collection or additional blood draws unless clinically indicated. 
8. A treatment course of oseltamivir phosphate or baloxavir marboxil will be provided to all subjects who do not have two consecutive 
negative NP swabs by qualitative RT-PCR for influenza by Study Day 6.  Treatment will begin as early as Study Day 6, or thereafter 
if indicated, or earlier if terminates study prior to Study Day 8.  

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9. Height and weight will be measured at Screening.  
10. Concomitant medications taken within 90 days prior to signing the ICF are collected. 
11. Clinical evaluation (including oral/pharyngeal, neck, lung and heart exam), and symptom evaluation daily while in the 
confinement unit. An otoscopic exam will be performed at baseline and as needed. 
12. Oral temperature, blood pressure, pulse, and respiratory rate will be assessed approximately every 8 hours while in the 
confinement unit (from 12:00AM – 11:59PM), while the subjects are awake, and as clinically indicated. 
13. Peripheral oxygen saturation (SpO2) on room air at screening, on admission, and when oral temperature, blood pressure, pulse, and 
respiratory rate are assessed through the confinement period and at follow-up visits. 
14. Self-assessment using FLU-PRO and Validation Diary to be performed after 3:00 pm on the Day -2 and after 3:00 pm each day of 
confinement and through Day 15. 
15. Modified Jackson Score to be obtained on the evening of Day -2 and in the morning and afternoon of each day of confinement 
(Approximately at 8:00 AM and after 3:00 PM) and on Day 15 during visit.  Assure AM assessment on Study Day 1 is performed pre-
challenge.  
16. Unsolicited AEs will be monitored for approximately 28 days post challenge. Review of FLU-PRO for indicators of possible 
severe influenza associated illness (signs, symptoms, or lab findings). 
17. ECG to be performed at screening, on Study Day 6, and as clinically indicated. Sites will consult a cardiologist if ECG is abnormal 
to determine clinically necessary work up.  
18. Among female subjects of childbearing potential, a repeat urine pregnancy test will be performed locally before CXR performed if 
> 7 days have passed since the negative serum pregnancy test was drawn.  
19. Negative drug urine toxicology result (amphetamines, cocaine, and opiates) required on screening and (amphetamines, cocaine, 
opiates) on admission to the challenge unit, unless the drug is deemed acceptable by the Investigator. 
20. Screening Safety/Eligibility Labs: white blood cells (WBCs), absolute lymphocyte count, hemoglobin, platelets, alanine 
transaminase (ALT), and creatinine (Cr). 
21. Follow-Up Safety Labs: Study Days 2, 4, and 8, and as clinically indicated: white blood cells (WBCs), absolute lymphocyte count, 
hemoglobin, platelets, and alanine transaminase (ALT), and creatinine (Cr). 
22. Including HAI, MN and NAI antibody titers on Visits 00C (Day -1) 8, 9, 10 and 11. 
23. Multiplex respiratory virus assay to include SARS-CoV-2 to be performed at each site at the time points listed above. Positive 
results prior to influenza virus challenge will result in subject exclusion from the study. No respiratory virus testing will be done on 
the day of challenge. Quantitative PCR will not be done at screening. 
24. If indicated.   
25. If terminates study < Study Day 8 and procedure not performed on day study terminated. 

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26. If terminates study early and procedure not scheduled to be performed on day study terminated or if missed study procedure on 
scheduled day.  
27. To achieve baseline data for physiological biomarkers (e.g., heartrate variability, sleep patterns, etc.). 
28. If the Garmin and/or Oura ring is not able to be distributed at screening this will not be a protocol deviation. 
29. Follow-up visits can be conducted by phone if in-person visit is not feasible. 
30. Nasal curettage will be an optional procedure. 
31. At UMB, on Study Days -1, 2, 4, 6 and 8, 1x106 freshly isolated PBMCs will be used to stain by mass cytometry. These specimens 
will be stained in conjunction with NLF cells isolated from individuals whose NLF cell counts trigger mass cytometry staining 
(>50,000 cells). No additional blood volumes or additional blood draws will be required. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1.3 
Study Schema 
Figure 1. Adaptive Dosing Schedule 
 
 See Section 4.1 for a detailed explanation of the study schema.

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2. 
INTRODUCTION 
2.1 
Study Rationale 
Influenza is a seasonal respiratory disease that is a leading cause of hospitalizations, missed 
work, missed school and economic disruption. While there are currently a number of seasonal 
vaccines available across numerous vaccine platforms, efforts are still underway to develop 
either an improved seasonal vaccine with greater efficacy, or a universal vaccine. The human 
challenge model has proven a valuable tool to determine the effectiveness of a vaccine in a 
controlled setting. This study is designed to determine the optimal infectious dose of the H3N2 
(A/Texas/71/2017 (H3N2), clade 3C3a) influenza virus challenge strain for use in future phase 1 
clinical trials evaluating vaccine candidates. 
2.2 
Background 
2.2.1 
Purpose of Study 
Despite current vaccination strategies and availability of antivirals, influenza remains an 
important cause of worldwide morbidity and mortality. Those particularly at risk include older 
adults, young children, the immunocompromised, and individuals with chronic respiratory, 
cardiac, or metabolic diseases. The young adult population was particularly vulnerable during the 
2009 H1N1 pandemic. Therefore, improved prevention and management approaches for seasonal 
and pandemic influenza across all populations are urgently needed, including the development of 
new universal vaccines that offer broad and robust protection. 
One of the key components in NIAID’s strategic plan for universal influenza vaccine 
development is to expand the capacity to conduct human challenge studies with relevant 
influenza challenge viruses.[1] After recent completion of the DMID sponsored 18-0010 in-
patient study (A Controlled Human Infection Study of Influenza A/Bethesda/MM2/H1N1 Virus 
(A/California/04/2009/H1N1-like) in Healthy Subjects to Assess the Effect of Pre-Existing 
Immunity on Symptomatic Influenza Virus Infection) BB-IND 19213, the Collaborative 
Influenza Vaccine Innovation Centers (CIVICs) are now planning dose-ranging studies for 
recombinant H3N2 and other H1N1 strains to be provided by DMID.  The CIVICs program is 
part of NIAID’s universal influenza vaccine strategic plan and aims to support development of 
novel influenza vaccines that provide broader and more robust protection and immunity through 
coordinated multi-disciplinary efforts toward discovery, product development, and pre-clinical 
and clinical evaluation in human challenge studies.[2] 
Human challenge models provide a powerful tool to assess prevention and treatment options for 
influenza in a shorter timeframe and with a smaller number of subjects compared to typical 
clinical trials, using a well-characterized virus strain. The controlled time and inoculum of virus 
exposure in challenge studies allow for targeted collection of clinical samples, including prior to 
infection and in short timeframes following inoculation. Human challenge models may be 
particularly useful to evaluate novel influenza vaccines that do not target the HA head region, 
and therefore cannot be evaluated based on current regulatory criteria for licensure of inactivated 
influenza vaccines.  In addition, human challenge models offer the unique opportunity to further 
study influenza pathogenesis, host immune responses, and correlates of protection, which remain 

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unclear. Such experimental models are conducted in a well-controlled challenge setting in which 
virus exposure, infection rate, virus shedding, and controlled sampling are monitored in real-
time. Such parameters are next to impossible to control for in a “real world” setting.  
Importantly, the setting of a controlled experimental model offers the opportunity to 
simultaneously evaluate the clinical, molecular, physiological and immunological outcomes and 
responses across the time course of disease and treatment. 
The overall objective of the present study is to conduct a dose-ranging human challenge study 
with two CIVICs clinical programs to determine the safety and optimal infectious dose of a 
recombinant H3N2 (A/Texas/71/2017 (H3N2), clade 3C3a) influenza virus.  The study will 
enroll and challenge up to 106 persons with live virus (plus approximately 8 persons with shams) 
healthy adult volunteers who will be prescreened for study inclusion to have serological HAI 
antibody titers of ≤1:40 against the challenge strain.  Eligible participants will be enrolled 
sequentially into challenge cohorts and will be randomly assigned to receive a single dose of 
either sham inoculum or a virus dose between 104 to 106 TCID50.  Dose titration will be 
conducted under an adaptive escalation schedule (Figure 1) whereby dosing will start at the 
lowest dose (104 TCID50) and only escalate to the next dose if a pre-determined symptomatic 
influenza attack rate is not met and the dose is determined to be safe and no pre-defined halting 
rule is met. In addition to safety, the attack rate (AR), defined as the percentage of subjects 
meeting shedding and symptom criteria to be classified as having symptomatic influenza 
infection (see clinical case definition in Section 4.1), will be determined for each challenge dose 
group in order to identify the optimal infectious dose (55%-80% AR).  This adaptive dose 
ranging approach allows adjustments to challenge dose group size and escalation dose schedule 
to be informed by the AR and safety results. 
2.3 
Risk/Benefit Assessment 
2.3.1 
Known Potential Risks 
The potential risks of participating in this trial are listed below. 
Symptoms and Complications of Influenza Illness 
Minor symptoms of common influenza illness include: 
• Body aches or pains 
• Chest congestion 
• Chest tightness 
• Chills 
• Congested or stuffy nose 
• Coughing 
• Diarrhea 
• Difficulty swallowing 
• Eyes sensitive to light 
• Fatigue 
• Feeling dizzy 
• Fever 
• Head Congestion 

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• Headache 
• Lack of appetite 
• Swollen lymph nodes 
• Nausea 
• Runny or dripping nose 
• Scratchy or itchy throat 
• Shivering 
• Sinus pressure 
• Sleeping more than usual 
• Sneezing 
• Sore or painful eyes 
• Sore or painful throat 
• Stomachache 
• Sweating 
• Teary or watery eyes 
 
Complications or severe signs or symptoms of influenza illness, which are unlikely to occur in 
healthy subjects who participate in this study, include: 
 
Moderate complications 
• Sinus infections 
• Ear infections 
Severe complications (for additional clarification on any of the below, please refer to study-
specific SOP CIVICs 001) 
• Pneumonia 
• Severe dehydration 
• Severe bronchitis 
• Myocarditis 
• Myocardial infarction 
• Pericarditis 
• Guillain Barré Syndrome/ Bell’s Palsy 
• Transverse myelitis 
• Encephalitis 
• Worsening of chronic health conditions 
• Admission to hospital 
• Hypoxemia/respiratory failure 
• Arrhythmia/cardiac arrest  
• Death 
Though all subjects will be vaccinated, there is a risk of being exposed to SARS-CoV-2 as 
subjects will be evaluated at a screening visit and will be confined in an enclosed setting with 
other volunteers for an extended period of time.  False negative RT-PCR upon study entry or 
introduction of COVID-19 by staff or other study personnel is possible. 

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Symptoms and Complications of COVID-19 Illness  
• Fever or chills 
• Cough 
• Shortness of breath or difficulty breathing 
• Fatigue 
• Muscle or body aches 
• Headache 
• New loss of taste or smell 
• Sore throat 
• Congestion or runny nose 
• Nausea or vomiting 
• Diarrhea  
• Respiratory failure 
• Blood clots 
• Death 
There may be other risks, discomforts, or side effects that are unknown at this time.  There is a 
small amount of risk to subjects that report they are in good health, but who have an unknown 
health problem at the time of screening. This trial will screen by medical history, ECG, Chest X-
ray, vital signs and physical examination. Subjects will be screened for the presence of infection 
with HIV, hepatitis B and hepatitis C, and SARS-CoV-2.  Women of childbearing potential will 
be screened to ensure they are not pregnant. Clinical screening labs for white blood cells (WBC), 
hemoglobin (Hgb), platelets (PLT), alanine aminotransferase (ALT), absolute lymphocyte count 
(ALC), and creatinine (Cr) will be drawn and obtained prior to enrollment and throughout the 
study. In addition, subjects will be pre-screened for serum HAI titer. 
Risks of Nasal Lavage and Nasopharyngeal Swabs 
Obtaining nasal lavage fluid or a nasopharyngeal swab can cause discomfort in the nares, 
nasopharynx, a gag reflex, epistaxis, watery eyes, or coughing at the time of specimen collection. 
Risks of Blood Draw  
Drawing blood may cause transient discomfort and fainting. Fainting is usually transient and 
managed by having the subject lie down. Bruising at the blood draw site may occur but can be 
prevented or lessened by applying pressure to the draw site for several minutes. Drawing blood 
may also cause infection. The use of aseptic technique will make infection at the site where 
blood will be drawn extremely unlikely. Risks for blood drawn may include anemia, however the 
risk is low since the total amount drawn over the entire study period is about the same as a half-
liter of blood (i.e., ~534 mL). The blood volumes anticipated for specimen collection are listed in 
Appendix C.  
Risks of oseltamivir phosphate (TAMIFLU) antiviral  
Adverse events reported in at least 1% of adult and adolescent subjects treated with oseltamivir 
phosphate included nausea (10%), vomiting (9%), diarrhea (7%), bronchitis (2%), abdominal 

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pain (2%), dizziness (2%), headache (2%), cough (1%), insomnia (1%), vertigo (1%), and 
fatigue (1%).[3]  
Influenza A virus isolates with reduced susceptibility to oseltamivir carboxylate have been 
recovered by serial passage of virus in cell culture in the presence of increasing concentrations of 
oseltamivir carboxylate, from clinical isolates collected during treatment with oseltamivir, and 
from viral isolates sampled during community surveillance studies. Reduced susceptibility of 
influenza virus to inhibition by oseltamivir carboxylate may be conferred by amino acid 
substitutions in the viral neuraminidase and/or hemagglutinin proteins. Changes in the viral 
neuraminidase that have been associated with reduced susceptibility to oseltamivir 
carboxylate.[3]  
Risks of baloxavir marboxil (XOFLUZA) antiviral  
Adverse events reported in at least 1% of adult and adolescent subjects treated with baloxavir 
marboxil included diarrhea (3%), bronchitis (2%), nasopharyngitis (1%), headache (1%) and 
nausea (1%).[4] 
Influenza A viruses with treatment-emergent amino acid substitutions at positions associated 
with reduced susceptibility to baloxavir marboxil in cell culture have been observed in clinical 
studies. It is theoretically possible that baloxavir marboxil treatment can cause treatment-
emergent baloxavir marboxil resistant viruses. The overall incidence of treatment-emergent 
amino acid substitutions associated with reduced susceptibility to baloxavir marboxil in two prior 
clinical trials was 2.7% (5/182) and 11% (39/370).[4] 
Risks of Delaying Influenza Vaccination Until 60 Days Post-challenge  
In the US, routine annual influenza vaccination is recommended for all persons, with an 
emphasis placed on vaccination of high-risk groups and their caregivers. In this study, subjects 
are asked to delay 2021-2022 influenza season vaccination through approximately 57 days post-
challenge. If the influenza season begins before this time, the subject is at risk of developing 
influenza illness.  
Risks Associated with the Garmin and Oura Wearable Devices 
There are minor risks associated with the Garmin and Oura wearable device in this study. For the 
Garmin Vivosmart 4 and Oura, subjects with pacemakers or other internal electronic devices 
should consult a physician before using any heart rate monitor. This study excludes subjects with 
internal cardiac devices, minimizing this risk. The Garmin and Oura ring also emit green light 
and flash occasionally. Subjects with epilepsy or sensitivity to flashing lights should also consult 
their physician before use. The device operating temperature is -4 to 122°F while charging 
temperatures are 32 to 113°F. These temperature ranges should be observed in order to minimize 
heat and/or fire associated battery risks. Lithium-ion batteries should not be disassembled, 
removed or modified. They should not be exposed to fire or explosions, or there is a risk of fire, 
chemical burn, electrolyte leak, and/or injury. 
Risks Associated with the Faros Wearable Device 

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The Bittium Faros 180 is an FDA approved Class II medical device designated for research use 
in adults. As long as the exclusion criteria are met (excluding those subjects with pacemakers or 
other implanted electronic medical devices), there are no known physical risks associated with 
wearing the Faros 180. Long-term ECG monitoring is an established procedure that carries 
minimal risks. Holter monitors, which are ambulatory ECG devices, are often worn for days or 
weeks to monitor heart rhythm. The only known physical risk related to wearing these types of 
sensors relates to skin irritation caused by the electrodes. The electrodes have a clear tape that 
allows for visual monitoring of skin condition. The participant will be instructed to temporarily 
remove the device if any redness or irritation develops because of sensitivity to the adhesive. 
Risks of Chest Radiograph (CXR) 
According to the United Nations Scientific Committee on the Effects of Atomic Radiation, a 
single posterior-anterior chest radiograph (CXR) exposes the subjects to about 0.05 mSv, which 
is about the radiation dose people are exposed to naturally over the course of 10 days. Subjects in 
our study will have one CXR at screening (Days -45 to -3) and if deemed medically necessary as 
follow-up of an Adverse Event. Female subjects will undergo a serum pregnancy test, and an 
additional urine pregnancy test if more than 7 days have passed since the serum test, before 
CXR. 
Risks of Electrocardiogram (ECG) 
The electrodes of an electrocardiogram (ECG) may feel cold when applied; in rare cases, a rash 
or skin irritation develops where the patches are placed. This type of irritation usually resolves 
by itself, but topical medication is occasionally required. Subjects in our study will have one 
ECG at screening (Days -45 to -3), a repeat ECG at study Day 6, and additional ECGs if deemed 
medically necessary.  
Risks of Genetic Testing 
Genetic data may be stored and shared with other researchers through a controlled-access online 
repository, such as dbGaP. There may be a risk that genetic information while not linked with 
personal identifiable information, may potentially be reidentified and linked to a subject and 
could be misused for discriminatory purposes. However, such risk is perceived as low and state 
and federal laws provide some protections against genetic discrimination. Researchers who will 
have access to genetic information will take measures to maintain the confidentiality of the 
information. HLA genetic testing will be performed for research purposes only and will not be 
performed by a CLIA certified laboratory. Therefore, the results of HLA testing will not be 
shared with subjects.  
Risk of Loss of Confidentiality 
There is also a risk for loss of confidentiality. In order to minimize this risk, data will be stored 
in a password protected computer database. Samples will be labeled with barcodes prior to 
storing with a unique identifier. All biological samples will be stored in secure and monitored 
freezers accessible only to key personnel with the appropriate access. Personnel working on this 
study will have undergone training for responsible conduct of research with human subjects and 
are listed on the delegation log/key personnel list and their relevant institutions. 
 
2.3.2 
Known Potential Benefits 

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There is no direct benefit to the subjects. There is potential benefit to the greater public health 
and future studies that may result from information gained from this study. 
 
2.3.3 
Assessment of Potential Risks and Benefits 
The most cost effective and efficient method for assessing new influenza vaccines is through the 
use of controlled human influenza infection studies.  Towards that end, it is necessary to 
determine the optimal dose of the influenza challenge strain as is being done in this study. The 
study will significantly add to knowledge about the best dose of the challenge virus to use in 
future studies evaluating new vaccine products.  Although it is anticipated that subjects will 
develop mild influenza illness, the risk of any subject developing severe disease will be 
mitigated by carefully selecting subjects who are healthy.  Subjects will be closely monitored for 
signs or symptoms of severe influenza illness and will be offered antiviral therapy for influenza 
and other medical care if such should occur.  Likewise, subjects will be monitored carefully for 
any complications potentially arising from influenza. The potential risks therefore are thought to 
be acceptable given the potential benefits for society.   
 

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3. 
OBJECTIVES AND ENDPOINTS 
 
The overall objective of the study is to determine the safety and optimal infectious dose of 
recombinant H3N2 (A/Texas/71/2017 (H3N2), clade 3C3a) influenza challenge virus.  
 
OBJECTIVES 
ENDPOINTS 
(OUTCOME MEASURES) 
Primary  
 
1. To determine the optimal infectious dose1 
of a recombinant influenza virus 
(A/Texas/71/2017 (H3N2), clade 3C3a) to be 
used as a clinical challenge strain in future 
vaccine efficacy or intervention studies as 
assessed by viral shedding and clinical 
symptoms 
a. Percentage of subjects within a challenge dose group 
with detectable shedding in nasopharyngeal (NP) 
swab(s) by RT-PCR2 over any 2 days beginning 24 
hours post challenge through Day 8 and with symptom 
scores that meet the clinical case definition for 
symptomatic infection (Modified Jackson score). See 
section 4.1 for the clinical case definition for 
symptomatic infection. 
2. To describe viral detection by quantitative 
and qualitative RT-PCR from study subjects 
at baseline and post-challenge 
 
 
 
 
 
 
 
 
 
 
a. Percentage of subjects within a challenge dose group 
with detected viral shedding in NP swab(s) each day 
post-challenge from Day 2 through Day 8 using 
qualitative RT-PCR and quantitative RT-PCR  
b. Magnitude of virus shedding post-challenge in each 
subject defined as the peak viral load in NP swab(s) 
from day 2 through Day 8 post-challenge by 
quantitative RT-PCR  
c. Duration of viral shedding defined as the number of 
days from first positive RT-PCR to last positive RT-
PCR where virus is detected in NP swab(s) by 
quantitative or qualitative RT-PCR post challenge day 1 
for each subject 
3. To document clinical symptoms from self-
reported surveys and standardized symptom 
scales at baseline and post challenge 
a. Computed total symptom score from modified Jackson 
score through Day 8 and at Day 15. 
b. Percentage of subjects with total symptom scores that 
define symptomatic or asymptomatic categories based 
on modified Jackson score for each subject 
Secondary 
 
1. To assess the safety profile of a live 
recombinant influenza strain 
(A/Texas/71/2017 (H3N2), clade 3C3a) 
following challenge in healthy adult 
volunteers 
 
a. Number of adverse events (AE) reported from challenge 
through Day 29 
b. Percentage of subjects reporting any AE from challenge 
through Day 29 
c. Number of serious adverse events (SAE) from 
challenge through Day 57 
d. Percentage of subjects reporting an SAE at any time 
from challenge through Day 57 

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2. To describe the host serum 
hemagglutination inhibition and 
microneutralization antibody responses at 
baseline and post-challenge  
 
a.   Percentage of subjects with serological conversion 
defined as a minimum 4-fold rise in post-challenge 
serum antibodies (HAI and MN)3 to influenza 
infection at Days 8, 15, and 29, or latest time point 
available, post challenge compared to baseline (Day -
1). 
b.    HAI and MN antibody geometric mean titers (GMTs) 
at baseline (Day -1) and GMTs and Geometric Mean 
Fold Rise (GMFR) post-challenge at Days 8, 15, and 
29, or latest time point available  
3. To describe anti-HA-stalk antibody titer at 
baseline and post-challenge 
a. Percentage of subjects with serological conversion 
defined as a minimum 4-fold rise in post-challenge 
HA-stalk specific serum antibody response to HA 
group 2 stem domains at any day up to Day 29, or 
latest time point available, post challenge compared to 
baseline (Day -1). 
b. For HA-stalk specific response, the GMTs to HA 
group 2 stem domains at baseline (Day -1), and GMTs 
and GMFR at approximately Days 8, 15, and 29, or 
latest time point available. 
Exploratory 
 
1. To describe the host serum neuraminidase 
inhibiting antibody responses at baseline and 
post-challenge  
a.   Percentage of subjects with serological conversion 
defined as a minimum 4-fold rise in post-challenge 
serum antibodies (NAI)4 to influenza infection at Days 
8, 15, and 29, or latest time point available, post 
challenge compared to baseline (Day -1). 
b.   NAI antibody geometric mean titers (GMTs) at 
baseline (Day -1) and GMTs and Geometric Mean 
Fold Rise (GMFR) post-challenge at Days 8, 15, and 
29, or latest time point available 
2. To describe the host immune responses 
(innate, serum antibody, nasal IgA) at 
baseline and post-challenge in at least a 
subset of subjects 
 
a. GMT and GMFR responses for 
A/Texas/71/2017/H3N2-specific sIgA, non-normalized 
and normalized for total IgA content, in NLF at 
baseline (Day -1), and post-challenge Days 8, 15, and 
29. 
b. Percentage of subjects with a minimum of 4-fold 
increase in nasal IgA content over baseline. 
c. Levels of A/Texas/71/2017/H3N2 induced blood 
cytokines and chemokines at baseline (Days -2 and -1), 
and Days 2-8. 
d. Levels of A/Texas/71/2017/H3N2 induced cytokines 
and chemokines in NLF at baseline (Day -1), and 
post-challenge Days 2-8. 
3. To describe physiological responses from 
an influenza infection as assessed by 
wearable devices at baseline and post-
challenge 
a. Physiological data (heart rate variability, temperature, 
sleep) at baseline and post challenge Days 1-8 
associated with influenza infection. 

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4. To describe systemic transcriptional 
responses at baseline and post-challenge in at 
least a subset of subjects 
a. Peripheral blood gene expression profiling based on 
sequencing at baseline (Days -2 and -1) and post-
challenge Days 2-8, and 15 in a subset of subjects 
5. To compare the clinical features of 
symptomatic RT-PCR-positive and RT-PCR-
negative illness post-challenge 
a. Self- and investigator-solicited report of clinical 
symptoms and their severity as measured by 
component questions in FLU-PRO and Validation 
Diary and modified Jackson score by post-challenge 
Study Day 15 and qualitative RT-PCR positive status 
6. To explore alternative case definitions 
(e.g., MMID) for symptomatic RT-PCR-
positive influenza virus infection 
a. The combination of symptoms and symptom severity 
as determined by report using the FLU-PRO and 
Validation Diary or modified Jackson score which 
optimize sensitivity and/or specificity for classifying 
influenza virus infection based on qualitative RT-PCR. 
7.  To determine the symptomatic 
background within uninfected participants 
and between sites 
a. Compare symptoms from subjects inoculated with 
shams to subjects inoculated with virus 
8. To evaluate the plasmablast response post-
challenge in a subset of study subjects 
a. A/Texas/71/2017/H3N2-specific plasmablasts, their 
specific-antibody (e.g., anti-H3, -N2 and – H3N2 
virus) production and isotype (e.g., IgG, IgA) using an 
enzyme-linked immunospot (ELISpot) assay in 
PBMCs on Day 8 in a subset of subjects 
9. To describe mucosal and systemic human 
immune cells in blood and NLF and/or nasal 
curettage at baseline and post-challenge in at 
least a subset of study subjects 
a. The frequency, duration, timing and phenotypic 
characteristics of immune cells in the blood of 
subjects at baseline (Day -1) and at approximately 
on Days 4, 6, 8, and 29 in a subset of subjects 
b. The frequency, duration, timing and phenotypic 
characteristics of immune cells in the nasal 
specimens in subjects at baseline (Day -1) and Days 
2, 4, 6 and 8 in a subset of subjects 
10. T cell activation in cells from nasal 
curettage in a subset of study subjects 
a. Frequency of activated T cells after challenge (Days 
-2, 15, 29, and 57) in a subset of subjects 
11. To assess the association of human 
leukocyte antigen (HLA) class I and II alleles 
with clinical, immune and viral responses in 
at least a subset of subjects 
a. The frequency of HLA class I and II alleles as 
measured by genetic testing at baseline (Day -1) in a 
subset of subjects 
b. Associations of subject HLA class I and II alleles 
with development of infection post-challenge 
through Day 8 and magnitude and breadth of the 
elicited immune responses post-challenge in a subset 
of subjects 
12. To evaluate cellular immune response at 
baseline and post-challenge in at least a 
subset of subjects 
a. Frequency of influenza-specific B cell subsets in 
circulation (PBMC) at baseline (Day -1), and at 
approximately on Days 2, 8, 15, 29, and 57 in a 
subset of subjects 
b. Magnitude of influenza-specific T cell responses in 
circulation (PBMC) to conserved T cell epitopes 
and inactivated virus at baseline (Day -1), and at 
approximately on Days 2, 8, 15, 29, and 57 in a 
subset of subjects 

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c. Presence of hemagglutinin (HA)-specific CD4 T 
cells focusing on Tfh cells on Day 8 on a subset of 
subjects 
13. To describe viral detection by 
quantitative culture from study subjects at 
baseline and post-challenge  
a.   Percentage of subjects within a challenge dose group 
with detected viral shedding in NLF and/or NP 
swab(s) each day post-challenge from Day 2 through 
the end of the confinement period using quantitative 
viral culture. 
b.   Magnitude of virus shedding post-challenge in each   
subject defined as the peak viral load in NLF 
and/or NP swab(s) from Day 2 through the end of 
the confinement period by quantitative viral 
culture. 
1 The optimal infectious dose is the minimal infectious dose that is considered safe and maximizes the AR 
and symptoms among the doses assessed 
2 RT-PCR = reverse transcriptase – polymerase chain reaction 
3 HAI = hemagglutination inhibition; MN = microneutralization 
4 NAI = neuraminidase inhibition 
 
 

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4. 
STUDY DESIGN 
4.1 
Overall Design 
This is an exploratory Phase 1, blinded, randomized, dose-finding influenza virus challenge 
study in approximately between 62-114 (targeted maximum sample size of 106 active plus 8 
shams) males and non-pregnant females, 18 to 45 years old, inclusive, who are in good health 
and meet all eligibility criteria. The clinical study is designed to determine the optimal infectious 
dose and safety profile of a recombinant influenza A/Texas/71/2017 (H3N2) clade 3C3a strain in 
healthy adult volunteers.      
 
The study will enroll approximately between 13-39 subjects in each of up to 3 challenge dose 
groups at 2 sites (Duke University [DU] and University of Maryland [UMD]) for approximately 
between 62-114 subjects total for the study.  We plan to enroll each challenge dose group 
according to an adaptive dose-escalation schedule (Figure 1) which allows study results (see 
Section 9.5.6) to be analyzed between challenge cohorts to inform the sample size and virus dose 
for the next escalation cohort(s).  The study timeline anticipates 4-6 weeks of lead-time for study-
specific screening and start-up activities for the initial cohort, and similarly, 4-6 weeks between 
subsequent cohorts at each site to evaluate study results and turn-over of the clinical unit.  
However, to compress the overall dosing timelines, challenge cohorts may be performed 
concurrently or around a 2-week timeline between the sites. We plan up to 2 weeks (Day -2 to 
Day 8) to complete the clinical in-patient component for each challenge cohort, thus altogether 
projecting the following to complete the dose-ranging clinical confinement component: up to 3.5 
to 4 months, if 3 challenge cohorts evaluated; 4 to 4.5 months, if 4 challenge cohorts evaluated; 5 
to 6 months if 5 challenge cohorts evaluated; and 5.5 to 6.5 months if 6 challenge cohorts 
evaluated.  Each participant has an additional 7 weeks of follow-up post confinement.  The study 
period could range from approximately 7.5 months to one year, depending upon the number of 
challenge cohorts enrolled.   
 
Potentially eligible study subjects may be pre-screened under an active screening protocol 
(DMID 20-0004) to identify those with HAI antibody titers ≤1:40 specific to the challenge virus 
strain. 
 
During the study screening period of this protocol (Day -45 to Day -3), participants who have not 
received a seasonal influenza vaccine within the past 4 months and for whom there are no safety 
concerns from standard safety assessments and screening labs, are eligible for enrollment to the 
study.  Backup subjects will be confined under respiratory isolation prior to challenge to ensure 
sufficient number of volunteers undergo challenge, given the potential for dropouts, development 
of exclusion criterion, and/or pre-challenge evidence of viral respiratory infection among 
subjects. The number of backup subjects will depend on site-specific considerations, such as 
anticipated drop-out rates, anticipated development of new exclusionary criteria, and respiratory 
virus circulation in the community. Safety assessments and safety labs include a medical history 
review, physical examination, ECG, a chest X-ray (CXR), HIV and hepatitis B/C testing, 
baseline SpO2 saturation, and testing for WBCs, ALC, hemoglobin, platelets, ALT, and 
creatinine. Women of childbearing potential must have a negative serum pregnancy test at 
screening and a negative urine pregnancy test on Day -2.  Likewise, participants (including 

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backup subjects) must have a negative drug urine toxicology test (unless the drug is deemed 
acceptable by the Investigator), negative multiplex respiratory viral test, and a negative SARS-
CoV-2 RT-PCR test at screening and on Day -2.  After a standard physical exam to exclude 
active infection or other illness, participants will begin confinement on Day -2 and remain 
confined for up to 12 days (Days -2 to 10). Backup subjects will also begin confinement on Day -
2 (either within the confinement facility or at an alternate location under respiratory isolation). 
They will be discharged prior to challenge if they are not needed. All backup subjects replacing 
discharged subjects will be admitted to the confinement unit prior to viral challenge and remain 
confined in the confinement unit through Day 10.   
 
On Day 1, subjects will be randomized and administered a single challenge dose of either a sham 
inoculum or a virus dose between 104 and 106 TCID50.  The challenge inoculum will be 
administered using the MAD Nasal Intranasal Mucosal Atomization Device (Teleflex, 
Morrisville NC) attached to a 1cc syringe.  Approximately 500 μL of study product will be 
delivered in each nostril with the subject in a recumbent position.  Subjects, clinical study staff, 
administrators of challenge inoculum, and clinical evaluators will be blinded as to whether the 
participant received either sham or virus challenge inoculum.  The dosing plan will follow an 
adaptive dose-escalation schedule (Figure 1) that will include up to 8 different challenge cohorts.  
This dosing plan is designed to optimize subject safety, starting with a low dose and allowing 
flexibility to make informed adjustments between challenge cohorts from outcome results of the 
previous challenge cohort.  For the first exposure in humans, the initial low dose challenge will 
be conducted in a group of a targeted total of 13 subjects (including 1 sham) to assure the virus is 
safe with no study-related or unanticipated adverse events observed or reported before further 
use.  Dose escalation to the next (higher) dose and subsequent higher doses, if necessary, will be 
informed by both safety and AR outcomes given a pre-specified decision algorithm (see section 
9.3.2) for dose-escalating. 
 
Clinical outcomes and pre-defined halting criteria will be used to determine whether the trial 
moves forward to the higher dose of the challenge strain. 
 
Clinical Outcomes 
  
Symptomatic influenza virus infection criteria will be based on the virologic assessment and the 
modified Jackson score.  A subject will be defined as having symptomatic influenza virus infection 
if the following criteria are met: 
• Viral shedding detected in NP swab(s) by qualitative RT-PCR on at least two days 
beginning 24 hours after challenge until Study Day 8. 
• A cumulative symptom score ≥ 6 from daily component symptoms, including documented 
fever, computed across any consecutive 5-day window through Day 8 beginning on Day 2 
post challenge using a modified Jackson score. Highest point score per day per sign or 
symptom will be used. Component signs and symptoms include fever, lymphopenia (< 
1000 cells/mL), runny nose, stuffy nose, sneezing, sore throat, headache, cough, malaise, 
body ache, chills, feverish, shortness of breath, and earache ranked on a 4-point severity 
scale (0-3). (Appendix A for Temperature). Temperature will be measured three times a 
day and symptoms will be collected twice daily on the modified Jackson score.   

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Note: A documented fever and feeling feverish will count as one point on the cumulative 
symptom score.     
 
Signs and symptoms will also be collected via the FLU-PRO and Validation Diary instrument 
which will be completed once daily after 3:00 pm through confinement. Subjects will continue to 
complete the FLU-PRO daily after discharge through Day 15. The modified Jackson score and 
FLU-PRO will be compared in exploratory analyses. 
• Component signs and symptoms of the FLU-PRO and Validation Diary include 
runny/dripping nose, congested/stuffy nose, sinus pressure, scratchy or itchy throat, sore 
or painful throat, difficulty swallowing, teary or watery eyes, sore or painful eyes, eye 
sensitivity, trouble breathing, chest congestion, chest tightness, dry or hacking cough, wet 
or loose cough, nausea, stomach ache, dizziness, head congestion, headache, lack of 
appetite, excessive sleepiness, body aches or pains, weakness or tiredness, chills or 
shivering, feeling cold, feeling hot, and sweating. 
 
Safety Threshold 
The safety threshold is defined as meeting one of the protocol-defined halting criteria 
outlined in Section 7.1.1 which includes the occurrence of severe influenza complications in any 
study subject (Section 2.3.1).  If a subject has a severe influenza complication(s), the study staff 
and subject will be unblinded as to receipt of challenge virus or sham and as to the results of 
influenza RT-PCR testing for safety evaluation and potential therapeutic intervention with an 
approved influenza antiviral agent.  
In this adaptive schema, the initial cohort (Cohort 1A) for the lowest challenge dose 
group will include a targeted total of 12 subjects receiving 104 TCID50 of the challenge strain and 
one subject receiving sham placebo.  If fewer than 80% of subjects meet the case definition for 
influenza in Cohort 1A, the dose will escalate to 105 TCID50 to include Cohort 2A.  If 80% or 
more subjects in Cohort 1A meet the case definition for influenza, the lowest challenge dose 
group will be expanded to include a targeted total of 12 additional subjects receiving 104 TCID50 
of the challenge strain and one subject receiving sham placebo to comprise Cohort 1B.  
Cumulatively, if fewer than 80% of subjects in Cohorts 1A and 1B meet the case definition for 
influenza, the dose will escalate to 105 TCID50 to include Cohort 2A. If 80% or more of the 
subjects in Cohorts 1A and 1B meet the case definition for influenza than the lowest challenge 
dose group will be expanded to include a targeted total of 12 additional subjects receiving 104 
TCID50 of the challenge strain and one subject receiving sham placebo to comprise Cohort 1C.  
Cumulatively, if fewer than 80% of subjects in Cohorts 1A, 1B, and 1C meet the case definition 
for influenza the dose will escalate to 105 TCID50 to include Cohort 2A. If 80% or more of the 
subjects in Cohorts 1A, 1B, and 1C combined meet the case definition for influenza then the 
optimal dose of 104 TCID50 will be selected.  
The 105 TCID50 challenge dose group similarly includes up to three cohorts each with a 
targeted total of 12 subjects receiving 105 TCID50 of the challenge strain and one subject 
receiving sham placebo with the thresholds for dose escalation to 106 TCID50 being < 70% 
subjects for Cohorts 2A, 2B, and 2C cumulatively moving from Cohort 2A to Cohort 2B and 
Cohort 2C, respectively.  The highest challenge dose group to be evaluated in this study is 106 

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TCID50, with each of two challenge cohorts including a targeted total of 17 subjects receiving 
106 TCID50 of the challenge strain and one subject receiving sham placebo. 
Following virus challenge, subjects will be monitored daily for up to 7 days post-challenge 
(Days 2-8). During this confinement period, time-series collections of biological samples and 
clinical information will occur daily from pre-challenge (baseline) through Study Day 8.  The 
different specimen types and clinical assessments are shown in (Table 1).  Whole blood samples 
will be processed into component parts (e.g., PBMCs, plasma, sera, RNA, DNA) to evaluate 
multiple host response systems including the immune, molecular, and cellular responses in the 
circulation and nasal mucosa.  Additionally, subjects will be provided wearable devices for 
continuous monitoring of physiological measures including heart rate, ECG, body temperature, 
and sleep habits.   
Examples of the devices used in the study include those listed below. Other devices approved for 
consumer and/or clinical use may also be used, provided they are fully non-invasive and do not 
present any additional risk to study subjects: 
• Garmin Vivosmart 4: This commercially available product is intended for training and 
recreational use. It is worn on the wrist and uses photoplethysmography (PPG) technology along 
with mechanical sensors to measure heart rate, oxygen saturation, activity, etc.  The Garmin will 
transfer the subject’s data via Bluetooth to the smartphone where the compatible app has been 
installed. These apps will then upload the data to cloud-based servers maintained by the 
individual device manufacturers. Data will then be uploaded to a secure server at the study sites. 
• Oura Ring: This commercially available product is intended for training and recreational 
use. It is worn on the finger and uses photoplethysmography (PPG) technology along with 
mechanical sensors to measure heart rate, body temperature, activity, etc.  The Oura Ring will 
transfer the subject’s data via Bluetooth to the smartphone where the compatible app has been 
installed. These apps will then upload the data to cloud-based servers maintained by the 
individual device manufacturers. Data will then be uploaded to a secure server at the study sites. 
• Bittium Faros 180: This FDA approved and CE marked medical device is available for 
clinical use; specifically for outpatient monitoring or occupational therapy after a cardiac event. It 
has an ECG sensor and accelerometer and can be worn using cable/electrode connections, or over 
the chest with a fabric strap.  Data collected by the Faros 180 will be manually uploaded (via 
USB) to a secure cloud server maintained by the sites.  
For safety assessments, ECG, and safety lab results will be evaluated at pre-specified 
timepoints as well as daily targeted physical exams during confinement.  Viral shedding will be 
measured by quantitative RT-PCR and culture, as well as qualitative multiplex RT-PCR tests 
using the BioFire or Luminex respiratory viral panel performed daily on nasal swab(s).  In 
parallel to specimen collections, subjects will provide self-reported symptom diaries once daily 
through Study Day 15 using a standard symptom instrument, FLU-PRO and Validation Diary.  
The modified Jackson score will be administered by study investigators twice daily through 
confinement and once at the Study Day 15 visit.  On Study Day 8, subjects may be discharged 
once they meet all of the following discharge criteria (subjects meeting discharge criteria may 
leave after receipt of qualitative RT-PCR results for the day):  
• Have two qualitative RT-PCR tests on NP swabs (that are collected 12 hours apart on 
Study Day 6 or thereafter on consecutive days) that are negative for influenza A using a 
multiplex respiratory virus assay (BIOFIRE® FILMARRAY® respiratory panel by 
bioMérieux or Luminex xTAG®).  

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• Are afebrile (< 100.6°F/38.1°C). 
• SpO2 ≥ 95% on room air 
• Show no signs or symptoms of moderate or severe influenza (Section 2.3.1) by clinical 
evaluation. 
• Are clinically and hemodynamically stable for at least 48 hours (per the evaluation of the 
licensed study clinician listed on the Form FDA 1572). 
• All AEs and SAEs will be recorded on the appropriate DCF.  
 
Subjects and study staff performing clinical evaluations and assessments will remain blinded to 
results of RT-PCR testing for influenza only until Study Day 6 or upon early discharge. The 
study team will not be blinded to RT-PCR testing for other respiratory pathogens. Subjects will 
be offered a course of an antiviral (oseltamivir or baloxavir marboxil) if they remain 
symptomatic and/or have a positive influenza test by qualitative RT-PCR on Study Day 6. 
Treatment can begin as early as Study Day 6 after test results are received. Subjects will remain 
in confinement until they have two negative influenza tests by qualitative RT-PCR. The two tests 
must be collected at least 12 hours apart.  Subjects will return for three follow-up visits at 
approximately Days 15, 29 and 57 post-challenge to assess overall health and for additional 
specimen and clinical data collections. 
 
Schedule of assessments can be found in Section 1.2, Schedule of Activities 
Protocol Schema can be found in Section 1.3, Study Schema. 
Dose escalation or dose-ranging details are found in Section 6.1.2, Dose Escalation 
Full details of interim analysis are found in Section 9.5.6, Planned Interim and Early Analyses 
 
4.2 
Scientific Rationale for Study Design 
Eligible participants will sequentially be enrolled into challenge cohorts and will be randomly 
assigned to receive a single dose of either sham inoculum or a virus dose between 104 to 106 
TCID50.  This study uses an adaptive dose-escalation design in order to most efficiently 
determine the optimal infectious dose with an AR ranging from 55 to 85% depending on the dose 
and, at the same time, closely monitor subject safety. This adaptive dose-ranging allows 
adjustments to challenge dose group size and escalation dose schedule to be informed by the AR 
and safety results.  Also, the study design allows for the study to progress across 2 sites with one 
site lagging behind the other by a period of around two weeks in order to achieve efficiency in 
enrollment of cohorts.  Randomization to challenge virus or sham placebo is essential to 
facilitate objectivity in symptom reporting.  One sham per challenge cohort will be included such 
that it is a possibility for any study subject to receive sham placebo, and thereby, subjects and 
evaluators would be more likely to objectively report and rate their self-reported symptoms if it 
was possible that they could have received sham placebo.  Collecting clinical, virologic and 
immunologic data on enrolled subjects using a standardized timeline and collection instruments 
should provide valuable information to determine the optimal dose of the challenge strain.  
4.3 
Justification for Dose 

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Subjects will be randomized and administered a single challenge dose of either sham placebo or a 
virus dose between 104 to 106 TCID50.  Historically, a 104 TCID50 has been safely used as the 
lowest dose tested in challenge studies[5-7]. There is no indication from non-clinical studies of the 
recombinant influenza A/Texas/71/2017 (H3N2), clade 3C3a virus in mouse, hamster and ferret 
models of infection that this virus will behave any differently in humans than the wild-type parent 
influenza virus. 
The dosing plan will follow an adaptive dose escalation schedule (Figure 1) that will include up 
to three different challenge dosing groups.  This dosing plan is designed to optimize subject safety, 
starting with a low dose and allowing flexibility to make informed adjustments between challenge 
cohorts from outcome results of the previous challenge cohort. The plan is guided by our 
experience and the results of our 3 previous human titration studies.  
For the first exposure in humans, the initial low dose challenge will be conducted in a group of a 
targeted total of 12 subjects (and 1 sham) to assure the virus is safe with no study-related or 
unanticipated adverse events observed or reported.  Dose escalation to the next dose level and 
subsequent higher doses, if necessary, will be informed by both safety and AR outcomes given a 
pre-specified decision algorithm for dose-escalating.  The decision to escalate or not escalate to the 
next challenge dose will involve the following criteria:  
• The tested dose is determined to be safe with no occurrence of halting criteria or severe 
influenza complications 
• An AR < 80% triggers dose escalation in the 104 TCID50 challenge dose group. 
• An AR < 70% triggers dose escalation in the 105 TCID50 challenge dose group. 
• An AR ≥ 80% in the 104 TCID50 challenge dose group would stop escalation, and a repeat 
of the presumed optimal dose for up to 3 total challenge cohorts: 1A, 1B and 1C. 
• An AR ≥ 70% in the 105 TCID50 challenge dose group would stop escalation, and a repeat 
of the presumed optimal dose for up to 3 total challenge cohorts: 2A, 2B and 2C. 
NOTE: AR is the percentage of participants meeting the pre-defined symptomatic influenza 
infection definition described in section 4.1.  
 
 

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5. 
STUDY POPULATION 
 
The study population will include male and female adult volunteers aged 18-45 years, inclusive, 
and generally in good health. 
5.1 
Inclusion Criteria 
Subjects eligible to participate in this study must meet all of the following inclusion criteria: 
1. Provide written informed consent prior to initiation of any study procedure 
2. Are able to understand and comply with planned study procedures and be available for 
all study visits 
3. Agree to remain an inpatient for at least 7 days after challenge AND until they have no 
viral shedding1, determined by qualitative RT-PCR beginning on Study Day 6  
 
1No viral shedding is defined as two negative RT-PCR tests 12 or more hours apart 
4. Healthy2 males and non-pregnant, non-breastfeeding females aged ≥ 18 and < 46 years 
of age at enrollment 
2Healthy is defined in inclusion criteria #11. 
NOTE:  Female subjects of childbearing potential must have a negative serum 
pregnancy test at screening, a negative urine pregnancy test upon admission to the 
confinement unit AND a negative pregnancy test before any CXR (if ≥ 7 days have 
passed since a serum pregnancy test). 
5. Women of childbearing potential3 must agree to use or have practiced true abstinence4 or 
use at least one acceptable primary form of contraception5 for at least 30 days prior to 
challenge 
3Not of childbearing potential - post-menopausal females (defined as having a history of 
amenorrhea for at least one year) or a documented status as being surgically sterile 
(hysterectomy, bilateral oophorectomy, tubal ligation/salpingectomy, or Essure® 
placement with history of documented radiological confirmation test at least 90 days 
after the procedure). 
4True abstinence is 100% of time no sexual intercourse (male’s penis enters the female’s 
vagina). (Periodic abstinence [e.g., calendar, ovulation, symptothermal, post-ovulation 
methods] and withdrawal are not acceptable methods of contraception). 
5Acceptable forms of primary contraception include monogamous relationship with a 
vasectomized partner who has been vasectomized for180 days or more prior to the 
subject receiving the influenza challenge virus, intrauterine devices, birth control pills, 
and injectable/implantable/insertable hormonal birth control products. Must use at least 
one acceptable primary form of contraception for at least 30 days prior to challenge and 
at least one acceptable primary form of contraception during the remainder of the study 
or approximately 57 days after confinement (Day -2). 

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NOTE:  These criteria are applicable to female subjects in a heterosexual relationship 
AND of child-bearing potential.  These criteria do not apply to subjects in a same sex 
relationship. 
6. Non-habitual smoker6 of tobacco, e-cigarettes or marijuana 
6Non-habitual smokers are those who smoke no more than four cigarettes, other tobacco 
products, e-cigarettes (to include vaping and Juuling products) or marijuana in a week 
and agree not to smoke cigarettes, other tobacco products, e-cigarettes and/or 
marijuana products during participation in the study. 
7. No self-reported or known history of alcoholism within the last 2 years and agrees to 
abstain from alcohol for at least one week before admission and throughout the 
confinement period 
8. No self-reported or known history of restricted drug use for at least 30 days prior to 
challenge and agrees to abstain from restricted drugs throughout the confinement period 
9. Negative drug urine toxicology result on screening (i.e., amphetamines, cocaine, and 
opiates) and on admission to the challenge unit (i.e., amphetamines, cocaine, and 
opiates)8 
8Select drug use may be allowed at Investigator’s discretion (e.g., prescribed 
amphetamines for ADHD).  
10. Agree not to use the listed prescription or over the counter medications9 within 7 days 
prior to  and through confinement period, unless approved by the investigator 
9Oseltamivir, zanamivir, peramivir, baloxavir marboxil, amantadine (generic) and 
rimantadine (Flumadine and generic), aspirin, intranasal steroids, decongestants, 
antihistamines, and other non-steroidal anti-inflammatory drugs (NSAIDs) 
11. In good health10, and do not have clinically significant medical, psychiatric, chronic or 
intermittent health conditions including those listed in Exclusion Criteria (Section 5.2) 
10Good health, as determined by medical history, medication use and physical 
examination to evaluate ongoing chronic medical or psychiatric diagnoses or conditions, 
defined as those that have been present for at least 90 days, which would not affect the 
assessment of the safety of subjects or the immunogenicity of challenge. These medical 
diagnoses or conditions should be stable for the last 90 days (no hospitalizations, 
emergency room (ER) or urgent care for condition (excluding musculoskeletal 
conditions) and not listed in Exclusion Criteria (Section 5.2).  
Subjects may be on medications only if the condition or disease is stable and not 
deteriorating, if the medical intervention (such as device or medication) was not 
available during the maximal inpatient period of time, medications are not listed in the 
Exclusion Criteria (Section 5.2) and pose no additional risk to subject safety or 
assessment of adverse events. This also includes no change in prescription medication, 
dose or frequency as a result of new symptoms or deterioration of the medical diagnosis 
or condition in the 90 days prior to enrollment. Any prescription change that is due to 
change of health care provider, insurance company, etc., or that is done for financial 
reasons, as long as in the same class of medication, will not be considered a deviation of 

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this inclusion criterion.  Any change in prescription medication due to improvement of a 
disease outcome (e.g., lowering of the dosage or frequency), as determined by the site 
principal investigator (PI) or designated clinician licensed to make medical diagnoses 
and listed on Form FDA 1572, will not be considered a deviation of this inclusion 
criterion. 
12. Vital signs as follows:  
• Pulse is 47 to 99 beats per minute, inclusive 
• Systolic blood pressure is 85 to 139 mmHg, inclusive 
• Diastolic blood pressure is 55 to 89 mmHg, inclusive 
• SpO2 > 95%; RR < 18 
• Oral temperature is less than 100.6°F 
 
13. Eligibility laboratory values (WBC, Absolute Lymphocyte Count, Hgb, PLTs, ALT and 
Cr) are within acceptable parameters11  
11Labs within normal range or grade 1 abnormalities deemed not clinically significant 
by a study investigator are considered acceptable 
14. Body mass index (BMI) >18.5 and <40 kg/m2 at screening 
15. Other screening tests (ECG and CXR) are within normal reference range or not deemed 
clinically significant by the PI or appropriate sub-investigator12 
12Designated clinician licensed to make medical diagnoses and listed on the Form FDA 
1572 
16. Negative test for HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) at screening 
17. Negative respiratory virus panel by BIOFIRE® FILMARRAY® respiratory panel by 
bioMérieux or by Luminex xTAG® on Day -2, and Day -1 
18. Negative RT-PCR test for SARS-CoV 2 on screening and Day -2 
19. HAI antibody titer ≤1:40 against influenza A/Texas/71/2017 (H3N2) at screening 
20. Receipt of the recommended number of doses of an EUA authorized or licensed COVID-
19 vaccine product ≥ two weeks prior to confinement (Day -2). 
5.2 
Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participating in the 
study: 
1. Presence of self-reported or medically documented significant medical or psychiatric 
condition(s)1 
1Significant medical or psychiatric conditions include but are not limited to: 
a. Respiratory disease (e.g., chronic obstructive pulmonary disease [COPD], 
asthma) requiring daily medications [Inhaled, oral or intravenous (IV) 
corticosteroids, leukotriene modifiers, long and short acting beta agonists, 
theophylline, ipratropium, biologics] or any treatment for respiratory disease 

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exacerbations (e.g., asthma exacerbation) within the last 5 years 
b. Presence of any febrile illness or symptoms suggestive of a respiratory infection 
within two weeks prior to challenge 
c. Significant cardiovascular disease (e.g., congestive heart failure, 
cardiomyopathy, ischemic heart disease) or history of myocarditis or pericarditis 
as an adult. 
d. Neurological or neurodevelopmental conditions (e.g., epilepsy, stroke, seizures, 
encephalopathy, focal neurologic deficits, Guillain-Barre syndrome, 
encephalomyelitis or transverse myelitis). 
e. Ongoing malignancy or recent diagnosis of malignancy in the last five years 
(excluding basal cell carcinoma of the skin) 
f. 
Presence of an autoimmune disease. 
g. Immunodeficiency of any cause. 
h. History of diabetes. 
2. Presence of immunosuppression or any medications that may be associated with impaired 
immune responsiveness2 
2Including, but not limited to, corticosteroids exceeding 10 mg/day of prednisone 
equivalent, allergy injections, immunoglobulin, interferon, immunomodulators, cytotoxic 
drugs, or systemic corticosteroids or other similar or toxic drugs during the preceding 
12-month period prior to screening. Low dose topical and intranasal steroid 
preparations used for a discrete period of time are permitted (Section 6.5). 
3. Known allergy or intolerance to treatments for influenza (including any neuraminidase 
inhibitors or baloxavir marboxil). 
4. Known allergy to two or more classes of antibiotics (e.g., penicillins, cephalosporins, 
fluoroquinolones, or glycopeptides). 
5. Known allergy to excipients3 in the challenge virus inoculum. 
 
3 Sucrose, KH2PO4, K2HPO4, L-glutamic acid (in SPG diluent) 
6. Receipt or planned receipt of any investigational drug/investigational vaccine/licensed 
vaccine, except for a licensed or emergency use authorized COVID-19 vaccine product, 
within 30 days prior to the date of challenge. 
7. Prior enrollment in an influenza virus challenge study with an influenza virus of the same 
subtype within the past 2 years. 
8. Currently enrolled in any investigational study or intends to enroll in such a study within 
the ensuing study period.4 
4Co-enrollment in an observational study, an investigational study in a follow-up/post-
vaccination stage, or a study involving a licensed drug or biologic may be allowed at the 
investigator’s discretion. 
9. Receipt of any influenza vaccine four months prior to challenge or plans to receive 
influenza vaccine during the study (approximately 57 days after challenge). 
10. History of a previous severe allergic reaction to any drug or biologic with generalized 

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urticaria, angioedema, or anaphylaxis. 
11. Receipt of blood or blood products during the six months prior to the planned date of 
challenge. 
12. Plans to donate blood or blood products during the study (approximately 102 days). 
13. Any condition (including medical and psychiatric conditions) that, in the opinion of the 
Investigator, might interfere with the safety of the subject and/or study objectives. 
14. An ongoing symptomatic condition for which subject has had or has ongoing medical 
investigations but has not yet received a diagnosis or treatment plan e.g., ongoing fatigue 
without a diagnosis. 
15. Known close contact with anyone known to have or suspected to have a respiratory viral 
illness within 7 days prior to challenge. 
16. Significant abnormality altering anatomy of nose/nasopharynx (including significant 
nasal polyps), clinically significant nasal deviation, or nasal/sinus surgery within 180 
days prior to challenge. 
17. History in the last five years of chronic or frequent intermittent sinusitis. 
18. Recent history (180 days) of epistaxis or anatomic or neurologic abnormality impairing 
the gag reflex or contributing to aspiration. 
19. Currently using an internal cardiac device such as a pacemaker or other implanted 
electronic medical devices. 
 
5.2.1 
Exclusion of Specific Populations 
For safety purposes, we are targeting a young, healthy population.  We are excluding persons 
over 45 years of age and children and adolescents under 18 years of age.  We are also excluding 
pregnant women and women who are planning to become pregnant from 30 days prior to 
challenge through the end of the study. 
 
5.3 
Inclusion of Vulnerable Participants 
Not applicable. 
 
5.4 
Lifestyle Considerations 
During this study, participants are asked to: 
• Remain in a confinement unit for a minimum of 7 days following challenge (total of 10 
days) or until they have at least 2 consecutive negative influenza tests performed 12 hours 
apart starting on Study Day 6 of the study. 
• Refrain from drinking alcohol or restricted drug use for one week prior to confinement 
and during the confinement period. 

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• Refrain from taking any forbidden medications including over-the-counter medications 
(including non-steroidal anti-inflammatory medications) for one week prior to 
confinement and during the confinement period (unless approved during confinement). 
• Refrain from enrolling in any other investigational study throughout the duration of the 
study. 
• Refrain from donating blood or blood products throughout the duration of the study and 
for 2 months prior to challenge. 
• For women of childbearing potential, avoid getting pregnant and use contraception within 
30 days from challenge. 
5.5 
Screen Failures 
After the screening evaluations have been completed, the investigator or designee will review the 
inclusion/exclusion criteria and determine the subject’s eligibility for study randomization. 
Subjects who are found to be ineligible will be told the reason for ineligibility and will be 
replaced.  Individuals who do not meet the criteria for participation in this study (screen failure) 
because of a current respiratory illness may be re-screened after 7 days.   
5.6 
Strategies for Recruitment and Retention 
5.6.1 
Recruitment 
Potential participants may be selected from the existing cohort that were pre-screened for 
influenza antibody titers in the screening protocol DMID 20-0004 and from other existing 
participant registries, through advertising or by word of mouth. Potentially eligible participants 
for this study must consent to the repository protocol, DMID 19-0025, prior to initiation of any 
screening activities. Recruitment staff at all sites will contact those individuals enrolled in 20-
0004 and 19-0025 to assess their eligibility for this study. It is anticipated that the demographic 
composition of the 20-0004 cohort and subsequent subjects for this study will be representative 
of the respective study site region. 
 
The IRB will approve the recruitment process and all materials provided prior to any recruitment 
to prospective subjects directly. 
 
Screening will begin with a brief discussion with study staff. Some people will be excluded 
based on demographic data and medical history (i.e., pregnant, recently vaccinated, etc.). 
Information about the study will be presented to potential subjects and questions will be asked to 
determine potential eligibility.  
5.6.2 
Retention 
Retention of subjects in this trial is very important for determining the primary endpoint. As 
such, after discharge from confinement, participating subjects will be reminded of subsequent 
study visits and every effort will be made to accommodate the subject’s schedule to facilitate 
follow-up within the specified visit window. Additionally, there are many circumstances that 
influence the ability to obtain outcome information after discharge. Follow-up visits may be 
conducted by phone if in person visits are not feasible. Lastly, compensation strategies will be 
implemented to encourage subjects to return for follow-up assessments and sample collections.  

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5.6.3 
Compensation Plan for Subjects 
Compensation will be determined locally and in accordance with IRB requirements, and subject 
to IRB approval. Compensation will be described in the site-specific informed consent. 
5.6.4 
Costs 
There is no cost to subjects for the research tests, procedures, and study product while taking part 
in this trial.  However, procedures and treatment for clinical care may be billed to the subject, 
subject’s insurance or third party.   
 
 

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6. 
STUDY PRODUCT 
6.1 
Clinical Challenge Strain 
A GMP lot of reverse-genetics (RG) derived, recombinant influenza A/Texas/71/2017 (H3N2) 
virus for the purpose of conducting human influenza virus challenge studies has been 
manufactured by Charles River Laboratories (CRL), Malvern, PA. As part of the GMP 
manufacturing process, a number of tests have been performed to assure the virus is 
genotypically and phenotypically accurate, including genomic sequencing of the GMP-grade 
virus. In addition, pre-clinical animal testing has been performed in ferrets, mice, and hamsters to 
demonstrate that manufactured virus behaves similarly in animals to the wild-type strain from 
which it was derived. Non-clinical testing results are included in the Investigator’s Brochure (IB) 
for the Live Influenza Virus RG-A/Texas/71/2017 (H3N2). 
The challenge virus is nearly identical to circulating influenza A/Texas/71/2017 (H3N2) viruses 
that are susceptible to FDA-approved neuraminidase inhibitors oseltamivir and zanamivir.  Full 
genomic sequencing was performed on the Live Influenza Virus RG-A/Texas/71/2017 (H3N2) 
(Lot1507-232149) and did not indicate any mutations that confer resistance to these antivirals. 
The 2017 H3N2 viruses are resistant to the amantadine class of influenza antivirals [8]. There is 
no indication that the Live Influenza Virus RG-A/Texas/71/2017 (H3N2) challenge virus would 
behave any differently in humans than the wild-type influenza A/Texas/71/2017 (H3N2) virus, 
based on the available in vitro and in vivo data.  
The diluent for the challenge strain is 1X SPG (7.4% sucrose, 3.8 mM KH2PO4, 7.2 mM 
K2HPO4, 5.4 mM L-glutamic acid), Lot #141701, produced using GMP manufacturing processes 
at CRL, Malvern, PA. 
6.1.1 
Dosing and Administration 
 
Table 2. Dosing and Administration 
Product Name 
Doses 
Route 
Frequency of 
Administration 
Live Influenza Virus RG-
A/Texas/71/2017 H3N2 
1.0 mL (0.5 mL per nostril) 
of approximately 104, 105, 
or 106 TCID50 each dose * 
Intranasal by 
atomizer delivery 
Challenge virus will be 
administered once on Day 1 
Sham (SPG) inoculum 
1.0 mL (0.5 mL per nostril) 
of vehicle inoculum 
Intranasal by 
atomizer delivery 
Inoculum will be administered 
once on Day 1 
* TCID50, median tissue culture infectious dose 
 
6.1.2 
Dose Escalation 
The dose escalation schema is provided in Figure 1.  
6.1.3 
Dose Modifications 
There will be no individualized dose modifications of virus inoculum.   

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6.2 
Preparation/Handling/Storage/Accountability 
6.2.1 
Acquisition and Accountability 
Clinical challenge virus stock and SPG diluent will be sourced by DMID Clinical Material 
Services (CMS).   
Records will be maintained that document receipt, release for dosing, disposal, or return to the 
sponsor.   
Upon request by DMID study products 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 
 
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.  Refer to the protocol-specific MOP for details on storing study 
medications. 
 
Unused study product 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. Used study product will be 
disposed of per the MOP. Documentation of study product destruction will be available to 
monitors upon request. 
 
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 
institution’s SOP/policy mandates it. If local SOPs allow/require destruction of used study 
product containers, then the used vials can be destroyed per the site’s SOPs with a second staff 
member’s observation and signed verification (two signatures) that the used vials were 
destroyed. 
 

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Refer to the protocol-specific MOP for details should any vials need retained for viral titer 
testing.   
 
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, Packaging, and Labeling 
Live Influenza Virus RG-A/Texas/71/2017 (H3N2)  
The final product was vialed at approximately 1.86 X 106 TCID50/mL of Influenza A Human 
Challenge Virus (H3N2) in 1X SPG (7.4% sucrose, 3.8 mM KH2PO4, 7.2 mM K2HPO4, 5.4 mM 
L-glutamic acid). Each vial has a fill volume of 0.6 mL.  
Each vial was individually labeled with the product name, description, and caution statement. 
The label was affixed to 2 mL cryogenic vials prior to final fill with Live Influenza Virus RG-
A/Texas/71/2017 (H3N2) Virus [Charles River Laboratories, Malvern PA] (Lot #1507-232149). 
Diluent and Sham Product 
1X SPG (7.4% sucrose, 3.8 mM KH2PO4, 7.2 mM K2HPO4, 5.4 mM L-glutamic acid).  Each vial 
has a fill volume of 1.2 mL. The diluent will also be used as a sham inoculum. Each vial was 
individually labeled with the product name, description, and caution statement. The label was 
affixed to 2 mL cryogenic vials prior to final fill with 1X SPG [Charles River Laboratories, 
Malvern PA] (Lot #141701). 
6.2.3 
Product Storage and Stability 
Live Influenza Virus RG-A/Texas/71/2017 (H3N2)  
The human challenge virus will be shipped to the sites and stored at ≤ -65˚C in single-use vials. 
Additional details of product storage and stability can be found in the MOP.  
Diluent/Sham Product 
The 1X SPG will be shipped to the sites and stored at -20°C  10C in single-use vials. 
Additional details of product storage and stability can be found in the MOP.  
MAD Nasal Intranasal Mucosal Atomization Device (Teleflex, Morrisville NC) 
The MAD Nasal Intranasal Mucosal Atomization Device (Teleflex, Morrisville NC) will be 
stored at room temperature (15-30°C). 
6.2.4 
Preparation and Administration 
Live Influenza Virus RG-A/Texas/71/2017 (H3N2)  
The Live Influenza Virus RG-A/Texas/71/2017 (H3N2) is an infectious influenza virus and 
requires handling at Biosafety Level 2. All steps in preparing the syringes should be performed 
with aseptic technique in a biosafety cabinet.  
The challenge virus will be administered intranasally, 0.5 mL in each nostril of a recumbent 
participant using the MAD Nasal Intranasal Mucosal Atomization Device attached to a 1 mL 

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syringe.  Detailed instructions for challenge virus inoculum preparation and administration are 
provided in the MOP.  
6.3 
Measures to Minimize Bias: Randomization and Blinding 
6.3.1 
Study Product Assignment Procedures 
Subjects will be randomized to receive either Live Influenza Virus RG-A/Texas/71/2017 (H3N2) 
challenge virus or sham placebo within each challenge dose group. Subject will receive one of 
three doses of challenge virus depending upon the challenge cohort into which they enroll using 
the adaptive trial design.  The ratio of subjects randomized to receive either challenge virus or 
sham placebo is described below.   
Challenge 
Cohort 
Challenge Dose Group 
  
Challenge virus:Sham 
placebo 
1A, 1B, 1C 
104 TCID50 
Targeted 12:1 
2A, 2B, 2C 
105 TCID50 
Targeted 12:1 
3A, 3B   
106 TCID50 
Targeted 17:1 
 
6.3.2 
Randomization and Blinding 
Eligible subjects for each challenge cohort will be randomized to receive either challenge virus 
or sham. Enrollment into the active challenge cohort will be completed before the next challenge 
cohort is opened to enrollment. The subsequent challenge cohort will be selected and opened for 
enrollment after safety review, as shown in Figure 1. To compress the overall dosing timelines, 
challenge cohorts may be split between the 2 sites. Subjects that drop out less than 48 hours prior 
to challenge will not be replaced, but additional randomization slots will be included (at the same 
targeted allocation ratios for each cohort) to allow for the possibility of enrollment beyond the 
targeted cohort sizes. 
The list of randomized treatment assignments will be prepared by statisticians at The Emmes 
Company, LLC. and included in the enrollment module of Emmes’ Internet Data Entry System 
(IDES).  IDES will assign each subject a treatment code from the list after demographic and 
eligibility data have been entered into the system.  A designated individual at each site will be 
provided with a treatment key, which links the treatment code to the actual treatment assignment, 
which will be kept in a secure place. 
Instructions for use of the enrollment module are included in the IDES User’s Guide.  Manual 
back-up randomization procedures are provided in the MOP for use in the event that the site 
temporarily loses access to the Internet or the online enrollment system is unavailable. 
6.3.3 
Blinding and Masking Procedures 
Study product will be prepared by an unblinded pharmacist but administered by a blinded 
administrator. The subjects, the study personnel who perform study assessments after 
administration, data entry personnel at the sites, and laboratory personnel performing 
immunologic assays will be blinded to treatment assignment.  The SMC will receive data in 
aggregate by cohort and challenge dose group, and they may be unblinded to individual study 

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product assignments, as needed, to adequately assess safety issues.  Refer to the MOP for 
unblinding procedures. 
6.4 
Study Intervention Compliance 
Subjects 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 on the 
Treatment Administration Record and entered into the eCRF. 
6.5 
Concomitant Therapy 
All concomitant medications taken within 90 days prior to signing the ICF will be reviewed with 
subjects to determine stability of chronic diseases and eligibility. Medications reported in the 
eCRF are limited to those taken within 30 days prior to challenge. Concomitant medications will 
be reviewed at every study visit through the end of the study. Women of childbearing potential in 
a heterosexual relationship must agree to use true abstinence or use at least one acceptable 
primary form of contraception through the end of the study. 
 
Receipt of any influenza vaccine during the 2019/2020 and/or 2020/2021 influenza vaccine 
seasons, regardless of the date of receipt, will be documented. Subject receipt of non-seasonal 
influenza vaccine, including those that are experimental, product type, vaccine virus strains and 
approximate date of previous two season’s vaccination will be documented. Prior participation in 
any influenza challenge study will be documented.  Note that prior enrollment in any influenza 
challenge study within the prior two years and/or receipt of any influenza vaccine four months 
prior to challenge is exclusionary.   
 
The following prescription or over-the-counter medications cannot be used within 7 days prior to 
admission to and through the confinement period, unless approved by the investigator: 
oseltamivir, zanamivir, peramivir, baloxavir marboxil, amantadine (generic) and rimantadine 
(Flumadine and generic), aspirin, intranasal steroids, decongestants, antihistamines, and other 
non-steroidal anti-inflammatory drugs (NSAIDs).  
 
Subjects who use asthma medications including inhaled, oral, or IV corticosteroids, leukotriene 
modifiers, long and short acting beta agonists, theophylline, ipratropium, biologics, will be 
excluded from the study.  
 
Any medications that may be associated with impaired immune responsiveness including, but 
not limited to, corticosteroids exceeding 10 mg/day of prednisone equivalent, allergy injections, 
immunoglobulin, interferon, immunomodulators, cytotoxic drugs, or systemic corticosteroids or 
other similar or toxic drugs cannot be used during the preceding 12-month period prior to 
screening. Low dose topical and intranasal steroid preparations used for a discrete period of time 
are permitted.  
 
Subjects should not have received any investigational drug/investigational vaccine/licensed 
vaccine within 30 days prior to the planned date of challenge, with the exception of an EUA 
authorized or licensed COVID-19 vaccine product ≥ two weeks prior to admission.  
 

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Subjects should not have received influenza vaccine four months prior to challenge and must not 
plan to receive an influenza vaccine during the study period (approximately 57 days).  
 
Subjects should not have received blood or blood products during the six months prior to the 
planned date of challenge. 
6.5.1 
Non-Research Standard of Care 
Oseltamivir phosphate (TAMIFLU) or baloxavir marboxil (XOFLUZA) will be offered to 
any subject who has not had two consecutive days of negative qualitative RT-PCR tests for 
influenza virus by Study Day 8.  Oseltamivir is a neuraminidase inhibitor indicated for the 
treatment of acute uncomplicated influenza A and B in patients 2 weeks of age and older who 
have been symptomatic for no more than 2 days, and for prophylaxis of influenza in patients 1 
year and older. Baloxavir is a polymerase acidic (PA) endonuclease inhibitor indicated for the 
treatment of acute uncomplicated influenza in patients 12 years and older who have been 
symptomatic for no more than 48 hours.  
 
Subjects who continue to shed virus beyond Day 8 will undergo routine daily evaluations as 
described in Sections 5 and 8. The licensed study clinician listed on the Form FDA 1572 may 
also treat the subject with symptom-directed, over-the-counter therapies as permitted by the 
study protocol. If the subject meets criteria for escalation of care, evaluation and treatment will 
be at the discretion of the consulting physician. Escalation of care may result from the 
development of serious related or unrelated signs or symptoms during confinement period. 
 
 

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7. 
STUDY INTERVENTION DISCONTINUATION AND 
PARTICIPANT DISCONTINUATION/WITHDRAWAL 
7.1 
Halting Criteria and Discontinuation of Study Intervention 
7.1.1 
Study Halting Criteria 
Additional enrollment and interventions/administration of study products will be paused for 
Safety Monitoring Committee (SMC) review/recommendation if any of the following events are 
reported: 
1. Any subject experiences laryngospasm, bronchospasm, or anaphylaxis within one day 
after challenge. 
2. Two or more subjects experience generalized urticaria (defined as occurring at more than 
two body parts) within three days after challenge. 
3. Any subject experiences a related SAE during the confinement unit period.  
o This includes any subject who develops a severe influenza complication (Section 
2.3.1) during the confinement unit period and requires admission to an Intensive 
Care Unit (ICU), and/or receipt of ICU level of care 
4. Two or more subjects per challenge cohort (or four or more subjects per dosing group) 
experience the same related grade 3 AE of any kind after challenge through the 
confinement unit period and related to study product. 
5. Any medically significant safety issue that the PI determines should halt the study. 
 
The SMC is external to the DMID and comprises at least 3 voting members. The SMC will 
consist of members with appropriate expertise to contribute to the interpretation of the data from 
this study. Its activities will be delineated in a SMC charter that will describe the membership, 
responsibilities, and the scope and frequency of data reviews. The SMC will operate on a 
conflict-free basis independently of the study team. The DMID or the SMC may convene ad hoc 
meetings of the SMC according to protocol criteria or if there are concerns that arise during the 
study.  
 
As defined in the charter, the SMC will review data at specified times during the course of the 
study for subject and overall study progress and will conduct ad hoc reviews as appropriate when 
a halting rule is met or for immediate concerns regarding observations during this study. The 
SMC will hold the following meetings; an organizational meeting prior to subject enrollment, 
any ad hoc meetings when a halting rule is met or due to specific safety issues as the SMC deems 
necessary, and a final meeting to review cumulative safety data. 
7.2 
Participant Withdrawal from the Study and Replacement 
Participants are free to withdraw from participation in the study at any time upon request. 
Subjects may also withdraw voluntarily from receiving the study intervention for any reason.  
Subjects will have approximately a 48-hour window to decide if they would like to drop out of 
the study and leave the confinement unit before challenge. The investigator should be explicit 
regarding study follow-up (e.g., safety follow-up) that might be carried out once the subject 
undergoes challenge but decides to leave the confinement unit before discharge criteria are met.  

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Subjects will be strongly discouraged from withdrawing from the study post-challenge. Any 
subjects who withdraw from the study post-challenge but prior to meeting the discharge criteria 
outlined will be asked to sign a document stating that they are aware of the potential risks of 
developing influenza complications, they are aware of the risks of transmitting influenza virus to 
high-risk populations, and that they will stay sequestered in their homes and avoid contact with 
others until they have two consecutive negative tests for influenza virus by qualitative RT-PCR 
conducted at least 12 hours apart. The PI, co-investigator or study team will counsel the subject 
on infection control practices to minimize any community transmission. Subjects without two 
consecutive days of negative tests for influenza virus by qualitative RT-PCR will be given one 
dose of baloxavir marboxil or a 5-day course of oseltamivir at the time of premature withdrawal 
from the study. For the purposes of safety and serological testing, the subject will also be asked 
to come for all follow-up visits. If the subject leaves the confinement unit prior to Study Day 8, 
whether or not they are continuing to shed virus, the subject will be encouraged to return for 
follow-up visits per outpatient schedule (i.e., Visits 9, 10, and 11). If the subject does not return 
for scheduled follow-up visits, extensive effort (i.e., at least three documented contact attempts 
via phone calls, e-mails, text message, or private social media communication, etc., as 
voluntarily provided by the subject and based on IRB recommendations, made on at least 3 
separate days and followed by a certified letter if unsuccessful) will be made to locate the subject 
and reiterate that follow-up visits are strongly encouraged for safety reasons. These efforts will 
be documented in the subjects’ records. See the MOP for Subject Contact Information. The 
investigator will inform the subject that already collected data will be retained and analyzed even 
if the subject withdraws from this study. If a subject withdraws or is withdrawn prior to 
completion of the study, the reason for this decision must be recorded in the case report forms 
(CRFs).  
An investigator may discontinue or withdraw a participant from the study for the following 
reasons: 
• Subject becomes pregnant.  
• Study non-compliance that, in the opinion of the investigator, poses an increased risk 
(i.e., missing safety labs), or compromises the validity of the data.  
• 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. 
• Any subject that experiences a related SAE during the confinement unit period.  
o This includes any subject who develops a severe influenza complication (Section 
2.3.1) during the confinement unit period and requires admission to an Intensive 
Care Unit (ICU), and/or receipt of ICU level of care 
• If the participant met an exclusion criterion for participation in the study (either newly 
developed or not previously recognized) that precludes further study participation 
• 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 
subject, interfere with the subject's successful completion of this study, or interfere with 
the evaluation of responses 
• Subject becomes lost to follow-up. 

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If the subject agrees, every attempt will be made to follow all AEs through resolution. 
Subjects who withdraw, or are withdrawn from this study, or are lost to follow-up after signing 
the informed consent form (ICF) and administration of the study product will not be replaced.  
Subjects who withdraw, or are withdrawn from this study, or are lost to follow-up after signing 
the ICF but before randomization may be replaced.  
The reason for participant discontinuation or withdrawal from the study will be recorded on the 
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 and Outcome Procedures 
8.1.1 
Screening Procedures 
Potentially eligible study subjects may be pre-screened under an active screening protocol 
(DMID 20-0004) to identify those with HAI antibody titers ≤1:40 specific to the challenge virus 
strain.  Additionally, potentially eligible subjects may be recruited through other site registries, 
advertisements or by word-of-mouth.  During the study screening period (Day -45 to Day -3), 
participants who meet all inclusion criteria and none of the exclusion criteria are eligible for 
enrollment to the study.  Participants will be screened during the screening period and again at 
the time of confinement (Day -2).    
After the informed consent, the following assessments are performed to determine eligibility and 
obtain baseline data: 
• Take a focused medical history, including the following information: 
• History of any chronic medical conditions related to inclusion and exclusion 
criteria 
• Review of receipt of 2020/2021 influenza vaccine and 2021/2022 influenza 
vaccine (note that receipt of 2020/2021 influenza vaccine and receipt of 
2021/2022 influenza vaccine within the past 4 months or within approximately 57 
days post-challenge is exclusionary) 
• Concomitant medications taken within 90 days prior to signing the ICF will be 
reviewed with subjects to determine stability of chronic diseases and eligibility 
• Women of childbearing potential must have a negative serum HCG pregnancy test at 
screening and a negative urine HCG pregnancy test on Day -2.   
• Participants must have a negative drug urine toxicology test (amphetamines, cocaine, and 
opiates) at screening and on Day -2 (amphetamines, cocaine, and opiates), unless the drug 
is deemed acceptable by the Investigator. 
• Physical exam (standard exam at screening and targeted exam on Day -2) 
• Height measurement  
• Weight measurement  
• Vital signs measurement (including RR, HR, BP, oral temperature) 
• SpO2 measurement 
• ECG  
• Baseline posterior anterior (PA) and lateral CXR (Among women of childbearing 
potential, a repeat urine pregnancy test will be performed locally if >7 days have passed 
since the negative serum pregnancy test was drawn. The result of the urine pregnancy test 
must be negative before the CXR is performed.) 
• Blood for laboratory evaluations (approximately 15 mL at screening only): 
• Serology at screening: HIV, Hepatitis B surface antigen, Hepatitis C antibody, 
• Chemistry at screening: alanine transaminase (ALT) and creatinine (Cr) 
• Hematology at screening: white blood cells (WBCs), absolute lymphocyte count, 
hemoglobin, and platelets 

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• Blood for HAI titer 
• Participants must have a negative qualitative multiplex respiratory virus assay 
(BIOFIRE® FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) 
including a test for SARS-CoV-2 on NP swab on Day -2 in order to be eligible for 
influenza challenge 
• Nasopharyngeal swab for virologic assessments 
 
Clinical screening laboratory evaluations and the HAI titer will be performed locally by the site 
laboratory. The volume of venous blood to be collected is presented in Appendix C. 
 
The overall eligibility of the subject to participate in the study will be assessed once all screening 
values are available. 
8.1.2 
Discharge from Quarantine 
Subjects will be discharged from quarantine on or after Study Day 8 once they meet all of the 
following discharge criteria (subjects meeting discharge criteria may leave after receipt of 
qualitative RT-PCR results for the day). Subjects who do not meet discharge criteria on Study 
Day 8 will remain in the confinement unit until the criteria are met. Study procedures for the 
confinement unit will be the same as on Study Day 8, except no additional blood draws unless 
clinically indicated. 
• Have two consecutive qualitative RT-PCR tests on NP swabs (collected 12 hours apart on 
Study Day 6 or thereafter on consecutive days) that are negative for influenza A using a 
multiplex respiratory virus assay (BIOFIRE® FILMARRAY® respiratory panel by 
bioMérieux or Luminex xTAG®).  
• Are afebrile (< 100.6°F/38.1°C). 
• SpO2 ≥ 95% on room air 
• Show no moderate or severe influenza signs or symptoms by clinical evaluation. 
• Are clinically stable for at least 48 hours (per the evaluation of the licensed study 
clinician listed of the Form FDA 1572). 
• 
All AEs and SAEs will be recorded on the appropriate DCF.  
8.1.3 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
If a physiologic parameter, e.g., vital signs, is outside of the protocol-specified range, then the 
measurement may be repeated once if, in the judgment of the investigator, the abnormality is the 
result of an acute, short-term, rapidly reversible condition (e.g., stress, anxiety, or “white coat 
syndrome”). A physiologic parameter may also be repeated if there is a technical problem with 
the measurement caused by malfunctioning, or an inappropriate measuring device (i.e., 
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A subject may be re-screened if there is a transient disease status (e.g., subject 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 re-
screening 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 subject may be screened more than twice for the same challenge cohort due to a screening 
failure result as defined above. However, subjects that screen fail for one challenge cohort can be 
screened in the future for another challenge cohort. 
Participants will be provided the results of any abnormal clinical laboratory results and will be 
referred to their primary healthcare provider. Research laboratory results will not be provided to 
the participant.  
 
8.1.4 
Outcome / Immunogenicity / Genetic Assessments 
The specific timing of procedures/evaluations to be done at each study visit are captured in 
Section 1.2, Schedule of Activities (SoA).  All labs in this section will be completed in research 
labs, thus not requiring CLIA certification.  Instructions for specimen preparation, handling, and 
storage are included in the protocol-specific MOP. Specimen shipment to designated research 
laboratories for outcome/immunogenicity/genetic assessments will occur at intervals during the 
course of this study following all applicable International Air Transport Association (IATA) 
requirements and according to the specifics for storage temperature and documentation as 
detailed in the protocol-specific MOP.  
  
8.1.4.1 
Outcome Evaluations 
Clinical assessments 
• Symptomatic influenza infection for determining AR will be assessed using a Modified 
Jackson score collected by study staff twice a day (approximately 8 AM and after 3 PM) 
from Day -2 until the time of discharge. The investigator will also complete the Modified 
Jackson Score at the Day 15 visit to assess any ongoing symptoms.  The Modified 
Jackson score will be calculated after evaluation of vital signs, laboratory results, and the 
Modified Jackson score across multiple days. The score will be calculated during analysis 
for safety reporting by the SDMCC. 
 
• An additional validated, patient-reported outcome (PRO) measure to standardize 
assessment of influenza symptoms in clinical research (i.e., FLU-PRO Survey Instrument 
and Validation Diary) will be completed by subjects once a day (after 3:00 PM) from 
Day -2  through Day 15.    The investigators will review the FLU-PRO and Validation 
Diary at the Day 15 visit to assess any ongoing symptoms.   
 
• Wearable devices 
o The Garmin uses PPG technology along with mechanical sensors to measure heart 
rate, oxygen saturation, activity, etc. 
o The Oura Ring uses PPG technology along with mechanical sensors to measure 
heart rate, body temperature, activity, etc. 

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o The Faros device has an ECG sensor and accelerometer and can be worn using 
cable/electrode connections. 
 
Virologic assessments 
Virologic assessments will be done at screening (Days -45 to -3), baseline (Days -2 and -1) and 
will be repeated daily from Days 2-8 during the confinement period or for a longer period if still 
RT-PCR positive for influenza on Days 7 or 8 until two negative swabs are confirmed, at least 12 
hours apart, consecutively.  Only qualitative RT-PCR will be performed at the screening visit. 
• 
Qualitative multiplex respiratory virus assay (BIOFIRE® FILMARRAY® respiratory 
panel by bioMérieux or Luminex xTAG®) on NP swab(s). 
• Quantitative RT-PCR for influenza from NP swab(s) specimen. 
• Qualitative and quantitative RT-PCR for influenza from NLF specimens may be 
performed.  
• Quantitative viral culture from NP swab(s) and/or NLF specimens may be performed.  
 
8.1.4.2 
Immunogenicity Assessments 
• HAI, MN, NAI, anti-HA-stalk antibody titers from serum 
• sIgA titers from NLF  
• Systemic cytokines and chemokines from serum 
• Mucosal cytokines and chemokines from NLF or nasal curettage specimens 
• Transcriptional responses from whole blood 
• Plasmablasts (ASC) in circulation (from PBMC) 
• Influenza-specific B and T cell immune responses from PBMCs 
• T cell activation may be performed in cells isolated from nasal curettages 
• Immunophenotyping of innate, B and T cell subsets in NLF or nasal curettage specimens 
Please see Appendix C for venipuncture volumes. 
 
8.1.4.3 
Genetic/Genomic Analyses 
• Human leukocyte antigen (HLA) class I and II alleles from blood 
• Transcriptional responses from whole blood 
Please see Appendix C for venipuncture volumes. 
 
Genetic Privacy and Confidentiality  

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Genetic data and health information may be stored and shared with other researchers through a 
controlled-access repository, such as dbGaP. There may be a risk that information resulting from 
research genetic testing could be misused for discriminatory purposes. However, state and 
federal laws provide some protections against genetic discrimination. Researchers who will have 
access to genetic information will take measures to maintain the confidentiality of the 
information. HLA genetic testing will be performed for research purposes only and will not be 
performed by a CLIA certified laboratory. Therefore, the results of HLA testing will not be 
shared with subjects. 
 
Management of Results and Genetic Counseling 
Subjects will be contacted if a clinically actionable gene variant is discovered either as part of 
this protocol or a related sub-study. Clinically actionable findings for the purpose of this study 
are defined as disorders appearing in the American College of Medical Genetics and Genomics 
recommendations for the return of incidental findings that is current at the time of primary 
analysis. Subjects will be contacted at this time with a request to provide a blood sample to be 
sent to a CLIA certified laboratory. If the research findings are verified in the CLIA certified lab, 
the subject will be offered the opportunity to come back to have genetic education and 
counseling to explain this result. If the subject does not want to come back, a referral to a local 
genetic healthcare provider will be provided (at their expense). 
This is the only time during the course of the study that incidental findings will be returned. No 
interrogations regarding clinically actionable findings will be made after the primary analysis. 
8.2 
Safety and Other Assessments 
Study procedures are specified in the SoA. A study clinician licensed to make medical diagnoses 
and listed on the 1572 will be responsible for all trial-related medical decisions.  Specimens for 
clinical screening and safety laboratory evaluations will be transported from the participating 
clinic sites or confinement units to the respective site’s certified clinical laboratories or on-site 
certified laboratories for analysis.  
 
Physical Examination 
A physical exam will be conducted at screening (Day -45 to -3) by a licensed study clinician 
listed on the Form FDA 1572. A more targeted physical examination will be conducted at pre-
challenge screen (Day -2), pre-challenge baseline (Day -1), viral challenge (Days 1-8), and 
follow-up (Days 15, 29, 57). The targeted physical exam will include evaluation of the 
oral/pharyngeal, neck, lung, and heart exams. An otoscopic exam will be performed as part of 
the targeted physical exam at baseline (Day -1) and as needed. Additional clinical evaluation will 
be performed if clinically indicated. 
 
Vital Signs 
Vital signs will be assessed at screening (Day -45 to -3), pre-challenge screen (Day -2), pre-
challenge baseline (Day -1), pre-challenge baseline (Day 1 prior to challenge), post-challenge 
(Days 1-8) approximately within each 8-hour period (i.e., three times per day), and follow-up 
(Days 15, 29, 57). Vital signs will include pulse, systolic blood pressure, diastolic blood 
pressure, respiratory rate, and oral temperature. 

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SpO2 
SpO2will be assessed at screening (Day -45 to -3), pre-challenge screen (Day -2), pre-challenge 
baseline (Day -1), pre-challenge baseline (Day 1 prior to challenge), post-challenge (Days 1-8) 
approximately within each 8-hour period (i.e., three times per day) and follow-up (Days 15, 29, 
57).  
 
Electrocardiograms (ECGs) 
A 12-lead ECG will be performed at screening (Day -45 to -3), post-challenge (Day 6), and as 
clinically indicated and confirmed by the PI or designated clinician licensed to make medical 
diagnoses and listed on Form FDA 1572.  Screening ECGs must be within normal reference 
ranges or deemed not clinically significant. 
 
Chest X-Ray (CXR) 
A PA and lateral CXR will be performed at screening (Day -45 to -3).   
 
Clinical Laboratory Evaluations 
• Serology at screening: HIV, Hepatitis B surface antigen, Hepatitis C antibody, and HAI 
titer  
• Hematology (at screening and study Days 2, 4, and 8): white blood cells (WBCs), 
absolute lymphocyte count, hemoglobin, and platelets  
• Chemistry at (as screening and study Days 2, 4, and 8): alanine transaminase (ALT) and 
creatinine (Cr) 
• A urine toxicology test (amphetamines, cocaine, and opiates,) at screening and on the day 
of admission to the confinement unit (amphetamines, cocaine, and opiates) 
• Serum HCG pregnancy test at screening 
• Urine HCG pregnancy test (for female subjects of childbearing potential) to be done 
before the baseline CXR (if > 7 days have passed since the negative serum pregnancy test 
was drawn) and on Day -2, to be performed locally. 
• Qualitative multiplex respiratory virus assay (BIOFIRE® FILMARRAY® respiratory 
panel by bioMérieux or Luminex xTAG®), including SARS-CoV-2 RT-PCR, on NP 
swab(s) at screening and on Day -2 and Day -1 in order to remain on the unit for 
challenge and daily from Day 2 through the confinement period. 
 
8.2.1 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
The site PI or designated clinician licensed to make medical diagnoses and listed on Form FDA 
1572 is responsible for recording all AE/SAEs that are observed or reported during this study, 
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, using a local laboratory as necessary. In determining eligibility, refer to Section 
5.1 and 5.2, and the protocol-specific MOP. 
 

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Signs, symptoms, laboratory findings that are part of mild to moderate influenza disease 
collected as part of the Modified Jackson Score, in addition to mild or moderate fever and 
lymphopenia from the time of challenge through 7 days post-challenge, will not be considered 
adverse events (AEs). These signs, symptoms, and laboratory findings (mild and moderate) 
include the following: runny nose, stuffy nose, sneezing, sore throat, headache, cough, malaise 
(tiredness), body aches, chills, feverish, shortness of breath, earache, fever, and lymphopenia.  
Mild to moderate fever is defined in Appendix A and mild to moderate lymphopenia is defined 
in Appendix B.  Influenza infection related symptoms that are deemed by the investigator to be 
severe, in addition to severe fever as defined in Appendix A and severe lymphopenia as defined 
in Appendix B, will be considered as adverse events.  Symptoms as assessed by the FLU-PRO 
will not be assessed as adverse events unless deemed by the investigator to be severe.  
 
Safety will be assessed by the frequency and severity of: 
1. Study product-related serious adverse events occurring from the time of the challenge 
through the end of the study (approximately 2 months post-challenge). 
2. Clinical safety laboratory adverse events occurring from the time of challenge through 
Study Day 8. Parameters to be evaluated include: white blood cells (WBCs), absolute 
lymphocyte count, hemoglobin, platelets, and alanine transaminase (ALT), and creatinine 
(Cr). 
3. Adverse Events – non-serious adverse events occurring from the time of challenge 
through approximately 28 days post-challenge. See below for how adverse events are 
defined. 
8.3 
Adverse Events and Serious Adverse Events 
8.3.1 
Definition of Adverse Events (AE) 
ICH E6 defines an AE as any untoward medical occurrence in a patient or clinical investigation 
subject administered a pharmaceutical product regardless of its causal relationship to the study 
treatment. FDA defines an AE as any untoward medical occurrence associated with the use of a 
drug in humans, whether or not considered drug 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) 
product. The occurrence of an AE may come to the attention of study personnel during study 
visits and interviews of a study recipient presenting for medical care, or upon review by a study 
monitor.  
In this study, solicited symptoms of symptomatic influenza infection will be collected from the 
time of challenge through 7 days post-challenge and will not be considered adverse events. 
Objective clinical examination findings consistent with the solicited symptom, such as 
oropharyngeal erythema or lymphadenopathy, will also not be considered AEs. Any influenza 
signs, symptoms or lab findings determined by the clinician to have exceeded the expected 
severity or any moderate or severe complications, as listed in Section 2.3.1, will be considered 

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unsolicited adverse events and captured on the appropriate data collection form and electronic 
case report form (eCRF). Events that are not consistent with illness due to influenza will be 
considered unsolicited AEs as well. Events that occur after administration of antivirals will also 
be considered unsolicited AEs.  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 study will be documented appropriately regardless of relationship. AEs will be 
followed through resolution. Resolution of an AE is defined as the return to pretreatment status 
or stabilization of the condition with the expectation that it will remain chronic.  
Any chronic or stable medical condition that is present at the time that the subject is screened 
will be considered as baseline and not reported as an AE. However, if the severity of any pre-
existing medical condition increases, it should be recorded as an AE. 
8.3.1.1 
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.  
8.3.2 
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 subject 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. 
SAEs will be:  
• Assessed for severity and relationship to study product and alternate etiology (if not 
related to study product) by a licensed study clinician listed on the Form FDA 1572 or by 
the Institution as the site Principal Investigator or Sub-Investigator.  
• Recorded on the appropriate SAE data collection form and eCRF.  

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• Followed through resolution by a licensed study clinician (for investigational new drug 
application [IND] studies, a licensed clinician listed on the Form FDA 1572 as the site 
Principal Investigator or Sub-Investigator).  
• Reviewed and evaluated by DMID, an Independent Safety Monitor (ISM) (as deemed 
necessary), the Safety Monitoring Committee (SMC) (periodic review unless related), 
and the IRB/IEC. 
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 
reasonably possible but is not listed in the Investigator Brochure, Package Insert or 
Summary of Product Characteristics. 
8.3.2.2 
Severity of Event 
All AEs or SAEs will be assessed for severity, according to the toxicity grading scales in 
Appendices A and B.  
 
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 subject’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 affect 
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. 
8.3.2.3 
Relationship to Study Intervention 
The licensed study clinician’s assessment of an AE’s relationship to study product is part of the 
documentation process, but it is not a factor in determining what is or is not reported in this 
study. If there is any doubt as to whether a clinical observation is an AE, the event should be 
reported. The relationship to study product must be assessed for AEs using the terms: related or 
not related. In a challenge, the study product must always be suspect. To help assess, the 
following guidelines are used:   
• 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. 

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• 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. 
8.3.3 
Time Period and Frequency for Event Assessment and Follow-Up 
In this study, solicited symptoms of influenza infection will be collected from the time of 
challenge through 7 days post-challenge and will not be considered adverse events.  
Any influenza signs, symptoms or lab findings determined by the clinician to have exceeded the 
expected severity of a mild or moderate influenza symptom or sign will be considered 
unsolicited adverse events and captured on the appropriate data collection form and electronic 
case report form (eCRF). Events that are not consistent with illness due to influenza will be 
considered unsolicited AEs as well. Unsolicited AEs will be captured starting at the time of 
challenge through 28 days post-challenge.  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 study will be documented appropriately regardless of relationship. AEs will 
be followed through resolution. Resolution of an AE is defined as the return to pretreatment 
status or stabilization of the condition with the expectation that it will remain chronic.  
Any chronic or stable medical condition (Section 5.1) that is present at the time that the subject is 
screened will be considered as baseline and not reported as an AE. However, if the severity of 
any pre-existing medical condition increases, it should be recorded as an AE. 
8.3.4 
Adverse Event Reporting 
8.3.4.1 
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 should 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 should 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. 
8.3.5 
Serious Adverse Event Reporting 
8.3.5.1 
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 

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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 site will send a copy of the SAE report(s) 
to the ISM (as deemed necessary) when they are provided to the DMID Pharmacovigilance 
Group.   The DMID Medical Monitor will review and assess the SAE for regulatory reporting 
and potential impact on study subject 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. 
8.3.5.2 
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 
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.   
8.3.6 
Reporting of Pregnancy 
Pregnancies occurring in study subjects will be reported via Advantage electronic data capture 
system (Advantage eClinical) on the Pregnancy Report form. With the subject’s permission, a 
venous blood sample for serological assays will be collected per protocol; however large volume 
blood samples for cellular immunological assays will be discontinued, and the subject will 
continue to be followed for safety for the duration of this study.  
8.4 
Unanticipated Problems 
8.4.1 
Definition of Unanticipated Problems (UP) 

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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 of 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. 
8.4.2 
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 
8.3.5 Serious Adverse Event Reporting within 24 hours of the investigator becoming 
aware of the event.  
• Any other UP will be reported to the IRB and to the DCC/study sponsor within three days 
of the investigator becoming aware of the problem.  
 
 

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9. 
STATISTICAL CONSIDERATIONS 
9.1 
Introduction 
The goal of this study is to find a challenge virus dose that is safe and can achieve a symptomatic 
influenza AR that will be sufficiently high for utilization in future vaccine or intervention 
studies.  The optimal dose of the three considered is broadly defined as the minimum challenge 
virus dose that elicits the highest AR without meeting safety-stopping criteria.  Additionally, 
viral recovery, clinical symptoms, and immune responses over the post-challenge period will be 
described by challenge dose group. 
9.2 
Statistical Hypotheses 
The study is not designed to achieve predetermined levels of power or precision to address the 
primary, secondary, or exploratory objectives.  It is of interest to utilize more of the available 
sample size at higher doses, unless there is adequate evidence that one of the lower doses is 
sufficiently infectious.  This is further described in Section 9.3.2.  General methodology planned 
to address study objectives is described in Section 9.5, and the specific null and alternative 
hypotheses and details about planned tables, figures, and listings will be given in a separate 
statistical analysis plan (SAP). 
9.3 
Sample Size Determination 
9.3.1 
Study Design 
This is a challenge study of A/Texas/71/2017 (H3N2), clade 3C3a virus infection to assess viral 
dynamics, clinical and immunological response, and safety.  A targeted maximum of114 
(targeting 106 active plus 8 shams) participants will be enrolled at the clinical sites and will 
receive one of 3 doses (104, 105, or 106 TCID50) of A/Texas/71/2017 (H3N2), clade 3C3a virus 
or sham placebo.  There will be a targeted maximum of 36 active subjects enrolled into each 
challenge dose group.  For each challenge cohort, a target of 1 sham placebo subject will also be 
included, with the primary intent of reducing the rate of false positive symptoms due to subjects 
not being certain whether they are receiving active virus. 
 
Initially, a targeted total of 12 subjects will be enrolled to receive a dose of 104 TCID50, as well 
as 1 sham placebo subject.  While it is unexpected that this dose would be highly infective, this 
initial cohort will allow for a safety assessment of the virus and study challenge procedures.  The 
criteria for determining the dose received by the subsequent cohort will then depend on the 
percentage of those initial subjects meeting the symptomatic influenza criteria, and whether the 
study stopping-safety criteria have been met.  The doses received by each subsequent challenge 
cohort will be determined in a similar manner, as depicted in Figure 1.  At the highest dose, both 
challenge cohorts would include a target of 17 active subjects and 1 sham, in order to increase 
the total sample size at what are assumed to be doses more likely to meet the criteria for 
optimality.  Escalation will stop when the maximum sample size is reached. 
 
9.3.2 
Dose Escalation and Sample Size Allocation 

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The study is planned to target enrolling a maximum of 114 subjects (targeting 106 active plus 8 
shams), at one of 3 challenge virus doses or sham placebo. While the study is not designed to test 
any specific null hypothesis or reach a pre-determined level of estimation precision, this section 
gives the precision for estimates and power for hypothesis tests of interest, based on this 
proposed sample size. Adjustments to p-values in the presence of multiple comparisons may be 
made, depending on the objective, but the calculations below do not account for this possibility. 
Details on adjustment for multiple comparisons are given in Section 9.5.1 and will be described 
further in the SAP. 
 
A targeted total of 13 participants (12 active plus 1 sham) will be planned to enroll for each 
cohort at the two lowest dose levels, and a targeted total of 18 participants (17 active plus 1 
sham) per cohort for the highest dose level.  Additional randomization slots will be included to 
allow for the possibility of enrolling beyond the targeted cohort sizes. These calculations allow 
for 2 subjects per cohort (of the targeted sizes) to withdraw prior to challenge, and if that does 
not happen, or if more than the targeted number of subjects are enrolled in a given cohort or dose 
group, the power and precision would increase (detectable difference would decrease) compared 
to what is presented in this section. Correspondingly, if more than 2 subjects withdraw the power 
and precision would decrease. 
 
The dose escalation decision rules have been constructed with the intent of safely escalating 
doses until there is compelling evidence of a sufficiently high AR at a given dose.  Since it is 
assumed that the AR will increase with the dose, the symptomatic influenza thresholds for dose 
escalation from one cohort to the next were chosen to increase the expected sample size at the 
higher doses and improve the precision for estimating the true AR for those doses. 
 
Figure 2 shows the probability of meeting the infection threshold for further enrollment at a 
dose, given varying numbers of subjects enrolled at that dose (10 enrolled in Cohorts 1A or 2A, 
15 at Cohort 3A, or 20 in Cohorts 1A+1B or 2A+2B), varying true infection probabilities, and 
the thresholds for further enrollment. 
 
 

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Figure 2. Probabilities of further enrollment at the current dose, given varying true 
infection probabilities, numbers of subjects enrolled at the current dose, and decision 
thresholds 
 
For illustration, with n=10 subjects enrolled in Cohort 1A at 104 TCID50, the probability of 
achieving 8 infections (solid line, top left panel) would be only 0.01 if the true infection 
probability were 0.4, compared to 0.67 if the true infection probability were 0.8. 
 
Depending on the number of infections observed at each dose level, the final sample size for 
each dose group will be approximately 10, 15, 20 or 30.  Figure 3 shows the estimated infection 
probabilities and associated exact 95% confidence intervals that would result from observing 
varying numbers of symptomatic influenza events at a given dose level.  The maximum CI width 
occurs at 50% observed events.  With 30 subjects enrolled at the optimal dose, this would 
correspond to observing 15 symptomatic influenza events and an estimated CI of (31.3%, 
68.7%). 
  

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Figure 3. Estimated symptomatic influenza probabilities and associated exact 95% CIs, 
given varying numbers of symptomatic influenza events observed and varying sample sizes 
 
Table 3 gives the probability of observing one or more safety events, such as a solicited 
symptom or an SAE of a particular classification in varying total dose group sizes, given 
underlying true event probabilities. 
 
Table 3. Probability (%) of observing at least one safety event, given varying underlying 
event probabilities and dose group sizes 
True Event Probability 
Dose group Size 
N=10 
N=15 
N=20 
N=30 
0.01 (Rare) 
0.1 
0.15 
0.2 
0.3 
0.1 (Uncommon) 
0.996 
1.49 
1.98 
2.96 
1 (Common) 
9.56 
14 
18.2 
26 
10 (Very Common) 
65.1 
79.4 
87.8 
95.8 
9.4 
Populations for Analyses 
• The Safety (SA) population will include all subjects who received study influenza 
challenge. 
• The Intent-to-Treat (ITT) population will consist of all subjects who received study 
influenza challenge. 
• The Per-Protocol (PP) population will include subjects in the ITT population with the 
following exclusions of data: 
o All data from any subjects who withdraw prior to the end of the challenge period 
o Data from any visit that occurs substantially out of window 

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o Data from all visits subsequent to major protocol deviations 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 investigational drug/investigational vaccine/licensed 
vaccine 
▪ Receipt of blood or blood products, or blood donation 
Certain analyses may be done on subsets of these populations, such as infected subjects, or those 
receiving what is determined to be the optimal dose. 
9.5 
Statistical Analyses 
Clinical (symptom and infection) and safety data through the last follow-up at Day 57 will 
comprise the primary clinical database for this study. Once the last subject has completed the 
final visit, the primary clinical database will be cleaned, monitored, and locked. The clinical 
study report (CSR), comprised of the final analyses of safety, clinical, and available 
immunological data will be completed by the Statistical and Data Management Coordinating 
Center (SDMCC) after the primary clinical database is locked and the separate statistical analysis 
plan (SAP) has been finalized. The CSR will be completed when all primary and secondary 
safety, clinical, and immunological endpoint data are available. 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.  
Some analyses may be conducted on an expedited basis prior to the completion of the study and 
included in a “topline” report, to aid in decision-making regarding future studies.  These analyses 
would not affect the conduct of this trial; subjects would be described by group but subject-level 
unblinding will not occur until the clinical database is locked.  The topline results would be 
considered final and included in the CSR as well. 
The formal SAP will elaborate on the analyses described here and will define which analyses 
would be included in an expedited report. 
9.5.1 
General Approach 
For descriptive analyses, continuous variables will be summarized by the non-missing sample 
size, mean, standard deviation, and the minimum, median, 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 with geometric means, first across replicates to compute 
one value for each individual sample and then applied again across subjects within the same 
group. 
Generally, summaries will be presented by challenge dose group, with sham placebo subjects 
pooled across all challenge cohorts into one group.  For safety endpoints, summaries will include 
a column for all subjects receiving active challenge virus as well.  Other groupings used for some 
analyses may include symptomatic/asymptomatic, shedding virus yes/no (i.e., RT-PCR+/RT-
PCR-), and symptomatic influenza+/symptomatic influenza- or MMID+/MMID-. 
Data listings will be presented sorted by clinical site or other grouping variable, subject, and then 
by visit number within subject, where applicable. All tables will be annotated with the total 

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population size relevant to that table, including any missing observations.  The analysis 
population for each exhibit will be clearly indicated. 
While the study was not designed for any formal group comparisons, hypothesis testing will be 
done for some endpoints and will be considered exploratory.  For hypothesis testing related to 
safety endpoints, two-sided tests at the α=0.05-level will be made without adjusting for multiple 
testing.  For hypothesis testing of clinical endpoints, initially a global, two-sided test will be used 
to determine if there are differences between any groups.  If the global test is significant at the 
α=0.05-level, the Bonferroni-Holm stepdown procedure [9] will be used to control the type-I 
error across pairwise two-sided tests, but no adjustment will be made across endpoints; a 
familywise 5% error rate will be used to determine significance.  For immunological, genomic, 
or physiological analyses involving hypothesis testing, more stringent thresholds for significance 
may be used, depending on the number of hypothesis tests comprising a related set of analyses. 
For hypothesis tests of continuous endpoints, ANOVA will be preferred for tests across multiple 
groups and t-tests for pairwise tests.  If histograms of the analysis data do not appear 
approximately normal even after log- or other transformation and the sample sizes for 
comparisons groups are too small for the application of the central limit theorem (which states 
that in sufficiently large samples, the sample mean is approximately normal), Wilcoxon rank-
sum tests or other nonparametric methods may be utilized instead.  For tests of categorical 
endpoints, exact binomial tests or Fisher’s exact tests will be used.  
Exact Clopper-Pearson confidence intervals will be computed for binary variables, where 
applicable, and for continuous variables confidence intervals will be computed based on the t-
distribution with n-1 degrees of freedom, where n denotes the number of observations.   
If other analysis approaches than those listed here are used, they will be clearly stated and 
justified in the SAP and/or CSR. 
9.5.2 
Analysis of the Primary Endpoints 
The three primary objectives for this study involve characterizing the infectivity and the 
symptom profile elicited by increasing doses of the study challenge virus, as well as determining 
the optimal dose for use in future challenge and vaccine studies. 
 
The primary endpoints are: 
• The percentage of subjects within a challenge dose group with detectable shedding by 
qualitative RT-PCR and symptom scores that meet the clinical case definition for 
symptomatic infection by the Modified Jackson Instrument score 
• The percentage of subjects within each challenge dose group with detected viral 
shedding, the magnitude of viral shedding from Day 2 through Day 8, and the duration of 
viral shedding 
• The computed symptom score from the modified Jackson symptom score, and the 
percentage of subjects with total symptom scores meeting criteria for symptomatic or 
asymptomatic categories 
 
The probability of symptomatic influenza will be estimated for each challenge dose group, along 
with associated 95% exact confidence intervals.  The optimal infectious dose will be the 
challenge dose group with the greatest estimated probability of symptomatic influenza which is 

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safe, as determined by the study halting criteria.  If doses with similarly high observed AR have 
substantially different group sizes (e.g., 10 vs. 30), the lower limit of the estimated 95% CIs for 
the AR may be considered as well. 
 
Viral shedding will be summarized by the number and percentage of subjects in each challenge 
dose group with positive qualitative and/or quantitative RT-PCR each day from Day 2 through 
the end of the challenge period (Day 8 or later, in the case of a subject not meeting discharge 
criteria on Day 8) excluding safety results reported after receipt of antiviral therapy.  The 
probability of viral shedding at any point during the challenge (i.e., incidence) will be estimated 
along with the associated exact 95% CI, separately by challenge dose group.  Peak viral shedding 
will be calculated for each subject as the maximum shedding from Day 2 through Day 8 or 
discharge, and total viral shedding will be calculated via the area under the curve (AUC) with 
study day on the x-axis and shedding on the y-axis.  These will be calculated separately for 
quantitative RT-PCR and quantitative culture.  Mean peak and total shedding based on the AUC 
will be calculated for each group along with 95% CIs, and comparisons will be made as 
described for continuous variables in Section 9.5.1.  A Wilcoxon signed-rank test will be used to 
compare distributions of these variables between quantitative RT-PCR and quantitative culture 
across all subjects, to assess if these methods of viral quantification are consistent.  Scatterplots 
will also be presented, along with Spearman rank-order correlation estimates.  Duration of viral 
shedding will be calculated for each subject as the number of days (including non-consecutive) 
determined by qualitative or quantitative RT-PCR to be positive from Day 2 until discharge; 
means and 95% CIs will be calculated for each group and comparisons will be made. 
 
Solicited influenza symptoms will be presented by the frequency and percentage of subjects 
reporting each symptom, severity, day, and challenge dose group.  The self-reported (FLU-PRO) 
and investigator-assessed symptoms will be tabulated separately.  Total symptom scores will be 
computed for each subject and each scoring algorithm.  Mean scores for both algorithms will be 
calculated for each group, along with 95% CI, and the groups will be compared as described in 
Section 9.5.1.  Additionally, the number and percentage of subjects meeting each scale’s 
definition of symptomatic and asymptomatic will be cross-tabulated by challenge dose group.  A 
Wilcoxon rank-sum test will be used to compare the distributions of each set of scores, across all 
subjects that received active challenge, to determine the agreement between the two symptom 
reporting methods. 
 
Clinical endpoints (including the primary endpoints) will be assessed in the ITT population.  
Subjects who withdraw during the challenge period prior to meeting discharge criteria will be 
included in summaries where data are available.  Subjects who withdraw during the challenge 
period and have yet to meet the relevant clinical case definition will be excluded from analyses 
of aggregate endpoints such as the incidence of symptomatic influenza. 
 
If at least 10% of the analysis data would be dropped according to PP population exclusions 
described in Section 9.4, the PP population will be used as a sensitivity analysis for the primary 
ITT analysis. 
 
9.5.3 
Analysis of the Secondary Endpoints 

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9.5.3.1 
Analysis of Secondary Endpoints 
Assessing the safety profile of the challenge virus is a secondary objective of the study. This will 
be measured by the number of adverse events reported and the percentage of subjects reporting 
adverse events through Day 29, and the number of serious adverse events and the percentage of 
subjects reporting adverse events through Day 57.   
 
Influenza signs and symptoms and clinical lab findings determined to have exceeded the 
expected severity will be considered unsolicited adverse events.  Events that are not consistent 
with illness due to influenza and abnormal vital signs will be considered unsolicited AEs as well.  
Solicited signs and symptoms of influenza as described in Section 4.1 will not be considered 
AEs.  The severity (mild, moderate, or severe) of abnormal vital signs and clinical laboratory 
values will be presented according to the toxicity grading scales in Appendices A, B and C.  
Other events will be reported according to the criteria in Section 8.3.2.1.  Relationship with 
receipt of the challenge virus will be assessed as described in Section 8.3.2.2. 
 
The frequencies of AEs through Day 29 and SAEs through Day 57 will be presented by 
MedDRA® system organ class (SOC) and preferred term (PT), severity, relationship to 
challenge virus, challenge dose group, and across all subjects receiving active virus.  Incidence 
will be assessed by the number and percentage of subjects in each challenge dose group ever 
reporting an event, along with associated 95% CI for the probability of the event.  To further 
assess the safety of the challenge virus, the incidence of any related moderate or severe AE and 
any SAE will be calculated for each challenge dose group and compared as described in Section 
9.5.1. 
 
Adverse events and clinical laboratory results will be presented in data listings.  Analyses of 
safety endpoints will be performed for the Safety population. 
 
9.5.3.2 
Analysis of Secondary Immunological Endpoints 
Secondary immunological objectives include describing the host serum antibody (HAI and MN) 
responses and anti-HA-stalk antibody responses at baseline and post-challenge.   
 
For these titer data, GMTs and the proportions of subjects achieving seroconversion, defined as a 
minimum 4-fold rise in titer post-challenge, will be presented by challenge dose group and visit. 
Geometric mean fold-rise (GMFR) for anti-HA-stalk antibody titers will be included for post-
challenge visits as well. Confidence intervals corresponding to these statistics will be computed 
for each group and visit as well. Hypothesis testing may be performed for select immunological 
endpoints and time points, which will be pre-specified in the SAP. 
9.5.4 
Analysis of Exploratory Endpoints 
Exploratory objectives for this study include further characterizing the immunological responses, 
assessing the impact of genetic variability on the immune, clinical, and viral responses, and 
further examining the clinical and physiological responses post-challenge. 
 

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9.5.4.1 
Exploratory Immunological Endpoints 
For analysis of exploratory immunological endpoints, immune responses will be described by 
visit and challenge dose group.  For antibody titer data, geometric means will be computed for 
each group at each time point (GMTs), and seroconversion 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 as well, but formal 
hypothesis testing is not planned for descriptive summaries of exploratory endpoints.   
 
Additional aggregate (i.e., encompassing data across multiple visits) immunological endpoints 
may include the duration of response, the timing and magnitude of the peak response, and the 
total immune response as measured by the area under the curve (where the study day is on the x-
axis and the immune response is on the y-axis). 
 
Neuraminidase inhibition antibody (NAI) responses will be analyzed similarly to HAI and MN 
assays, as described in 9.5.3.2. Other immunological endpoints will include frequencies of cell 
subsets, which will be described by mean frequencies within each group and the associated 95% 
CIs.  Further details for data types not covered here will be given in the standalone SAP. 
 
Exploratory immunological endpoints will be analyzed for the ITT population, with the PP 
population used for sensitivity analysis if at least 10% of analysis data would be excluded for the 
reasons described in Section 9.4.  Subjects who withdraw prior to being evaluable for certain 
endpoints may be excluded from specific analyses in the ITT population, but all available data 
will be included in descriptive analyses. 
 
9.5.4.2 
Exploratory Genomic Endpoint 
The frequencies of HLA class I and II alleles at baseline will be tabulated by challenge dose 
group and overall.  The ability of pre-challenge HLA class I and II alleles to modulate the 
magnitude (as measured by serological conversion defined as a minimum 4-fold rise in post-
challenge) of the immune response will be assessed using a two-sided Fisher’s exact test. The 
relationship between HLA alleles and pre-existing immune status (baseline HAI, NAI, MN titer) 
will be evaluated via one-way ANOVA. The association between HLA class I and II alleles and 
peak shedding and total viral shedding for quantitative RT-PCR and quantitative culture will be 
assessed using a two-sided Wilcoxon Rank-Sum test. Depending on the number of observed 
alleles, multiple testing adjustment may be carried out using the Benjamini Hochberg 
procedure[10]. 
 
9.5.4.3 
Exploratory Transcriptomic Endpoint 
For the transcriptomics exploratory analysis, RNA sequencing data will be pre-processed by 
removing adapters and low-quality reads and mapping sequences to the latest human reference 
genome using splice-aware alignment software such as HISAT2. Gene expression quantification 
will be carried out by using the Subread software using the latest Ensembl [11] gene model 
annotations as a reference. Systematic differences in sequence coverage between samples will be 
accounted for using the TMM method [12] as implemented in the edgeR [13] R package. 
Principal component analysis, non-metric multidimensional scaling, and hierarchical clustering 

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analysis will be used to identify potential global outliers and systematic batch effect. Negative 
binomial models as implemented in edgeR [13] will be used to identify genes for each post-
challenge day that were differentially expressed (DE) compared to pre-challenge. To control for 
testing multiple genes, the false-discovery rate (FDR) based on the Benjamini-Hochberg 
procedure[10] as implemented in the p.adjust R function will be applied. Genes with a FDR-
adjusted p-value < 0.05 will be deemed to be significantly DE. The pvclust [14] R package will 
be applied to identify co-expressed DE gene clusters and cluster time trends will be visualized. 
Pathway enrichment analysis based on the latest MSigDB [15] and KEGG [16] databases as well 
as Blood Transcription Modules [17] accounting for gene length bias will be carried out using 
the implementation provided by the goseq [18] R package. Pathway maps color-coded by 
challenge effect will be provided for significantly enriched KEGG pathways. In addition, for 
each enriched pathway, time trends of the median of the average pathway response will be 
visualized. Additional details/analyses will be described in the SAP. 
 
9.5.4.4 
Exploratory Physiological and Additional Clinical Endpoints 
Additional exploratory analysis of clinical endpoints will include cross-tabulations of symptoms 
as collected by each of the two scoring systems, separately by viral shedding status (as 
determined by RT-PCR), by receipt of active challenge virus or placebo, and across all subjects.  
To examine the ability of each scoring algorithm to accurately capture symptomatic 
influenza/MMID, the symptom scores (i.e., severity) for each symptom will be summarized by 
RT-PCR status.  These summaries will be made on a by-visit level and for the maximum 
severity/score of each symptom over the entire challenge period.  
 
Alternative case definitions for symptomatic influenza virus infection will be explored via 
development of clinical scoring algorithms based on each of the scoring scales.  A training and 
testing approach will be utilized to determine multi-symptom models predictive of RT-PCR 
positivity, using the maximum severity observed over the challenge period for each symptom.  
This approach will be applied separately for each scoring scale, and the operating characteristics 
of the final multi-symptom models will be compared. 
 
Physiological data, as collected by the wearable devices, will be summarized by dose group and 
by symptomatic influenza status, separately.  Further exploratory analysis of these data will be 
described in the SAP. 
 
9.5.5 
Baseline Descriptive Statistics 
Baseline and demographic characteristics will be summarized by clinical site for each challenge 
dose group as well as the placebo group with summary statistics as described in Section 9.5.1.  
These characteristics will include age, sex, ethnicity, race, and prior receipt of seasonal 
vaccination. 
 
9.5.6 
Planned Interim and Early Analyses 
After each cohort has finished the challenge period of the study, the symptomatic influenza AR 
will be analyzed and the decision to keep the same dose or escalate to the next dose for the 

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subsequent cohort will be made according to the criteria given in Figure 1.  A safety monitoring 
committee (SMC) will be convened, and the SMC will meet and review these data at scheduled 
time points or ad hoc as needed during this study, as delineated in the SMC charter. 
 
A set of analyses may be included in an expedited report prior to final database lock, as 
described in Section 9.5.  Care would be taken to ensure that dissemination of results prior to the 
end of follow-up would not impact study conduct. 
 
9.5.6.1 
Interim Safety Analyses 
An interim safety review may include enrollment and demographic information, clinical 
laboratory tests, and AE/SAEs. Additional data may be requested by the SMC.  The review(s) 
will be blinded to active virus or placebo, with unblinded study assignments available upon SMC 
request. 
As an outcome of each review, the SMC will make a recommendation to continue, modify, or 
terminate this study. The trial will also be monitored to determine if any of the halting rules in 
Section 7.1.1 are met, and if so, the SMC will be convened to review. 
 
9.5.6.2 
Interim Review to Determine the Dose for the Subsequent Cohort 
After all subjects from each challenge cohort have been discharged from the challenge unit, the 
infection, symptom, and safety data will be cleaned to prepare for the interim review to 
determine the dose for the next challenge cohort enrolled. 
 
If the minimum symptomatic influenza AR threshold given in Figure 1 is met and no halting 
criteria for safety have been met, the next group of subjects will be enrolled at the same dose 
level.  If the threshold is not met and no halting criteria have been met, the next cohort will be 
enrolled at the next highest dose level.  If the halting criteria are met, the SMC will review to 
determine whether the study should be halted due to safety concerns. 
 
Data will be considered in aggregate as part of the interim reviews for dose selection, in order to 
minimize the potential for unblinding. 
 
9.5.6.3 
Early Analyses 
As described in Section 9.5, some analyses may be completed prior to the end of follow-up 
and/or the clinical database lock, to facilitate planning for future studies.  The analyses intended 
for inclusion in an expedited topline report will be delineated in the SAP, along with operational 
details for maintaining the blind and the integrity of the rest of the study.   
 
9.5.7 
Sub-Group Analyses 
No explicit sub-group analyses beyond those relating to the dose of study challenge received are 
planned.  However, exploratory analysis may be conducted to examine potential impacts of 
baseline characteristics such as sex or baseline HAI, NAI, or MN titers. 

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9.5.8 
Tabulation of Individual Participant Data 
Safety data, clinical data, and immunological data will be presented in listings, in addition to the 
planned analyses by dose group. 
 
 

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10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
10.1 
Regulatory, Ethical, and Study Oversight Considerations 
Each institution engaged in this research will hold a current Federal Wide Assurance (FWA) 
issued by the Office for Human Research Protections (OHRP) for federally funded research. and 
provide the FWA number to DMID. 
 
A single IRB will conduct the ethical review and approval for participating sites.  Each site PI 
will obtain IRB approval for this protocol to be conducted at his/her research site(s) and send 
supporting documentation to the DMID before initiating recruitment of subjects. The investigator 
will submit applicable information to the IRB/IEC on which it relies for the review, to conduct 
the review in accordance with 45 CFR 46, ICH E6 GCP, and as applicable, 21 CFR 56 
(Institutional Review Boards) and 21 CFR 50 (Protection of Human Subjects), other federal, 
state, and local regulations. The IRB/IEC must be registered with OHRP as applicable to the 
research. DMID must receive the documentation that verifies IRB/IEC-approval for this 
protocol, associated informed consent documents, and upon request any recruitment material and 
handouts or surveys intended for the subjects, prior to the recruitment and enrollment of subjects.  
 
Any amendments to the protocol or consent materials will be approved by the IRB/IEC before 
they are implemented. IRB/IEC review and approval will occur at least annually throughout the 
enrollment and follow-up of subjects and may cease if annual review is no longer required by 
applicable regulations and the IRB/IEC. The investigator will notify the IRB/IEC of deviations 
from the protocol and reportable SAEs, as applicable to the IRB/IEC policy. 
10.1.1 
Informed Consent Process 
Informed consent is a process that is initiated prior to an individual agreeing to participate in a 
trial and continuing throughout the individual’s trial participation.   
Pre-screening may occur under an active screening protocol (DMID 20-0004).  Informed consent 
for the current protocol will be obtained at the time of screening Days -45 to -3 and documented 
prior to performing any study procedures.   Subjects will receive a concise and focused 
presentation of key information about the study, verbally and with a written consent form. The 
explanation will be organized and presented in lay terminology and language that facilitates 
understanding why one might or might not want to participate.  Additional screening activities 
may occur by phone using an IRB approved process that ensures confidentiality. The information 
obtained from this phone call assessment will remain locally at the site and will not be entered 
into the Advantage EDC data base unless the subject is enrolled. 
An investigator or designee will describe the protocol to potential subjects face-to-face. The key 
information about the purpose of the study, the procedures and experimental aspects of the study, 
risks and discomforts, any expected benefits to the subject, and alternative treatment will be 
presented first to the subject. The subject will be asked to consent for future use of specimens for 
secondary research.   Please refer to Section 10.1.4. The subject will be asked to consent 
specifically to genetic testing (HLA testing) as well as transcriptomics planned for this study. 

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Subjects will also receive an explanation that the study involves research, and a detailed 
summary of the proposed study procedures and study interventions/products. This will include 
aspects of the study that are experimental, the probability for random assignment to treatment 
groups, any expected benefits, all possible risks (including a statement that the particular 
treatment or procedure may involve risks to the subject or to the embryo or fetus, if the subject is 
or may become pregnant, that are currently unforeseeable), the expected duration of the subject’s 
participation in the study, alternative procedures that may be available and the important 
potential benefits and risks of these available alternative procedures. 
Subjects will be informed that they will be notified in a timely manner if information becomes 
available that may be relevant to their willingness to continue participation in the study.  Subjects 
will receive an explanation as to whether any compensation and any medical treatments are 
available if injury occurs, and, if so, what they consist of, or where further information may be 
obtained. Subjects will be informed of the anticipated financial expenses, if any, to the subject 
for participating in the trial, as well as any anticipated prorated payments, if any, to the subject 
for participating in the trial. They will be informed of whom to contact (e.g., the investigator) for 
answers to any questions relating to the research project. Information will also include the 
foreseeable circumstances and/or reasons under which the subject’s participation in the trial may 
be terminated. The subjects will be informed that participation is voluntary and that they are free 
to withdraw from the study for any reason at any time without penalty or loss of benefits to 
which the subject is otherwise entitled.  The rights and welfare of the subject(s) will be protected 
by emphasizing to them that the quality of their medical care will not be adversely affected if 
they decline to participate in this study. 
Subjects will be informed that records identifying the subject 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 subject’s identity will remain 
confidential. Subjects will be informed whether private information collected from this research 
and/or specimens will be used for additional research, even if identifiers are removed.  
Subjects will be informed that the monitor(s), auditors(s), IRB, NIAID, and regulatory 
authority(ies) will be granted direct access to the subject’s original medical records for 
verification of clinical trial procedures and/or data without violating the confidentiality of the 
subject, to the extent permitted by the applicable laws and regulations, and that, by signing a 
written informed consent form, the subject is authorizing such access.  
Subjects 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. 
Informed consent forms will be IRB-approved, and subjects will be asked to read and review the 
consent form. Subjects must sign the informed consent form prior to starting any study 
procedures being done specifically for this trial. A copy of the signed consent form will be given 
to the subject(s) for their records.   
New information will be communicated by the site principal investigator to subjects who consent 
to participate in this trial in accordance with IRB requirements.  The informed consent document 
will be updated, and subjects will be re-consented per IRB requirements, if necessary.   

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10.1.1.1 
Other Informed Consent Procedures 
The rights and privacy of human subjects 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 individual study subjects and their 
families.  The consent will include whether individual subject 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 subjects will not be 
released in unrestricted databases.  Subjects 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 subject will be asked to consent for future use of specimens for secondary 
research; genetic testing on these samples may be performed specifically to genetic testing (HLA 
testing and transcriptomics) planned for this study. Please refer to Section 10.1.4.1. 
 
In addition to this protocol, subjects will be asked to consent to a separate biorepository protocol 
for the use of residual samples and associated data in future 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 subjects 
• Halting criteria met and it is deemed unsafe to proceed with further dosing with influenza 
challenge 
• Insufficient compliance with protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Decision by regulatory authorities 
 
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 assure appropriate follow-up for the subjects, as necessary. 
 
The sponsor will notify regulatory authorities as applicable.   
  
10.1.3 
Confidentiality and Privacy 
Subject 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 genetic tests, 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. Subject 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 polices 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 subject 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 in order to meet the requirements of the Federal 
Food and Drug Administration (FDA). 
A Certificate of Confidentiality does not prevent the subject from voluntarily releasing 
information about themselves or their 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 
subject’s consent, information that would identify the subject 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.   
The release of individual private information or specimens for other research will only occur if 
consent was obtained from the individual to whom the information, document, or biospecimen 
pertains, or for the purposes of other research that the release is in compliance with applicable 
Federal regulations governing the protection of human subjects in research.  
 
10.1.4 
Future Use of Stored Specimens and Data  
Secondary Human Subject 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.   

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10.1.4.1 
Samples for Secondary Research 
Samples will not be collected for planned secondary research.  However, leftover research 
samples after the laboratory testing specified in this protocol is completed 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.  The specifics of this testing 
will be in a separate protocol and will be reviewed by an IRB.  
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 are allowed to 
be stored and used for secondary research, and for which the research subject gave 
specific consent. 
Repository research sample- Samples will be collected with the subject’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, subjects will be asked to consent to a separate biorepository protocol 
for the use of residual samples and associated data in future research. 
To participate in this study, subjects 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 subjects’ health records or shared with 
subjects, unless required by law. Reports will not be sent to the specimen repository. 
The subject’s decision for secondary research can be changed at any time by notifying the study 
doctors or nurses in writing. If the subject 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 subject 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 

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available after deidentification. The Protocol, Statistical Analysis Plan, and Analytic Code will 
also be made available.  This data will be available immediately following publication, with no 
end date.   
The investigator may request removal of data on individual study subjects 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 
The study is sponsored by DMID. Decisions related to the study will be made by the protocol 
team, which includes representatives from the participating sites (PI), DMID (sponsor), and the 
SDMCC. Key study team members and roles are listed in the Manual of Procedures (MOP). 
 
10.1.6 
Safety Oversight 
This clinical study will utilize an internal safety review committee (ISRC), consisting of the 
medical monitor, medical officer, the PIs from both sites and a representative from the SDMCC.  
The ISRC will meet following Day 8 for each dosing cohort to review safety events and assure 
no halting criteria have been met and to assess the attack rate for each cohort to determine how to 
proceed with either remaining at the same challenge dose or escalating to the next highest dose.  
If one of the halting criteria has been met, the SMC will be convened. 
 
As noted above, this clinical study will utilize an SMC, which is an independent group of experts 
that advises the DMID. The primary responsibility of the SMC is to monitor subject safety. The 
SMC is external to the DMID and comprises at least 3 voting members. The SMC will consist of 
members with appropriate expertise to contribute to the interpretation of the data from this study. 
Its activities will be delineated in a SMC charter that will describe the membership, 
responsibilities, and the scope and frequency of data reviews. The SMC will operate on a 
conflict-free basis independently of the study team. The DMID or the SMC may convene ad hoc 
meetings of the SMC according to protocol criteria or if there are concerns that arise during the 
study.  
 
As defined in the charter, the SMC will review data at specified times during the course of the 
study for subject and overall study progress and will conduct ad hoc reviews as appropriate when 
a halting rule is met or for immediate concerns regarding observations during this study. The 
SMC will hold the following meetings: 
• An organizational meeting prior to subject enrollment 
• Any ad hoc meetings when a halting rule is met or due to specific safety issues as the 
SMC deems necessary 
• A final meeting to review cumulative unblinded safety data in a standard summary 
format. 
  
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 

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SMC will review blinded aggregate data in the open session of the SMC meetings.  Unblinded 
data (grouped by treatment) will be reviewed in the closed session only. 
 
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 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, each site will develop a Clinical Quality Management 
Plan (CQMP).  The CQMP will describe 
• Routine internal quality control (QC) and quality assurance (QA) activities  
o For the purposes of measuring, documenting and reporting study conduct, 
protocol adherence, human subjects’ protections, and reliability of the protocol-
driven data collected.  
• A process for addressing in a timely manner any data quality issues (i.e., collecting, 
recording and reporting data), systemic issues (i.e., protocol conduct, non-compliance, 
human subject protections), and implementation of 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 

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10.1.9.1 
Data Collection and Management Responsibilities 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness 
of the data reported.   
 
Data collection forms will be derived from the eCRFs and provided by the SDMCC to the sites 
to record and maintain data for each subject enrolled in the study.  All source documents should 
be completed in a neat, legible manner to ensure accurate interpretation of data.  Permanent ink 
is required to ensure clarity of reproduced copies.  When making a change or correction, the 
original entry should be crossed out with a single line, and the change should be initialed and 
dated.  Do not erase, overwrite, or use correction fluid or tape on the original. 
 
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 the data collection form 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.  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 reported in the eCRF should be consistent with the data collection form/source documents 
or the discrepancies should be documented.   
 
The sponsor and/or its designee will provide guidance to investigators on making corrections to 
the data collection forms and eCRFs. 
 
All data collection forms, and laboratory reports must be reviewed by the clinical team and data 
entry staff, who will ensure that they are accurate and complete.  Adverse events must be graded, 
assessed for severity and causality, and reviewed by the site principal investigator or designee.  
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site principal investigator.  During the study, the investigator must maintain complete and 
accurate documentation for the study. 
 
The Emmes Company, Inc., under subcontract to Digital Infuzion, Inc. will serve as the 
Statistical and Data Management Coordinating Center for this study, and will be responsible for 
data management, quality review, analysis, and reporting of the study data. 
10.1.9.2 
Study Record Retention 
Study related records, including the regulatory file, study product accountability records, consent 
forms, subject 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 

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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 subject 
research. Both sites must contact DMID for authorization prior to the destruction of any study 
records. 
10.1.9.3 
Source Records 
Source data are all information in original records (and certified copies of original records) of 
clinical findings, observations, or other activities in a clinical trial necessary for the 
reconstruction and evaluation of the trial. Source data should be attributable, legible, 
contemporaneous, original, accurate, and complete. Each participating site will maintain 
appropriate medical and research records for this trial, in compliance with ICH GCP, regulatory, 
and institutional requirements. For this trial, hardcopies of the study visit worksheets will be 
provided for use as source document worksheets.  Data recorded in the eCRF derived from 
source documents should be consistent with the data recorded on the source documents. 
 
Interview of subjects is sufficient for obtaining medical history. Solicitation of medical records 
from the subject’s primary care provider is not required.  
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 protocol-specific MOP, or GCP requirements or 
any critical study procedures with specific instructions in ancillary documents referenced in the 
protocol such as a protocol-specific MOP.  
The noncompliance may be either on the part of the subject, 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 subject 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 via Advantage eClinical (IDES). Protocol 
deviations must be sent to the IRB/IEC per their guidelines.  The site principal investigator and 
personnel are responsible for knowing and adhering to their IRB requirements. All deviations 
from the protocol must be addressed in study data collection forms.  A completed copy of the 
DMID Protocol Deviation Form must be maintained in the Regulatory File, as well as in the 
subject's chart if the deviation is subject specific.   
10.1.11 
Publication and Data Sharing Policy 
Following completion of the study, results of this research will be published in a scientific 
journal.  
 
Data will be available immediately following publication, with no end date, with data sharing at 
the discretion of the Sponsor. Sites may also obtain individual or country level data from the 
database for separate publications is desired. Publication may occur prior to completion of a final 
clinical study report for the entire trial. 

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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, 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 Policy  
This study will comply with the NIH Genomic Data Sharing Policy, which applies to all NIH-
funded research that generates large-scale human data, as well as the use of these data for 
subsequent research. 
10.1.14 
Publications 
Following completion of the study, the lead Principal Investigator is expected to publish the 
results of this research in a scientific journal.  This study will adhere to the following publication 
and data sharing policies and regulations: 
This study will comply with the NIH Public Access Policy, which 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 site principal investigator that an injury occurred to a subject as a direct 
result of the tests or treatments that are done for this study, then referrals to appropriate health 
care facilities will be provided to the subject. Study personnel will try to reduce, control, and 
treat any complications from this study. Immediate medical treatment may be provided by the 
participating site.  No financial compensation will be provided by the NIAID, NIH, or the federal 
government to the subject, for any injury suffered due to participation in this trial. 
 
 

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10.3 
Abbreviations 
Table 4. Commonly Used Abbreviations 
ADL 
Activities of Daily Living 
AE 
Adverse Event 
ALC 
Absolute Lymphocyte Count 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Transaminase 
ANC 
Absolute Neutrophil Count  
ANCOVA 
Analysis of Covariance 
AR 
Attack Rate 
BLA 
Biologics License Applications 
BMI 
Body Mass Index  
CAPA 
Corrective and Preventative Action Plan 
CFR 
Code of Federal Regulations 
CHI 
Controlled Human Infection 
CI 
Confidence Interval 
CIOMS 
Council for International Organizations of Medical Sciences 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
CMS 
Clinical Material Services 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
COPD 
Chronic Obstructive Pulmonary Disease 
CRF 
Case Report Form 
Cr 
Creatinine 
CRO 
Contract Research Organization 
CSR  
Clinical Study Report 
CXR 
Chest x-ray 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DMID 
Division of Microbiology and Infectious Diseases 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FWA 
Federal Wide Assurance 
GCP 
Good Clinical Practice 
g/dL 
Grams per Deciliter 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HA 
Hemagglutination 
HAI 
Hemagglutination Inhibition Test 
HBV 
Hepatitis B 

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HCV 
Hepatitis C 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
ICU 
Intensive Care Unit 
IDE 
Investigational Device Exemption 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ISRC 
Internal Safety Review Committee 
ITT 
Intention-To-Treat 
MedDRA 
Medical Dictionary for Regulatory Activities 
MMID 
Mild-to-Moderate Influenza Disease 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
N 
Number (typically refers to subjects) 
NDA 
New Drug Application 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NLF 
Nasal Lavage Fluid 
NSAID 
Non-Steroidal Anti-inflammatory Drug 
OHRP 
Office for Human Research Protections 
OHSR 
Office for Human Subjects Research 
PA 
Posterior anterior (refers to CXR view) 
PE 
Physical Exam 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
PPG 
Photoplethysmography 
QA 
Quality Assurance 
QC 
Quality Control 
RG 
Reverse genetics 
RR 
Respiratory Rate 
RT-PCR 
Reverse Transcription – Polymerase Chain Reaction 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SDMCC 
Statistical and Data Management Coordinating Center 
SE 
Standard Error 

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SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
SPG 
Sucrose phosphate glutamate  
SpO2 
Saturation of Peripheral Oxygen 
TCID50 
Median Issue Culture Infectious Dose 
ULN 
Upper Limit of Normal 
UP 
Unanticipated Problem 
US 
United States 
VTEU 
Vaccine and Treatment Evaluation Unit 
WBC 
White Blood Cell 
 
 

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10.4 
Protocol Amendment History 
 
Version 
Date 
Description of Change  
Brief Rationale 
2.0 
19Jul2021 
Subjects will be pre-screened for 
study inclusion to have 
serological HAI antibody titers of 
≤1:40 against the clinical 
challenge strain instead of ≤1:10. 
To expand enrollment efforts 
Potentially eligible study subjects 
may be pre-screened under an 
active screening protocol (DMID 
20-0004) instead of will. 
Administrative 
The MAD Nasal Intranasal 
Mucosal Atomization Device 
(Teleflex, Morrisville NC) will be 
sourced by the sites instead of by 
DMID Clinical Material Services 
(CMS). 
Administrative 
Deleted “The MAD Nasal 
Intranasal Mucosal Atomization 
Device (Teleflex, Morrisville NC) 
will be stored at room 
temperature (15-30°C).” as this 
sentence was repeated twice in 
the same Section 6.2.3. 
Administrative 
Detailed instructions for 
challenge virus inoculum 
preparation are provided in the 
MOP. 
Administrative 
Each challenge cohort will be 
enrolled at one of the clinical sites 
instead of in an alternating 
fashion. 
Administrative 
3.0 
20Sep2021 
Clarified eligibility criteria 
Administrative 
Added option for licensed study 
clinicians listed on the Form FDA 
1572 to conduct various study 
procedures 
Administrative 
Removed information regarding 
enrollment at one of the clinical 
sites 
Administrative 
Clarified approximate cohort 
sizes 
Administrative 

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Updated wearable device 
language to allow for another 
device 
Administrative 
4.0 
29Oct2021 
Updated venipuncture volumes 
and SoA to collect blood on Days 
5 and 7 
Procedural 
 
 
Removed additional multiplex 
requirement at 72 hours prior to 
confinement 
Procedural 
 
 
Modified exclusion criterion to 
exclude individuals that 
participated in flu challenge study 
with flu virus of same subtype 
within past 2 years 
Administrative 
 
 
Removed Modified Jackson score 
from appendices. Reference in 
Emmes DCFs 
Administrative 
 
 
Added language to allow for 
initiation of antiviral treatment 
course on Study Day 6 
Procedural 
5.0 
26Jan2022 
Updated exclusion criterion #9 to 
relax requirement from 6 months 
to 4 months for prior receipt of flu 
vaccination. 
Administrative 
 
 
Added UMB’s proposed mass 
cytometry work 
Procedural 
 
 
Clarified IP temperature storage 
language 
Administrative 
 
 
Clarified grading for abnormal 
lab ranges so that ULN and LLN 
will not be considered abnormal 
Administrative 
6.0 
17Feb2022 
Modified study product 
preparation sections to refer to the 
MOP for details.  
Procedural 
 
 
Updated venipuncture volumes 
for EDTA tubes.   
Administrative 
 
 
Corrected spelling, grammar, and 
formatting errors. 
Administrative 
7.0 
11Apr2022 
Updated inclusion criterion #8 
and #9 to allow for select drug 
use 
Administrative 
 
 
Updated language regarding 
confinement of backup subjects 
Administrative 
8.0 
02May2023 Updated objectives and endpoints 
to move NAI assay from 
Administrative 

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secondary to exploratory 
objectives and associated changes 
in text in Section 9. 
 
 
 

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11. 
REFERENCES 
 
1. 
NIAID, NIAID Strategic Plan for Biodefense Research. 2007. 
2. 
Erbelding, E.J., et al., A Universal Influenza Vaccine: The Strategic Plan for the National 
Institute of Allergy and Infectious Diseases. J Infect Dis, 2018. 218(3): p. 347-354. 
3. 
Genentech, Full Prescribing Information - TAMIFLU. 2012. 
4. 
Genentech, Full Prescribing Information - XOFLUZA. 2018. 
5. 
Carrat, F., et al., Time lines of infection and disease in human influenza: a review of 
volunteer challenge studies. American journal of epidemiology, 2008. 167(7): p. 775-
785. 
6. 
Memoli, M.J., et al., Validation of the wild-type influenza A human challenge model 
H1N1pdMIST: an A(H1N1)pdm09 dose-finding investigational new drug study. Clin 
Infect Dis, 2015. 60(5): p. 693-702. 
7. 
Han, A., et al., A Dose-finding Study of a Wild-type Influenza A(H3N2) Virus in a 
Healthy Volunteer Human Challenge Model. Clin Infect Dis, 2019. 69(12): p. 2082-2090. 
8. 
Hussain, M., et al., Drug resistance in influenza A virus: the epidemiology and 
management. Infect Drug Resist, 2017. 10: p. 121-134. 
9. 
Holm, S., A Simple Sequentially Rejective Multiple Test Procedure. Scandinavian Journal 
of Statistics, 1979. 6(2): p. 65-70. 
10. 
Benjamini, Y. and Y. Hochberg, Controlling the False Discovery Rate: A Practical and 
Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society. Series B 
(Methodological), 1995. 57(1): p. 289-300. 
11. 
Aken, B.L., et al., Ensembl 2017. Nucleic Acids Res, 2017. 45(D1): p. D635-D642. 
12. 
Robinson, M.D. and A. Oshlack, A scaling normalization method for differential 
expression analysis of RNA-seq data. Genome Biol, 2010. 11(3): p. R25. 
13. 
Robinson, M.D., D.J. McCarthy, and G.K. Smyth, edgeR: a Bioconductor package for 
differential expression analysis of digital gene expression data. Bioinformatics, 2010. 
26(1): p. 139-40. 
14. 
Suzuki, R. and H. Shimodaira, Pvclust: an R package for assessing the uncertainty in 
hierarchical clustering. Bioinformatics, 2006. 22(12): p. 1540-2. 
15. 
Liberzon, A., et al., Molecular signatures database (MSigDB) 3.0. Bioinformatics, 2011. 
27(12): p. 1739-40. 
16. 
Kanehisa, M. and S. Goto, KEGG: kyoto encyclopedia of genes and genomes. Nucleic 
Acids Res, 2000. 28(1): p. 27-30. 
17. 
Li, S., et al., Molecular signatures of antibody responses derived from a systems biology 
study of five human vaccines. Nat Immunol, 2014. 15(2): p. 195-204. 
18. 
Young, M.D., et al., Gene ontology analysis for RNA-seq: accounting for selection bias. 
Genome Biol, 2010. 11(2): p. R14. 
 
 
 
 
 
 

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12. 
APPENDIX A. SEVERITY GRADING FOR VITAL SIGNS 
• 
Oral temperature will be graded as follows: 
 
Systemic (Quantitative) 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Fever* - oral† 
38.1°C – 38.4°C 
100.6°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. 
†Subjects must not eat or drink anything hot or cold, or smoke within 10 minutes prior to 
taking oral temperature 
 
• Pulse, Blood Pressure, and Respiratory Rate will be graded as follows: 
 
Physiologic Parameter 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Bradycardia – beats per 
minute 
45 – 46  
40 – 44 
<40 
Tachycardia – beats per 
minute 
100 – 130  
131 – 155 
>155 
Hypotension (systolic) 
mmHg 
80 – 84  
75 – 79 
<75 
Hypotension (diastolic) 
mmHg 
50 – 54  
45 – 49 
<45 
Hypertension (systolic) 
mmHg 
140 – 155  
156 – 160 
>160 
Hypertension (diastolic) 
mmHg 
90 – 100  
101 – 110 
>110 
SpO2 (%) 
92-94 
89-91 
<89 
RR (increase) (bpm) 
21-24 
25-29 
≥30 
RR (decrease) (bpm) 
9-11 
6-8 
<6 
 
 

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13. 
APPENDIX B. SEVERITY GRADING FOR SAFETY 
LABORATORIES 
• 
Clinical safety laboratory values will be graded as follows: 
 
Hematology 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
WBC x103/µL (Decrease) 
2.50 – <LLN 
1.50 – 2.49 
<1.50 
WBC x103/µL (Increase) 
>ULN – 15.09 
15.10 – 20.00 
>20.00 
Absolute Lymphocyte count x 
103/µL (Decrease) 
0.50 – <LLN 
0.40 - 0.49 
<0.40  
Hgb g/dL (Decrease) (Female) 
10.1 – <LLN 
8.5 – 10.0 
<8.5 
Hgb g/dL (Decrease) (Male) 
11.0 – <LLN 
9.5 – 10.9 
<9.5 
Platelets cell x 103/µL (Decrease) 
EDTA 
120 – <LLN 
100 – 119 
<100 
Platelets x 103/µL (Increase) 
EDTA 
>ULN – 550 
551– 750 
>750 
Chemistry 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
ALT IU/L (Increase) (Female)  
>ULN – 100  
101 – 200 
>200 
ALT IU/L (Increase) (Male)  
>ULN – 138 
139 – 275 
>275 
Creatinine mg/dL (Increase)  
>ULN – 1.8 
1.9 – 2.2 
>2.2 
 
 
 

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14. 
APPENDIX C: VENIPUNCTURE VOLUMES 
 
Study 
Screen
ing 
Pre-
Challenge 
Viral 
Challenge 
Post- Challenge 
Cumulative 
Blood 
Volume 
Total (mL) 
Unplanned visits10 
Confinement period 
Follow-up 
 
Extended 
Confinement11 
Early 
termination 
Unscheduled 
Study Visit Number 
00A 
00B 
00C 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
 
 
 
 
Study Day  
D -45 
to  
-3 
D -2 
D -1 
D1 
D2 
D3 
D4 
D5 
D6 
D7 
D8 
D15 
± 3 
D29 
± 5 
D57 
± 7 
 
D9+ 
Blood for Safety Lab 
Evaluations (Screen)1 
18.5 
 
 
 
 
 
 
 
 
 
 
 
 
 
18.5 
 
 
 
Blood for Safety Lab 
Evaluations (Follow-
up)2 
 
 
 
 
8.5 
 
8.5 
 
 
 
8.5 
 
 
 
25.5 
8.5 
8.5 
 
HAI Serology Screen 
8.5 
 
 
 
 
 
 
 
 
 
 
 
 
 
8.5 
 
 
 
Serological Assays3 
 
 
20 
 
 
 
 
 
 
 
20 
20 
20 
209 
100 
10 
20 
20 
Blood for plasma and 
PBMC for T- and B- 
cell immunology4,5,6 
 
 
24 
 
 
 
 
 
 
 
24 
24 
24 
24 
120 
 
24 
24 
Blood – PBMC for 
plasmablasts7 
 
 
 
 
 
 
 
 
 
 
24 
 
 
 
24 
 
24 
 
Blood – PBMC for 
immunophenotyping8 
 
 
24 
 
24 
 
24 
 
24 
 
24 
 
24 
 
144 
 
24 
24 
Serum for cytokine 
assays 
 
5 
5 
 
5 
5 
5 
5 
5 
5 
5 
5 
 
 
50 
5 
5 
 
Blood – RNA 
transcriptomics 
 
5 
5 
 
5 
5 
5 
5 
5 
5 
5 
5 
 
 
50 
5 
5 
 
Blood – DNA for 
genetic testing 
 
 
4 
 
 
 
 
 
 
 
 
 
 
 
4 
 
 
 

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Study 
Screen
ing 
Pre-
Challenge 
Viral 
Challenge 
Post- Challenge 
Cumulative 
Blood 
Volume 
Total (mL) 
Unplanned visits10 
Confinement period 
Follow-up 
 
Extended 
Confinement11 
Early 
termination 
Unscheduled 
Study Visit Number 
00A 
00B 
00C 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
 
 
 
 
Study Day  
D -45 
to  
-3 
D -2 
D -1 
D1 
D2 
D3 
D4 
D5 
D6 
D7 
D8 
D15 
± 3 
D29 
± 5 
D57 
± 7 
 
D9+ 
Per Visit Blood 
Volume Total (mL)  
27 
10 
82 
0 
42.5 
10 
42.5 
10 
34 
10 
110.
5 
54 
68 
44 
544.5 
28.5 
110.5 
68 
1 HIV, Hepatitis B surface antigen, Hepatitis C antibody, White blood cells (WBCs), absolute lymphocyte count (ALC), hemoglobin (Hb), platelets (PLT), 
alanine transaminase (ALT), creatinine (Cr), serum HCG (in females only). 
2 White blood cells (WBCs), absolute lymphocyte count (ALC), hemoglobin (Hb), platelets (PLT), alanine transaminase (ALT), creatinine (Cr) 
3 HAI = hemagglutination inhibition; NAI = neuraminidase inhibition; MN = microneutralization; or anti-HA-stalk 
4 Frequency of influenza-specific B cell subsets in circulation 
5 Magnitude of influenza-specific T cell responses in circulation (PBMC) to conserved T cell epitopes and inactivated virus 
6 Presence of hemagglutinin (HA)-specific CD4 T cells with a focus on Tfh cells. This will be done on a subset of subjects. 
7 A/Texas/71/2017/H3N2-specific plasmablasts, their specific-antibody (e.g., anti-H3, -N2 and – H3N2 virus) production and isotype (e.g., IgG, IgA) 
8 The frequency, duration, timing and phenotypic characteristics of immune cells in the blood  
9 To be collected at this time point or latest available time point 
10 These collections will only be taken in place of those from planned visits.  The total volume will not be larger than the cumulative blood volume total shown. 
11 These collections will only occur if a subject remains in confinement past Study Day 8 and if clinically indicated.

Participant ID: 
Participant Initials:  
Date:  
/ 
/ 
 
 
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15. 
APPENDIX D: FLU-PRO – PARTICIPANT REPORTED TOOL 
FLU-PRO© 
 
 
What time is it?  
AM / PM (please circle) 
 
Please rate the extent to which you had each symptom during the past 24 hours. 
 
Not at 
all 
A little bit 
Somewhat 
Quite a 
bit 
Very much 
Runny or dripping nose 
☐ 
☐ 
☐ 
☐ 
☐ 
Congested or stuffy nose 
☐ 
☐ 
☐ 
☐ 
☐ 
Sinus pressure 
☐ 
☐ 
☐ 
☐ 
☐ 
 
Scratchy or itchy throat 
☐ 
☐ 
☐ 
☐ 
☐ 
Sore or painful throat 
☐ 
☐ 
☐ 
☐ 
☐ 
Difficulty swallowing 
☐ 
☐ 
☐ 
☐ 
☐ 
 
 
Teary or watery eyes 
☐ 
☐ 
☐ 
☐ 
☐ 
Sore or painful eyes 
☐ 
☐ 
☐ 
☐ 
☐ 
Eyes sensitive to light 
☐ 
☐ 
☐ 
☐ 
☐ 
 
 
Trouble breathing 
☐ 
☐ 
☐ 
☐ 
☐ 
Chest congestion 
☐ 
☐ 
☐ 
☐ 
☐ 
Chest tightness 
☐ 
☐ 
☐ 
☐ 
☐ 
Dry or hacking cough 
☐ 
☐ 
☐ 
☐ 
☐ 
Wet or loose cough 
☐ 
☐ 
☐ 
☐ 
☐ 
 
 
Felt nauseous (feeling like you 
wanted to throw-up) 
☐ 
☐ 
☐ 
☐ 
☐ 
People experience the flu in different ways. We would like to know about the symptoms you have 
been experiencing during the past 24 hours. For each symptom, please mark one box ☐ under the 
response that best matches your experience. Mark the “Not at all” box, if you did not have that 
symptom in the past 24 hours. 

Participant ID: 
Participant Initials:  
Date:  
/ 
/ 
 
 
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Stomachache 
☐ 
☐ 
☐ 
☐ 
☐ 
 
 
Felt dizzy 
☐ 
☐ 
☐ 
☐ 
☐ 
Head congestion 
☐ 
☐ 
☐ 
☐ 
☐ 
Headache 
☐ 
☐ 
☐ 
☐ 
☐ 

Participant ID: 
Participant Initials:  
Date:  
/ 
/ 
 
 
DMID/NIAID/NIH 
CONFIDENTIAL 
Page 101 of 102 
Please rate the extent to which you had each symptom during the past 24 hours. 
 
Not at 
all 
A little bit 
Somewhat 
Quite a 
bit 
Very much 
Lack of appetite 
☐ 
☐ 
☐ 
☐ 
☐ 
Sleeping more than usual 
☐ 
☐ 
☐ 
☐ 
☐ 
Body aches or pains 
☐ 
☐ 
☐ 
☐ 
☐ 
Weak or tired 
☐ 
☐ 
☐ 
☐ 
☐ 
Chills or shivering 
☐ 
☐ 
☐ 
☐ 
☐ 
Felt cold 
☐ 
☐ 
☐ 
☐ 
☐ 
Felt hot 
☐ 
☐ 
☐ 
☐ 
☐ 
Sweating 
☐ 
☐ 
☐ 
☐ 
☐ 
 
 
 
In the past 24 hours, how often have you had any of the following symptoms? 
 
Never 
Rarely 
Sometimes 
Often 
Always 
Sneezing 
☐ 
☐ 
☐ 
☐ 
☐ 
Coughing 
☐ 
☐ 
☐ 
☐ 
☐ 
Coughed up mucus or phlegm 
☐ 
☐ 
☐ 
☐ 
☐ 
 
 
0 times 
1 time 
2 times 
3 times 
4 or more 
times 
How many times did you 
vomit? 
☐ 
☐ 
☐ 
☐ 
☐ 
How many times did you have 
diarrhea? 
☐ 
☐ 
☐ 
☐ 
☐ 
 
 
 
 
 
 
 
 
 
 
 
 

Participant ID: 
Participant Initials:  
Date:  
/ 
/ 
 
 
DMID/NIAID/NIH 
CONFIDENTIAL 
Page 102 of 102 
Items to be asked in the daily diary through to Day 15 along with the Flu-PRO items. 
 
1. 
Overall, how severe were your flu symptoms today? (Please select one response only) 
 
0 No flu symptoms today 
 
1 Mild 
 
2 Moderate 
 
3 Severe 
 
4 Very severe 
 
2. 
[Skip this question if you answered 0 above]. Overall, how were your flu symptoms today compared to 
yesterday?  (Please select one response only) 
 
1 Much better 
 
2 Somewhat better 
 
3 A little better 
 
4 About the same 
 
5 A little worse 
 
6 Somewhat worse 
 
7 Much worse 
 
3. 
[Skip this question if you answered 0 in Question #1]. How much did your flu symptoms interfere with your 
activities today? (Please select one response only) 
 
1 Not at all 
 
2 A little bit 
 
3 Somewhat 
 
4 Quite a bit 
 
5 Very much 
  
4. 
Have you returned to activities today? 
 
0 No 
 
1 Yes  
 
5. 
In general, how would you rate your physical health today? (Please select one response only) 
 
1 Poor 
 
2 Fair 
 
3 Good 
 
4 Very Good 
 
5 Excellent 
 
6. 
Are you in your usual state of health today? 
 
0 No 
 
1 Yes