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
Phase: PHASE1
Conditions: Influenza
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

DMID Protocol 18-0010 
Version 3.0 
H1N1v Virus Challenge Study in Healthy Subjects 
04 November 2020  
 
DMID/NIAID/NIH 
1 
CONFIDENTIAL 
 
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 
DMID Protocol Number: 18-0010 
DMID Funding Mechanism: Vaccine and Treatment Evaluation Units 
IND Sponsor: Division of Microbiology and Infectious Diseases (DMID), National Institutes 
of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) 
Lead Principal Investigator: Kathleen Neuzil, MD, MPH 
DMID Clinical Project Manager: Kathy Ormanoski, MS  
 
Version Number: 3.0 
04 November 2020 
 
 
 
 
 

DMID Protocol 18-0010 
Version 3.0 
H1N1v Virus Challenge Study in Healthy Subjects 
04 November 2020  
 
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STATEMENT OF ASSURANCE 
Each Institution will hold a current Federal Wide Assurance (FWA) issued by the Office of 
Human Research Protections (OHRP) for federally-funded human subjects research. Each FWA 
will designate at least one Institutional Review Board (IRB)/Independent Ethics Committee 
(IEC) registered with OHRP, for which the research will be reviewed and approved by the 
IRB/IEC and will be subject to continuing review [45 CFR 46.103(b)]. The IRB/IEC designated 
under an FWA may include an institution’s IRB/IEC, an independent IRB/IEC, or an IRB/IEC of 
another institution after establishing a written agreement with that other institution.  
 

DMID Protocol 18-0010 
Version 3.0 
H1N1v Virus Challenge Study in Healthy Subjects 
04 November 2020  
 
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STATEMENT OF COMPLIANCE 
The study will be carried out in accordance with Good Clinical Practice (GCP) and as required 
by the following: 
• United States (US) Code of Federal Regulations (CFR) 45 CFR Part 46: Protection of 
Human Subjects 
• Food and Drug Administration (FDA) Regulations, as applicable: 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), 21 CFR 812 (Investigational 
Device Exemptions) 
• International Council for Harmonisation of Technical Requirements for Pharmaceuticals 
for Human Use (ICH): Good Clinical Practice (GCP) E6(R2) Good Clinical Practice: 
Integrated Addendum to ICH E6(R1) Guidance for Industry,” published in the Federal 
Register (83 Federal Register 8882 (2018) 
• National Institutes of Health (NIH) Office of Extramural Research, Research Involving 
Human Subjects, as applicable 
• National Institute of Allergy and Infectious Diseases (NIAID) Clinical Terms of Award, 
as applicable 
• Applicable Federal, State, and Local Regulations and Guidance 

DMID Protocol 18-0010 
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04 November 2020  
 
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SIGNATURE PAGE 
The signature below provides the necessary assurance that this study will be conducted 
according to all stipulations of the protocol, including all statements regarding confidentiality, 
and according to local legal and regulatory requirements and applicable US federal regulations 
and ICH E6 GCP guidelines. 
I agree to conduct the study in compliance with GCP and applicable regulatory requirements. 
I agree to conduct the study in accordance with the current protocol and will not make changes to 
the protocol without obtaining the Sponsor’s approval and IRB/IEC approval, except when 
necessary to protect the safety, rights, or welfare of subjects. 
Site Investigator Signature: 
 
Signed: 
 
Date: 
 
 
Name 
Title 
 
 
 
 

DMID Protocol 18-0010 
Version 3.0 
H1N1v Virus Challenge Study in Healthy Subjects 
04 November 2020  
 
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TABLE OF CONTENTS 
STATEMENT OF ASSURANCE....................................................................................................... 2 
STATEMENT OF COMPLIANCE .................................................................................................... 3 
SIGNATURE PAGE ............................................................................................................................ 4 
TABLE OF CONTENTS ..................................................................................................................... 5 
LIST OF TABLES.............................................................................................................................. 10 
LIST OF FIGURES ............................................................................................................................ 11 
LIST OF ABBREVIATIONS ............................................................................................................ 12 
PROTOCOL SUMMARY ................................................................................................................. 16 
 
KEY ROLES ............................................................................................................................. 23 
 
BACKGROUND AND SCIENTIFIC RATIONALE ............................................................ 25 
 
Background ..................................................................................................................... 25 
 
Scientific Rationale......................................................................................................... 27 
2.2.1 Purpose of Study................................................................................................ 27 
2.2.2 Study Population ............................................................................................... 30 
 
Potential Risks and Benefits .......................................................................................... 30 
2.3.1 Common Symptoms of Influenza Illness ......................................................... 30 
2.3.2 Potential Risks ................................................................................................... 31 
2.3.3 Potential Benefits............................................................................................... 33 
 
STUDY OBJECTIVES AND OUTCOME MEASURES ...................................................... 34 
 
STUDY DESIGN ...................................................................................................................... 35 
 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT ........................................... 38 
 
Study Product Description ............................................................................................. 38 
5.1.1 Formulation, Packaging, and Labeling ............................................................ 38 
5.1.2 Product Storage and Stability ........................................................................... 38 
 
Acquisition/Distribution ................................................................................................. 39 
 
Dosage/Regimen, Preparation, Dispensing and Administration of Study 
Intervention/Investigational Product ........................................................................... 39 
 
Pre-determined Modification of Study Intervention/Investigational Product for an 
Individual Subject ......................................................................................................... 40 
 
Accountability Procedures for the Study Intervention/Investigational Product(s) ..... 40 
 
SELECTION OF SUBJECTS AND STUDY ENROLLMENT AND WITHDRAWAL.... 42 

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Eligibility Criteria ........................................................................................................... 42 
6.1.1 Subject Inclusion Criteria.................................................................................. 42 
6.1.2 Subject Exclusion Criteria ................................................................................ 44 
 
Withdrawal from the Study, Discontinuation of Study Product, or Study Termination
........................................................................................................................................ 47 
6.2.1 Discontinuation of the Study Product .............................................................. 47 
6.2.2 Subject Replacement ......................................................................................... 47 
6.2.3 Study Termination ............................................................................................. 47 
6.2.4 Withdrawal during the Inpatient Period ........................................................... 47 
6.2.5 Withdrawal during the Follow-up Period ........................................................ 48 
6.2.6 Reasons for withdrawal might include, but are not limited to the following:
 ............................................................................................................................ 48 
 
STUDY PROCEDURES .......................................................................................................... 50 
 
Screening Period ............................................................................................................. 50 
7.1.1 Study Visit 00A (Day -30 to -3) ....................................................................... 50 
 
Inpatient Period ............................................................................................................... 51 
7.2.1 Study Visit 00B (Day -2) .................................................................................. 51 
7.2.2 Study Visit 00C (Day -1) .................................................................................. 52 
7.2.3 Study Visit 01 (Day 1) Before Challenge ........................................................ 53 
7.2.4 Study Visit 01 (Day 1) Post-challenge ............................................................. 53 
7.2.5 Study Visit 02 (Day 2) ...................................................................................... 54 
7.2.6 Study Visit 03 (Day 3) ...................................................................................... 54 
7.2.7 Study Visit 04 (Day 4) ...................................................................................... 55 
7.2.8 Study Visit 05 (Day 5) ...................................................................................... 56 
7.2.9 Study Visit 06 (Day 6) ...................................................................................... 56 
7.2.10 Study Visit 07 (Day 7) ...................................................................................... 57 
7.2.11 Study Visit 08 (Day 8) ...................................................................................... 57 
7.2.11.1 Unscheduled Inpatient Days After Day 8 .......................................... 59 
 
Follow-up Period ............................................................................................................ 60 
7.3.1.1 
Post-discharge (Approximately Day 9 to Day 14) ............................ 60 
7.3.1.2 
Study Visit 09 (Day 15 ± 3 Days)3Days ............................................ 60 
7.3.1.3 
Study Visit 10 (Day 29 ± 3 Days) ...................................................... 60 
7.3.1.4 
Study Visit 11 (Day 61 ± 5 Days) ...................................................... 61 

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7.3.2 Final Study Visit -- Telephone Follow-up (Day 91 ± 7 Days) ....................... 61 
 
Early Termination Visit .................................................................................................. 61 
 
Unscheduled Study Visits .............................................................................................. 62 
 
Protocol Deviations ........................................................................................................ 62 
 
DESCRIPTION OF CLINICAL AND LABORATORY EVALUATIONS ........................ 64 
 
Clinical Evaluations ........................................................................................................ 64 
8.1.1 Research Procedures ......................................................................................... 65 
8.1.2 Assessment of Concomitant Medications/Treatments other than Study 
Product ............................................................................................................... 66 
8.1.3 Assessment of Subject Compliance with Study Intervention/Investigational 
Product/Investigational Device......................................................................... 66 
8.1.4 Non-Research Standard of Care ....................................................................... 67 
8.1.5 Non-Research Standard of Care ....................................................................... 67 
 
Laboratory Evaluations .................................................................................................. 67 
8.2.1 Clinical Laboratory Evaluations ....................................................................... 67 
8.2.2 Research Assays ................................................................................................ 68 
8.2.2.1 
Laboratory Specimen Preparation, Handling, and Storage ............... 68 
8.2.2.2 
Laboratory Specimen Shipping .......................................................... 68 
 
ASSESSMENT OF SAFETY .................................................................................................. 69 
 
Assessing and Recording Safety Parameters ................................................................ 69 
9.1.1 Adverse Events (AEs) ....................................................................................... 69 
9.1.1.1 
Adverse Events Grading...................................................................... 70 
9.1.2 Serious Adverse Events (SAEs) ....................................................................... 73 
 
Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings ......................................................................................... 74 
 
Specification of Safety Parameters ................................................................................ 74 
 
Reporting Procedures ..................................................................................................... 74 
9.4.1 Reporting Serious Adverse Events ................................................................... 74 
9.4.2 Regulatory Reporting for Studies Conducted Under DMID Sponsored- IND
 ............................................................................................................................ 75 
9.4.3 Reporting of Pregnancy..................................................................................... 75 
 
Type and Duration of Follow-up of Subjects after Adverse Events............................ 76 
 
Halting Rules................................................................................................................... 76 

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9.6.1 Study Halting Criteria ....................................................................................... 76 
 
Safety Oversight ............................................................................................................. 76 
9.7.1 Safety Monitoring Committee (SMC) ............................................................. 76 
 
HUMAN SUBJECTS PROTECTION .................................................................................... 78 
 Institutional Review Board/Independent Ethics Committee ........................................ 78 
 Informed Consent Process.............................................................................................. 78 
 Consent for Future Use of Stored Specimens and Data ............................................... 80 
 Exclusion of Women, Minorities, and Children (Special Populations) ...................... 81 
 Subject Confidentiality ................................................................................................... 81 
 Certificate of Confidentiality ......................................................................................... 82 
 Costs, Subject Compensation, and Research Related Injuries ..................................... 82 
 
STATISTICAL CONSIDERATIONS..................................................................................... 83 
 Introduction ..................................................................................................................... 83 
 Study Hypotheses ........................................................................................................... 83 
 Sample Size Considerations ........................................................................................... 83 
11.3.1 Study Design ...................................................................................................... 83 
11.3.2 Sample Size........................................................................................................ 83 
 Planned Interim Analyses............................................................................................... 84 
11.4.1 Interim Safety Review....................................................................................... 85 
 Final Analysis Plan ......................................................................................................... 85 
11.5.1 Analysis Populations ......................................................................................... 85 
11.5.2 Safety and Clinical Data ................................................................................... 86 
11.5.3 Immunological Data .......................................................................................... 87 
11.5.1 Transcriptomic Data .......................................................................................... 88 
11.5.2 Genomics Data................................................................................................... 88 
11.5.3 Missing Values and Outliers ............................................................................. 88 
 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS .............. 89 
 
QUALITY CONTROL AND QUALITY ASSURANCE ..................................................... 90 
 
DATA HANDLING AND RECORD KEEPING................................................................... 91 
 Data Management Responsibilities ............................................................................... 91 
 Data Coordinating Center/Biostatistician Responsibilities .......................................... 91 
 Data Capture Methods .................................................................................................... 91 

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 Types of Data .................................................................................................................. 92 
 Study Records Retention ................................................................................................ 92 
 
CLINICAL MONITORING ..................................................................................................... 93 
 
PUBLICATION POLICY ........................................................................................................ 94 
 
LITERATURE REFERENCES ............................................................................................... 95 
 
APPENDICES ........................................................................................................................... 99 
 
Schedule of Events ............................................................................ 100 
 
Venipuncture Volumes (mL) ............................................................ 104 
 
High-Risk Medical Conditions for Severe Influenza ...................... 106 
 
Flu-PRO Survey Instrument and Validation Diary ......................... 107 
 
 
 

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LIST OF TABLES 
 
Table 1: 
Study Objectives and Outcome Measures..................................................................... 17 
Table 2: 
Study Intervention .......................................................................................................... 21 
Table 3: 
Doses Used in Previous Challenge Studies in Humans Since 1985 ............................ 28 
Table 4: 
Dose Escalation Results with A/Bethesda/MM2/H1N1 challenge strain ................... 30 
Table 5:  
Study Intervention Regimen .......................................................................................... 40 
Table 6: 
Clinical Laboratory Normal Ranges .............................................................................. 46 
Table 7: 
Severity Grading for Oral Temperature ........................................................................ 71 
Table 8: 
Pulse and BP Adverse Event Grading ........................................................................... 71 
Table 9: 
Clinical Safety and Laboratory AEs Grading ............................................................... 72 
Table 10: Power to detect odds ratios (OR) for a one SD increase in a continuous, 
normally distributed covariate in a logistic regression model at the α=0.05-
level ................................................................................................................................. 84 
Table 11: Probability (%) of observing at least one safety event, given varying 
underlying event probabilities and group sizes ............................................................. 84 
Table 12: Schedule of Events ....................................................................................................... 100 
Table 13: Venipuncture Volumes (all volumes are approximate) .............................................. 104 
 
 

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Version 3.0 
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LIST OF FIGURES 
Figure 1: 
Schematic of Study Design ............................................................................................ 22 
 
 

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LIST OF ABBREVIATIONS 
°C 
Degree Celsius 
°F 
Degree Fahrenheit 
ADL 
Activities of Daily Living 
AE 
Adverse Event/Adverse Experience 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Transaminase 
ANC 
Absolute Neutrophil Count 
ASC 
AST 
Antibody Secreting Cells 
Aspartate Transaminase 
BMI 
Body Mass Index 
BP 
Blood Pressure 
BUN 
Blood Urea Nitrogen 
CBC 
Complete Blood Count 
CFR 
Code of Federal Regulations 
CHI 
Controlled Human Infection 
CK 
Creatinine Kinase 
Cl 
Chloride 
cMRI 
Cardiac MRI 
CMS 
Clinical Material Services 
CO2 
Carbon Dioxide 
CoP 
Correlation of Protection 
COPD 
Chronic Obstructive Pulmonary Disease 
Cr 
Creatinine 
CRF 
Case Report Form 
CXR 
Chest X-Ray 
DBP 
Diastolic Blood Pressure 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, 
NIH, DHHS 

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DSMB 
Data and Safety Monitoring Board 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
eGFR 
Estimated Glomerular Filtration Rate 
ELISA 
Enzyme-linked Immunosorbent Assay 
EPT 
End Point Titers 
ER 
Emergency Room 
FDA 
Food and Drug Administration 
Flu-PRO Survey Instrument Influenza Patient Reported Outcome 
FWA 
Federal Wide Assurance 
GCP 
Good Clinical Practice 
g/dL 
Grams per Deciliter 
GMFR 
Geometric Mean Fold Rise 
GMP 
Good Manufacturing Practice 
GMT 
Geometric Mean Titer 
H1N1pdm09 
A/California/04/2009 (H1N1)-like Influenza A Virus 
HA 
Hemagglutinin 
HAI 
Hemagglutinin Inhibition Test 
HBV 
Hepatitis B Virus 
HCV 
Hepatitis C Virus 
HIV 
Human Immunodeficiency Virus 
HR 
Heart Rate 
iADL 
Instrumental Activities of Daily Living 
ICF 
Informed Consent Form 
ICH 
International Council for Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use 
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 

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IV 
Intravenous 
K 
Potassium 
Kg/m2 
Kilogram per Square Meter 
LAR 
Legally Authorized Representative 
LDH 
Lactate Dehydrogenase 
LID 
Laboratory of Infectious Diseases at NIAID 
M 
Molar 
MAD 
Mucosal Atomization Device 
mg 
Milligram 
mg/dL 
Milligrams per Deciliter 
mL 
Milliliter 
mm 
Millimeter 
mm3 
Cubic Millimeter 
mm Hg 
Millimeter of Mercury 
MMID 
Mild-to-Moderate Influenza Disease 
MN 
Microneutralization 
MOP 
Manual of Procedures 
MRI 
Magnetic Resonance Imaging 
N 
Number (typically refers to subjects) 
Na 
Sodium 
NA 
Neuraminidase 
NAI 
Neuraminidase Inhibition 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, 
DHHS 
NIH 
National Institutes of Health 
NLF 
Nasal Lavage Fluid 
NP 
Nasopharyngeal 
NSAID 
Non-Steroidal Anti-inflammatory Drug 
OHRP 
Office for Human Research Protections 
PBMC 
PE 
Peripheral Blood Mononuclear Cells 
Physical Exam 
PI 
Principal Investigator 

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PT 
Prothrombin Time 
PTT 
Partial Thromboplastin Time 
QA 
Quality Assurance 
QC 
Quality Control 
REDCap 
Research Electronic Data Capture 
RR 
Respiratory Rate 
RT-PCR 
Reverse Transcription – Polymerase Chain Reaction 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SBP 
Systolic Blood Pressure 
SE 
Standard Error 
sIgA 
Secretory Immunoglobulin A 
SMC 
Safety Monitoring Committee 
SpO2 
Saturation of Peripheral Oxygen 
TCID50 
Median Tissue Culture Infective Dose 
THC 
Tetrahydrocannabinol 
UA 
Uric Acid 
ULN 
Upper Limit of Normal 
US 
United States 
VTEU 
Vaccine and Treatment Evaluation Units 
WBC 
wt 
White Blood Cell 
Wild-type 
 
 

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PROTOCOL SUMMARY 
Title: 
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. 
Phase: 
1 (Controlled human infection study) 
Population: 
Up to 80 healthy adult subjects between the ages of 18 to 49 
years of age inclusive, who are in good health and meet 
eligibility criteria.  
Number of Sites: 
Four domestic vaccine and treatment evaluation unit (VTEU) 
sites. 
Study Duration: 
Approximately 180 days (from time of study activation to last 
subject’s last study day). 
Subject Participation 
Duration: 
Approximately 120 days. 
Estimated Time to 
Complete Enrollment: 
Approximately 60 days 
Description of Study 
Product: 
Influenza A/Bethesda/MM2/H1N1 virus at a total dose of 2 mL 
of approximately 5x106/mL tissue culture infective dose 
(TCID50). 
• Route of administration: Intranasal 
• Volume: 1 mL per nostril (2 mL dose total) 
Design of the Study: 
This is a study of a reverse-engineered, GMP grade, antiviral-
sensitive, influenza A/Bethesda/MM2/H1N1 virus 
(A/California/04/2009/H1N1-like) infection to assess the effect 
of pre-existing immunity on clinical and immunological 
responses. Up to 80 healthy adult subjects will undergo 
intranasal inoculation with A/Bethesda/MM2/H1N1 virus, and 
their clinical manifestations, viral shedding and immunological 
responses will be characterized. 
 
 
 

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Table 1: 
Study Objectives and Outcome Measures 
Primary Objective  
Primary Outcome Measure 
• To evaluate the association of 
symptomatic RT-PCR-positive 
influenza virus infection post-
challenge and pre-existing HAI 
antibody titers 
• Baseline A/Bethesda/MM2/H1N1 
hemagglutination inhibition (HAI) antibody 
GMT association with development of MMID 
post-challenge, with MMID defined as 
presence of both of the following assessed 
through Day 8: 
1. Viral shedding detected by any approved 
positive RT-PCR test from NP swab, and 
2. Any one or more of the following 
symptoms or signs or laboratory findings, 
as related to the study agent; Arthralgia, 
Chest tightness, Chills, Conjunctivitis, 
Nasal congestion, Sinus Congestion, 
Coryza, Decreased appetite, Diarrhea, Dry 
Cough, Dyspnea/Shortness of Breath, 
Fatigue/Tiredness, Fever (>38.0°C), 
Headache, Lymphopenia (<1000 cells/mL), 
Myalgia, Nausea, Oxygen Saturation 
Decrease by ≥3% from baseline, 
Productive Cough, Rhinorrhea, Sore 
Throat, and Sweats.  
Secondary Objectives 
Secondary Outcome Measures 
• To describe viral recovery by 
quantitative RT-PCR from study 
subjects at baseline and post-challenge 
• Frequency, timing, magnitude, and duration of 
viral shedding in NLF or NP swab collected 
daily using quantitative RT-PCR from baseline 
(Day -2 or -1) and daily from Day 2 through 
Day 8 
• To describe serum HAI and MN 
antibody responses post-challenge in 
healthy subjects by infection status 
• Baseline and post-challenge HAI and MN 
antibody GMTs from serum measured at 
baseline (Day -2) and at Days, 8, 29, and 61 by 
infection status (RT-PCR-confirmed 
symptomatic, RT-PCR-confirmed 
asymptomatic, RT-PCR-negative 
symptomatic) 
• Percentage of healthy subjects achieving HAI 
and MN seroconversion (defined as either a 
pre-challenge titer <1:10 and a post-challenge 
titer ≥1:40 or a pre-challenge titer ≥1:10 and a 
minimum four-fold rise in post-challenge 

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antibody titer) from serum measured at 
baseline (Day -2) and on Days 8, 29, and 61 
• To evaluate the association of 
asymptomatic RT-PCR-positive 
influenza virus infection (viral 
shedding) post-challenge and pre-
existing HAI antibody titers 
• Baseline A/Bethesda/MM2/H1N1 HAI 
antibody GMT measured at baseline (Day -2) 
association with asymptomatic viral shedding 
by RT-PCR through Day 8  
• To evaluate the association of 
symptomatic RT-PCR-negative status 
post-challenge and pre-existing HAI 
antibody titers  
• Baseline A/Bethesda/MM2/H1N1 HAI 
antibody GMT measured at baseline (Day -2) 
association with development of any Flu-PRO 
symptoms post-challenge without influenza 
virus detection through Day 8  
• To determine the frequency of serious 
adverse events (SAE) post-challenge  
• Frequency of serious adverse events (SAE) 
post-challenge through the inpatient stay  
• Frequency of serious adverse events (SAE) 
post- inpatient discharge through the duration 
of the study (approximately three months post-
challenge) 
Exploratory Objectives 
Exploratory Outcome Measures 
• To describe serum NAI antibody 
responses post challenge in healthy 
subjects by infection status 
• Baseline and post-challenge NAI antibody 
GMTs from serum measured at baseline (Day -
2) and at Days, 8, 29, and 61 by infection 
status (RT-PCR-confirmed symptomatic, RT-
PCR-confirmed asymptomatic, RT-PCR-
negative symptomatic) 
• Percentage of healthy subjects achieving NAI 
seroconversion (defined as either a pre-
challenge titer <1:10 and a post-challenge titer 
≥1:40 or a pre-challenge titer ≥1:10 and a 
minimum four-fold rise in post- challenge 
antibody titer) from serum measured at 
baseline (Day -2) and on Days 8, 29, and 61 
• To evaluate the association of 
symptomatic RT-PCR-positive 
influenza virus infection, 
asymptomatic RT-PCR-positive 
influenza virus infection (viral 
shedding only) and symptomatic RT-
PCR-negative illness and pre-existing 
neuraminidase inhibition (NAI) 
antibody titers 
• Baseline A/Bethesda/MM2/H1N1 
neuraminidase inhibition (NAI) antibody GMT 
measured at baseline (Day -2) association with 
infection status through Day 8 by 
symptom/shedding status 

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• To evaluate mucosal sIgA immune 
response, at baseline and post-
challenge  
• Positive, GMT and GMFR responses for 
A/Bethesda/MM2/H1N1-specific sIgA, 
nonnormalized and normalized for total IgA 
content, in NLF at baseline (Day -2), and Days 
8, 29, and 61 
• To evaluate serum and mucosal 
cytokine and chemokine responses at 
baseline and post-challenge 
• Determine A/Bethesda/MM2/H1N1 induced 
cytokines and chemokines in serum at baseline 
(Day -2), and Days 2, 4, 6, 8, and 15 
• Determine A/Bethesda/MM2/H1N1 induced 
cytokines and chemokines in NLF by study 
arm at baseline (Day -2), and approximately on 
Days 2, 4, 6, 8, and 15 
• To assess the effects of age, sex, 
baseline antibody titers and prior 
receipt of seasonal influenza vaccine 
on symptomatic RT-PCR-positive 
influenza infection post-challenge  
• Evaluate the association between baseline 
A/Bethesda/MM2/H1N1 hemagglutination 
inhibition (HAI) GMT, age, sex, and receipt of 
seasonal influenza vaccine in the previous 
year, and the development of MMID post-
challenge through Day 8 by multivariate 
logistic regression 
• To compare the clinical features of 
symptomatic RT-PCR-positive and 
RT-PCR-negative illness post-
challenge  
• Self-report of clinical symptoms and their 
severity as measured by component question in 
Flu-PRO Survey Instrument by post-challenge 
day and infection status  
• To explore alternative case definitions 
for symptomatic RT-PCR-positive 
influenza virus infection 
• The combination of symptoms and symptom 
severity as determined by self-report using the 
Flu- PRO instrument will be evaluated to 
develop clinical case definitions which 
optimize sensitivity, specificity, and both for 
influenza virus infection among subjects with 
evidence of RT-PCR-positive influenza virus 
infection post-challenge 
• To evaluate the plasmablast response 
post-challenge in a subset of study 
subjects 
• Determine the kinetics of 
A/Bethesda/MM2/H1N1-specific plasmablasts, 
their specific-antibody (e.g., anti-H1, -N1 and - 
H1N1 virus) production and isotype (e.g., IgG, 
IgA) using an enzyme-linked immunospot 
(ELISpot) assay in PBMCs on Days 4, 6, and 8 
in a subset of subjects 
• To describe systemic transcriptional 
responses post-challenge in a subset of 
study subjects 
• Determine changes in peripheral blood gene 
expression profiling based on sequencing on 

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Days 2, 4, 8, and 15 relative to baseline (Days -
2 and 1 pre-challenge) in a subset of subjects 
• To describe mucosal and systemic 
human immune cells in blood and 
NLF at baseline and post-challenge in 
a subset of study subjects 
• Determine the frequency, duration, timing and 
phenotypic characteristics of immune cells in 
the blood of subjects at baseline (Day -2) and 
at approximately on Days 4, 6, 8, 15, 29, and 
61 in a subset of subjects  
• Determine the frequency, duration, timing and 
phenotypic characteristics of immune cells in 
the NLF in subjects at baseline (Day -2) and 
Days 2, 4, 6 and 8 in a subset of subjects  
• To describe anti-HA-stalk antibody 
titer at baseline and post-challenge  
• For HA-stalk specific response, the GMTs and 
GMFR to group 1 stem domains will be 
measured at baseline (Day -2), and at 
approximately on Days 29 and 61  
• To assess the association of human 
leukocyte antigen (HLA) class I and II 
alleles with clinical, immune and viral 
responses  
• Determine the frequency of HLA class I and II 
alleles as measured by genetic testing. 
• To assess the association of subject HLA class 
I and II alleles with development of MMID 
post-challenge through Day 8, pre-existing 
immune status, and magnitude and breadth of 
the elicited immune responses post-challenge 
• To evaluate cellular immune response 
at baseline and post-challenge  
• Summarize the data on systemic and mucosal 
cytokines and chemokines induced by 
challenge and define their possible role in the 
process by which influenza exposure leads to 
infection, disease outcomes, and 
seroconversion at baseline and daily through 
Day 8 
• Frequency of influenza-specific B cell subsets 
in circulation (PBMC) at baseline (Day -2), 
and at approximately on Days 6, 8, 15, 29 and 
61 
• Magnitude of influenza-specific T cell 
responses in circulation (PBMC) to conserved 
T cell epitopes and inactivated virus at baseline 
(Day -2), and at approximately on Days 4, 6, 8, 
15, 29, and 61 
 
 

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Table 2: 
Study Intervention 
Sample size 
Intervention 
80 
Influenza A/Bethesda/MM2/H1N1 Challenge Virus 2 mL of approximately 
5x106 TCID50/mL  
 
 

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Figure 1: 
Schematic of Study Design 
 
 
 
N = up to 80 
Challenge Dose: 
A/Bethesda/MM2/H1N1 
virus challenge at a 2 
mL dose of 5x106 
TCID50/mL 
 
Screening for eligibility 
Day -30 to -3 
Day -2 
Enrollment:  
Eligible subjects admitted to the inpatient unit. Baseline evaluations performed. 
Confirm consent. Training on Flu-PRO questionnaire and Validation Diary. Baseline lab 
Day -1 
Day 1 
Day 2-7 
Remain in the inpatient unit. Daily evaluations. 
Discharge (if discharge criteria are met) 
Day 8 
Up to Day 91 
Follow Up 
Challenge 

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KEY ROLES 
 
Lead Principal 
Investigator: 
 
 
Site Principal  
Investigator (Cincinnati): 
 
 
Site Principal  
Investigator (Duke): 
 
 
 
Site Principal  
Investigator (SLU): 
 
 
DMID Clinical Project 
Manager: 
Kathleen M. Neuzil, MD, MPH 
Professor of Medicine and Pediatrics 
University of Maryland School of Medicine 
(410) 706-4946 
kneuzil@som.umaryland.edu 
 
David Bernstein, MD, MA 
Professor, Pediatrics 
Cincinnati Children's Hospital Medical Center 
(513) 636-7682 
david.bernstein@cchmc.org 
 
Chris Woods, MD, MPH 
Professor, Medicine and Global Health 
Duke University School of Medicine 
(919) 668-7174 
chris.woods@duke.edu 
 
Daniel Hoft, MD, PhD 
Professor 
 
Saint Louis University School of Medicine  
(314) 977-5500 
daniel.hoft@health.slu.edu 
 
Kathy Ormanoski, MS 

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Division of Microbiology and Infectious Diseases 
NIAID, NIH 
5601 Fishers Lane, Room 8E37  
Bethesda, MD 20892-9825 
(240) 627-3334 
Kathy.ormanoski@nih.gov 
 
 
HAI and MN Antibody 
Assays Laboratory 
  
Southern Research Institute 
2000 9th Avenue South 
Birmingham, AL 35205 
 
 

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BACKGROUND AND SCIENTIFIC RATIONALE 
 
Background  
The high morbidity and mortality associated with both pandemic and seasonal influenza, the 
continued threat of highly pathogenic avian influenza infections, the development and global 
spread of antiviral resistance, and the inadequacy of current influenza vaccines makes influenza a 
high research priority. Although prior influenza research has addressed numerous aspects of viral 
biology and pathogenesis, many questions remain unanswered. Advancing basic, translational 
and clinical research on influenza will inform the development of new and improved diagnostics, 
therapeutics, and vaccines [1]. 
The natural history, pathogenesis, and immunology of human influenza have not been well 
characterized and cannot be adequately studied in animal models. Even the best models such as 
those in pigs and ferrets, although useful, have demonstrated limitations in their relevance to 
human disease. Influenza human challenge models offer a unique opportunity to further study 
influenza pathogenesis and to overcome some of the limitations associated with animal models 
and/or natural history studies in humans. These models are conducted in a well-controlled 
challenge setting in which virus exposure, infection rate, virus shedding, and sampling are being 
monitored in real-time. Such parameters are challenging to control in a “real world” setting. 
Previous human challenge studies conducted in the US during the 1970s, 1980s, and 1990s 
advanced our understanding of the natural history, clinical characteristics, and immune responses 
following infection [2-14]. Such studies were also used to assess the efficacy of vaccines and 
were critical for advancing the clinical development of influenza antivirals. Although these 
studies have provided important information, their generalizability to current questions is limited 
due to the scope of the studies, the scientific techniques that were available at the time, the 
evolution of circulating virus strains and population immunity, and advances in antiviral and 
vaccine development [2-14]. 
Ensuring subject safety is a paramount concern for any clinical study, particularly human 
challenge studies. In the early 2000s, influenza human challenge studies in the US were stopped 
due to a clinical event occurring after a human challenge study that was investigating the use of 
the antiviral peramivir as a prophylactic agent [15]. The subject was a 21-year-old, previously 
healthy man with no prior cardiac history. Following receipt of peramivir and infection with an 
influenza B challenge strain, he had asymptomatic ECG changes (described as new T wave 
inversions of leads II, aVF, and v4-6) on day 4 of the study. Repeat ECG at day 15 had returned 
to baseline, and he had no cardiac symptoms or cardiac enzyme (CPK) elevation. He then 
traveled to Indonesia for 2 weeks and became ill with an upper respiratory infection. When he 
returned to the US, he had an echocardiography performed at 51 days post influenza challenge, 
since he had had initial ECG changes post-infection. The echocardiogram showed reduced 
ejection fraction, with left ventricular enlargement. The man remained asymptomatic. Over the 
next 5 months, repeat echocardiograms showed gradual improvement and return to normal 
ejection fraction. The cause of the dilated cardiomyopathy was not ascertained but deemed 

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unlikely to be due to the study drug and also deemed unlikely to be due to the challenge virus 
[15, 16].  
As a result of the clinical event described above, influenza challenge studies were not performed 
in the US for many years. A large and thorough review of 56 different influenza challenge 
studies, conducted in 2008 [4], confirmed that infection from an influenza challenge stock 
induced only mild disease, with one third of subjects having a fever and one fifth of subjects 
developing lower respiratory symptoms. Previous influenza challenge studies have demonstrated 
that the required dosing differs depending on the viral strain, subtype, and administration method 
(Table 3) [17]. Influenza challenge studies in Europe continued to be performed [18].  
Since 2012, the Laboratory of Infectious Diseases (LID) at NIAID has been working to re- 
establish a controlled influenza A/H1N1 challenge model in healthy subjects in the US. The first 
dose-ranging study of 49 healthy subjects using a Good Manufacturing Practice (GMP) influenza 
A (H1N1pdm09) challenge strain demonstrated that mild to moderate influenza illness could be 
induced in 70% to 80% of healthy subjects and mimicked natural infection both clinically and 
immunologically [19]. Clinical symptoms of influenza occurred post-challenge with doses 
ranging from 103 to 107 TCID50 but were most prevalent at the 106 and 107 TCID50 doses (Table 
4).  
NIAID’s second challenge study at LID using the H1N1 model evaluated anti-hemagglutinin 
(HA), anti-neuraminidase (NA) and anti-HA stalk antibodies as correlates of protection in H1N1 
influenza illness in 74 healthy subjects, finding that while higher HAI titers are indicative of 
some protection, NAI titers may be more predictive of protection from influenza infection and 
reduced disease [20, 21]. These studies have led to a number of ongoing laboratory studies using 
samples collected to address questions of intra-host evolution, gene expression, and immune 
response, many of which are expected to be published in the coming months. In clinical studies 
the model is now being implemented to test new therapeutics and vaccines (NCT02371668 and 
NCT03845231). This virus has been used in 3 published trials and 1 unpublished trial to date in 
approximately 400 participants. No significant safety issues have been identified, no severe or 
complicated cases of influenza have occurred, and no transmission events outside of the study 
have been observed [19, 20, 22]. 
These studies helped to define a clinical endpoint termed “mild-to-moderate influenza disease” 
(MMID) which included evidence of influenza A virus shedding by RT-PCR and any of a 
number of standardized signs, symptoms, and laboratory findings consistent with clinical 
influenza illness. MMID is assessed using a validated, patient-reported outcome (PRO) measure 
to standardize assessment of influenza (flu) symptoms in clinical research, i.e. Flu-PRO Survey 
Instrument [23]. MMID is defined as influenza A virus shedding plus any one or more of the 
following symptoms or signs: arthralgia, chest tightness, chills, conjunctivitis, nasal congestion, 
sinus congestion, coryza, decreased appetite, diarrhea, cough, dyspnea/shortness of breath, 
fatigue/tiredness, fever (>38.0°C), headache, lymphopenia (<1000 cells/mL), myalgia, nausea, 
oxygen saturation decrease by ≥3% from baseline, rhinorrhea, sore throat, and sweats. The 
license agreement with Leidos Biomedical Research to use the Flu-PRO Survey Instrument 
requires that an additional Validation Diary comprised of nine questions be completed by 
subjects and that data from both instruments be provided to Leidos for further Flu-PRO 

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quantitative validation exercises. Both Flu-PRO Survey Instrument and the Validation Diary are 
in Appendix D. 
One of the key components and gap-filling measures 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]. Human challenge models provide a powerful tool 
to assess prevention and treatment options for influenza in a shorter time frame, with a smaller 
number of subjects and against a known virus strain. Due to the known time of infection, they 
allow for targeted collection of clinical samples, including prior to infection and in short time 
frames 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 judged 
based on current regulatory criteria for current inactivated influenza vaccines. Through 
collaboration with both other academic and industry partners, these models have and will 
continue to serve as an important resource in the influenza research community. 
 
Scientific Rationale 
2.2.1 
Purpose of Study 
This proposed study will establish a H1N1 influenza virus controlled human infection (CHI) 
model at the NIAID Division of Microbiology and Infectious Diseases (DMID)-sponsored 
VTEU clinical study sites. The model has been developed and used by NIAID Division of 
Intramural Research. The proposed study will expand US research capacity to perform influenza 
CHI studies to advance the understanding of influenza pathogenesis and novel vaccine research 
and development.  
The scientific goal of this study is to conduct an influenza CHI study in healthy adults to 
evaluate the association of pre-existing HAI titers and the development of symptomatic RT-
PCR-positive influenza virus infection. The study will build upon the NIH Clinical Center’s 
influenza CHI model with influenza A/California/04/2009 (H1N1)-like virus [19]. A CHI of up 
to 80 healthy subjects will be performed using a total dose of approximately 1 x 107 TCID50, 
which has been shown to induce 60% or higher rates of MMID in individuals who had pre-
challenge HAI titers of ≤1:40 [19]. Additional study objectives will assess clinical 
manifestations, viral shedding and immunological responses to the challenge virus. 

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Table 3: 
Doses Used in Previous Challenge Studies in Humans Since 1985 
 
Reference 
Dose 
(log10 
TCID
50) 
Proportion of Subjects* 
Mean Peak Titer 
Virus Shed (log10 
TCID50 ± SE) 
Mean Duration of 
Shedding (Days ± 
SE) 
Proportion of Subjects with 
Infected† 
Shedding 
Virus 
Systemic 
Febrile 
Illness 
Respiratory 
Illness 
Lower 
Respiratory 
Illness‡ 
H1N1 
A/California/10/78 
Treanor et al. (1987) 
[24] 
Treanor et al. (1990) 
[25] 
Clements et al. (1986) 
[26] 
 
4.5 
4.5 
4.0 
 
8/9 
8/8 
11/15 
 
8/9 
8/8 
11/15 
 
1.98 
2.4±0.4 
3.8±1.6 
 
NR 
3.6±0.6 
6.8±1.5 
 
0/9 
2/8 
5/15 
 
5/9 
3/8 
3/15 
 
NR 
NR 
1/15 
A/Hong Kong/123/77 
Betts et al. (1985) [27] 
 
4.2 
 
15/16 
 
15/16 
 
4.2±2.1 
 
4.7±2.1 
 
8/16 
 
5/16 
 
NR 
A/Kawasaki/9/86 
Youngner et al. (1994) 
[28] 
Hayden et al. (1994) 
[29] 
 
7.0 
7.0 
 
NR 
16/16 
 
10/14 
16/16 
 
2.2±0.4 
2.8±0.5 
 
NR 
NR 
 
3/14 
1/16 
 
6/14 
NR 
 
NR 
2/16 
A/Texas/1/85 
Sears et al. (1988) [30] 
Sears et al. (1987) [31] 
 
6.4 
6.7 
 
26/28 
20/22 
 
26/28 
18/22 
 
3.1±1.9 
3.0±1.9 
 
4.5±2.4 
3.5±1.7 
 
8/28 
5/22 
 
11/28 
9/22 
 
NR 
1/22 
A/Texas/91 
Hayden et al. (1996) 
[32] 
 
5.0 
 
26/26 
 
24/26 
 
3.2±2.1 
 
2 (0-6) § 
 
10/26 
 
21/26 
 
7/26 
H3N2 
A/Bethesda/1/85 
Treanor et al. (1990) 
[25] 
Sears et al. (1988) [30] 
Reuman et al. (1989) 
[33] 
 
4.5 
7.0 
7.1 
 
5/5 
10/10 
18/19 
 
4/5 
10/10 
18/19 
 
2.4±0.6 
4.2±1.4 
3.5±0.5 
 
3.0±1.0 
5.9±1.3 
6.5 
 
2/5 
3/10 
NR 
 
2/5 
1/10 
11/19 
 
NR 
NR 
NR 
A/England/40/83 
Al-Nakib et al. (1986) 
[34] 
 
4.1 
 
31/31 
 
30/31 
 
4.0 
 
NR 
 
NR 
 
17/31 
 
NR 
A/Korea/1/82 
Snyder et al. (1988) [35] 
 
6.0 
 
12/14 
 
12/14 
 
3.4±0.5 
 
5.1±0.3 
 
1/14 
 
5/14 
 
NR 
A/Los Angeles/2/87 
Clements et al. (1992) 
[36] 
 
7.0 
 
22/24 
 
16/24 
 
1.5±0.3 
 
2.6±0.4 
 
4/24 
 
11/24 
 
NR 
A/Washington/897/80 
 
 
 
 
 
 
 
 

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Reference 
Dose 
(log10 
TCID
50) 
Proportion of Subjects* 
Mean Peak Titer 
Virus Shed (log10 
TCID50 ± SE) 
Mean Duration of 
Shedding (Days ± 
SE) 
Proportion of Subjects with 
Infected† 
Shedding 
Virus 
Systemic 
Febrile 
Illness 
Respiratory 
Illness 
Lower 
Respiratory 
Illness‡ 
Clements et al. (1986) 
[26] 
6.0 
25/27 
22/27 
3.8±1.6 
4.3±2.0 
10/27 
7/27 
3/27 
B 
B/Ann Arbor/1/86 
Clements et al. (1990) 
[37] 
 
7.0 
 
10/12 
 
8/12 
 
4±0.7 
 
5.4±1 
 
2/12 
 
5/12 
 
NR 
B/Texas/1/84 
Keitel et al. (1990) [38] 
 
7.1 
 
6/6 
 
6/6 
 
2.2±0.7 
 
5.0±0.6 
 
NR 
 
3/6 
 
NR 
B/Yamagata/16/86 
Treanor et al. (1993) 
[39] 
 
7.0 
 
10/10 
 
7/10 
 
2.3 
 
NR 
 
3/10 
 
7/10 
 
2/10 
Adapted from a previous publication [17]. NR = Not Reported; * Number with finding / number evaluated; † Virus shedding, serum antibody response, or both; ‡ Reported as 
“lower respiratory illness” or “cough”; § Expressed as median (range). 
 

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Table 4: 
Dose Escalation Results with A/Bethesda/MM2/H1N1 challenge strain 
Dose 
Male 
Female 
Total 
Symptoms 
Viral 
Shedding 
Both 
(MMID) 
Four-fold Rise 
in HAI Titer 
103 TCID50 
3 
2 
5 
2 (40%) 
0 
0 
1 (20%) 
104 TCID50 
4 
0 
4 
2 (50%) 
0 
0 
0 
105 TCID50 
2 
3 
5 
4 (80%) 
1 (20%) 
1 (20%) 
1 (20%) 
106 TCID50 
10 
9 
19 
17 (89%) 
9 (47%) 
9 (47%) 
16 (84%) 
107 TCID50 
9 
4 
13 
11 (85%) 
10 (77%) 
9 (69%) 
11 (85%) 
Total 
28 
18 
46 
36 (78%) 
20 (43%) 
19 (41%) 
29 (63%) 
Data are presented as N (%). 
Abbreviations: HAI, hemagglutinin inhibition; MMID, mild-to-moderate influenza disease; TCID50, median tissue culture 
infective dose. 
2.2.2 
Study Population 
The study population will be healthy subjects (male and non-pregnant, non-breastfeeding 
females) between the ages of 18 to 49 inclusive drawn from the general communities of each 
study site. They must be non-habitual smokers, including non-habitual smokers of marijuana or 
e-cigarettes. Subjects must not have medical conditions at high risk for severe complications of 
influenza virus infection, as defined by the US Centers for Disease Control and Prevention 
(CDC) (Appendix C). Up to 80 healthy subjects will undergo A/Bethesda/MM2/H1N1 virus CHI 
and their clinical manifestations, viral shedding, and immunological responses will be 
characterized.  
 
Potential Risks and Benefits 
2.3.1 
Common Symptoms of Influenza Illness 
• Common signs or symptoms of 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 
• Felt dizzy 
• Fever 
• Head congestion 
• Headache 
• Lack of appetite 

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• 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 
• Stomach ache 
• Sweating 
• Teary or watery eyes 
2.3.2 
Potential Risks 
Complications or severe signs or symptoms of influenza illness, which are unlikely to occur in 
healthy subjects who participate in this study, include: 
• Pneumonia 
• Dehydration 
• Difficult breathing 
• Severe bronchitis 
• Myocarditis 
• Myocardial infarction 
• Pericarditis 
• Guillain Barré Syndrome/ Bell’s Palsy 
• Transverse myelitis 
• Encephalitis 
• Sinus infections 
• Ear infections 
• Worsening of chronic health conditions 
• Admission to an intensive care unit (ICU) 
• Hypoxemia/respiratory failure 
• Arrhythmia/cardiac arrest  
• Death  
There may be other risks, discomforts, or side effects that are unknown at this time. 
Risks of Nasal Lavage or Nasopharyngeal Swabs 
Obtaining a nasal lavage fluid or a nasopharyngeal swab can cause discomfort in the nares, a gag 
reflex, epistaxis, watery eyes, or coughing at the time of collection. 
Risks of Blood Draw  

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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 sterile 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 liter of 
blood (i.e. 525 mL). The blood volumes anticipated for specimen collection are listed in 
Appendix B.  
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 -30 to -3), and if deemed medically necessary as 
follow-up of an Adverse Event. Female subjects will 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 -30 to -3), a repeat ECG at study Day 6, and additional ECGs if deemed 
medically necessary.  
Risks of Xofluza (Baloxavir Marboxil) 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%) [40]. 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) [40]. 
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 2019-2020 influenza season vaccination through 60 days post-challenge. If the 
influenza season begins before this time, the subject is at risk of developing influenza illness. 

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Symptoms of influenza are described in 2.3.1 and the potential risks of influenza are described 
above in this Section.  
Risks of Genetic Testing 
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 but 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.  
2.3.3 
Potential Benefits 
There is no direct benefit to the subjects. There is potential benefit to society and future patients 
resulting from insights gained from participation in this study.  

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STUDY OBJECTIVES AND OUTCOME MEASURES 
Refer to Table 1 Study Objectives and Outcome Measures 
 

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STUDY DESIGN 
This is a CHI study of Influenza A/Bethesda/MM2/H1N1 virus to assess clinical response, 
immunological response, and safety. The study population will be healthy subjects (male and 
non-pregnant, non-breastfeeding females) between the ages of 18 to 49, inclusive. Up to 80 
subjects will receive A/Bethesda/MM2/H1N1 virus CHI. Their clinical manifestations, viral 
shedding, and immunological responses will be characterized.  
Influenza A/Bethesda/MM2/H1N1 virus CHI will be performed using a 2 mL dose of 
approximately 5x106 TCID50/mL, which has been shown to induce 60% or higher rates of 
MMID [19]. The primary objective of the study will be to evaluate the association of MMID 
post-challenge and pre-existing virus-specific HAI titers in healthy subjects. MMID is defined as 
the presence of both of the following, assessed through day 8:  
• Viral shedding detected by any approved positive RT-PCR assay from a NP swab, and 
• Any one or more of the following symptoms or signs or laboratory findings, as related to 
the study agent; Arthralgia, Chest tightness, Chills, Conjunctivitis, Nasal congestion, 
Sinus Congestion, Coryza, Decreased appetite, Diarrhea, Dry Cough, Dyspnea/Shortness 
of Breath, Fatigue/Tiredness, Fever (>38.0°C), Headache, Lymphopenia (<1000 
cells/mL), Myalgia, Nausea, Oxygen Saturation Decrease by ≥3% from baseline, 
Productive Cough, Rhinorrhea, Sore Throat, and Sweats. 
During Study Days -30 to -3 (Screening Period), subjects will provide informed consent to 
participate in this study. They will undergo a review of medical history, physical examination by 
a study physician, and screening laboratory tests. During this period, subjects will also have a 
Posterioranterior (PA) and Lateral chest X-ray (CXR), and a baseline 12-lead ECG. 
After enrollment, subjects will be admitted to an inpatient challenge unit (Day -2). Each study 
site will perform CHI on up to 20 subjects unless otherwise directed by DMID to meet the total 
number to be enrolled. This may be done in one or two cohorts. Backup subjects will be admitted 
into the inpatient unit to ensure sufficient number of volunteers undergo CHI, given the potential 
for drop-outs, presence of new exclusionary criteria, and/or pre-challenge evidence of viral 
respiratory infection among subjects. The number of backups 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. A review of eligibility criteria, and 
baseline clinical assessments, safety blood tests, influenza virology, and immunology tests will 
be performed and the subjects will have approximately a 48-hour window to decide if they 
would like to drop out of the study and leave the inpatient unit before CHI. Subjects must have 
no evidence of a respiratory viral infection as determined by multiplex respiratory virus RT-PCR 
assay testing prior to CHI (positive results prior to CHI will exclude the subject from the 
challenge, and additional subjects may serve as replacements). On Day -1, subjects will be 

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trained on the influenza patient related outcomes Flu-PRO Survey Instrument and Validation 
Diary[41].  
On Day 1, eligibility and clinical status will be reviewed. If criteria for proceeding with 
challenge are met, subjects will receive A/Bethesda/MM2/H1N1 CHI. One mL of 
A/Bethesda/MM2/H1N1 will be delivered in each nostril of a recumbent subjects per a study 
SOP. Backups will be discharged from the inpatient unit prior to challenging other subjects if 
they are not needed to replace ineligible subjects or subjects declining to proceed with CHI.  
After CHI, subjects will complete daily self-assessments using the Flu-PRO Survey Instrument 
[41] and Validation Diary through 14 days post-challenge to generate a symptom severity score. 
Subject clinical status will be monitored closely. Safety labs will be performed at scheduled 
times. Subjects will undergo scheduled blood draws, nasal lavages, and nasal swab collections 
for immunology and virologic laboratory testing. Immunologic evaluations will include 
assessment of antibody and cytokine response, T and B cell responses, and systemic 
transcriptional responses. Quantitative and qualitative evaluation of influenza will be done after 
CHI on Days 2 through 8. Subjects will also undergo daily qualitative evaluations for evidence 
of influenza by multiplex respiratory virus RT-PCR assay. Subjects will remain blinded to the 
results of their respiratory virus testing until they meet discharge criteria. An ECG will be 
performed at Day 6 on all subjects, and as clinically indicated at any other time.  
Following CHI, subjects will remain in the inpatient unit for a minimum of seven additional days 
and will be discharged only if 1) they have two consecutive negative influenza tests (that are 
performed on days 7 and 8) for influenza A using a multiplex respiratory virus assay 
(BIOFIRE® FILMARRAY® or Luminex xTAG®), AND 2) have been afebrile (<38.0°C), have 
a room air SpO2 ≥95 and have been clinically and hemodynamically stable for at least 48 hours. 
Discharge evaluation and criteria will be further described in a study SOP. In addition, subjects 
may have residual and/or resolving symptoms of influenza illness. It is expected that most 
subjects will shed the challenge virus for three to five days post exposure.  
If subjects are still shedding virus on Day 8, they will remain in the challenge unit, be offered 
one dose of baloxavir marboxil (a full treatment course), and they will continue to have 
nasopharyngeal (NP) swabs tested daily until they have two negative influenza tests performed 
on consecutive days and meet clinical discharge criteria described above.  
Any subject who develops serious related or unrelated signs or symptoms during inpatient stay 
will receive appropriate and as clinically necessary escalation of care (e.g., cardiologist 
consultation for cardiac symptoms or transfer for evaluation at the Emergency Department). Any 
subjects who experience complications or sequelae due to CHI will be followed until resolution 
and/or stable, and they will be referred for appropriate specialized care as clinically necessary. 
SOPs will be utilized to standardize evaluation and care for select known complications of 
influenza virus infection. All subjects will be followed for approximately 90 days post-challenge. 
Additional follow-up will be performed at in-person Visits 9 (Day 15), 10 (Day 29) and 11 (Day 

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61). Subjects will also undergo scheduled blood draws and, nasal lavages for immunology and 
virologic laboratory testing during this follow up period. A final follow-up by telephone will 
occur on Visit 12 (Day 91). 

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STUDY INTERVENTION/INVESTIGATIONAL 
PRODUCT 
 
Study Product Description 
5.1.1 
Formulation, Packaging, and Labeling 
The study agent in this protocol is a biologic (live virus) and requires special storage and 
handling. The A/Bethesda/MM2/H1N1 human challenge virus was manufactured in certified 
Vero cells under Good Manufacturing Practice (GMP) conditions by Charles River Laboratories 
in Malvern, PA from a reverse-genetics (RG) derived virus seed stock produced by Dr. Matthew 
Memoli and his laboratory team in the Viral Pathogenesis and Evolution Section of the 
Laboratory of Infectious Diseases (NIAID). The rescued RG derived virus was passaged six 
times in certified Vero cells to produce the seed virus.  
The final product was vialed at approximately 5 X 106 TCID50/mL of Influenza A Human 
Challenge Virus (H1N1) in 1X SPG (sucrose, 0.218 M; KH2PO4, 0.0038 M; K2HPO4, 0.0072 M; 
L-glutamic acid, 0.0054 M). Each vial has a fill volume of 1.1 mL. The filled vials were snap 
frozen in a dry ice-ethanol bath and stored at -80°C ± 15°C.  
Each vial will be individually labeled with the product name, description, and caution statement. 
The label was affixed to 2 mL cryogenic vials prior to final fill with A/Bethesda/MM2/H1N1 
Virus [Charles River Laboratories] (Lot #MM001). 
The Challenge virus is on an on-going stability and potency program and the most recent 
potency assessment performed in June 2019 is 5x106 TCID50/mL. CHI will be performed using a 
2 mL dose of approximately 5x106 TCID50/mL virus and will be administered intranasally. 
Intranasal CHI will be carried out using the MAD Nasal sprayer device (Wolfe-Tory Medical, 
Salt Lake City, Utah) attached to a 1 mL Luer lock syringe. One mL of virus will be delivered in 
each nostril of a recumbent subject. 
5.1.2 
Product Storage and Stability 
Influenza A/Bethesda/MM2/H1N1)  
The human challenge virus will be stored at -80°C ± 15°C in single-use vials. Additional details 
of product storage and stability can be found in the MOP. Preparation and dosing of the agent 
must take place within three hours of removing the virus from the freezer. The MAD Nasal 
sprayer device (Wolfe-Tory Medical, Salt Lake City, Utah) will be stored at room temperature 
(15 -30°C). 

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Acquisition/Distribution 
Influenza A/Bethesda/MM2/H1N1 
The human challenge virus will be stored and distributed by the DMID Clinical Material 
Services (CMS) to designated VTEU sites. The study agent will be handled by study personnel 
and administered only in the inpatient unit. The MAD Nasal sprayer device (Wolfe-Tory 
Medical, Salt Lake City, Utah) will be obtained and distributed by the DMID-Clinical Material 
Services (CMS) to designated VTEU sites. 
 
Dosage/Regimen, Preparation, Dispensing and Administration of 
Study Intervention/Investigational Product 
Influenza A/Bethesda/MM2/H1N1  
All steps in preparing the syringes and attaching MAD nasal atomizer to each syringe should be 
performed with careful sterile technique in a biosafety cabinet BSL Level 2 laboratory. 
CHI will be performed using a 2 mL dose of approximately 5x106 TCID50/mL virus and will be 
administered intranasally. Intranasal CHI will be carried out using the MAD Nasal sprayer 
device (Wolfe-Tory Medical, Salt Lake City, Utah) attached to a 1 mL syringe. Two vials will be 
needed per subject. The time that the 2 vials are withdrawn from the freezer will be documented 
in the Accountability Log. Once thawed 1.0 mL of virus will be drawn from each of vial and 
kept on wet ice until administered. Prior to administration the syringe will be attached to the 
MAD nasal atomizer. The total time from freezer to administration will not exceed 3 hours. One 
mL of virus will be delivered in each nostril of a recumbent participant. CHI will be performed 
according to the study MOP. 
The challenge virus is nearly identical to circulating influenza A H1N1pdm09 viruses. This virus 
is susceptible to FDA-approved neuraminidase inhibitors oseltamivir and zanamivir and 
sequencing does not indicate any mutations that confer baloxavir marboxil resistance [42, 43]. 
The 2009 H1N1pdm viruses are resistant to the adamantane class of influenza antivirals [43]. 
Full genomic sequencing was performed on the 2009 H1N1 Challenge Virus (Lot MM#001). A 
total of 4 nucleic acid changes post manufacturing were observed, 1 each in HA, PA, and NA, as 
well as one synonymous change. The three nonsynonymous changes were found as the dominant 
base in a double peak during sequencing along with the original wild-type (wt) nucleotide. There 
is no indication that the 2009 H1N1 Challenge Virus would behave any differently in humans 
than the wt influenza A/California/04/2009 (H1N1) virus, based on the in vitro and in vivo data.  
 

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Table 5:  
Study Intervention Regimen  
Product Name 
Dose 
Route 
Frequency of 
Administration 
A/Bethesda/MM2/H1N1 
2 mL of 
approximately 
5x106 
TCID50/mL 
Intranasal 
Challenge virus will be 
administered once on Day 1 
TCID50, median tissue culture infective dose 
 
Pre-determined Modification of Study Intervention/Investigational 
Product for an Individual Subject 
Not applicable. DMID will not permit modification of study product dose or regimen. 
 
Accountability Procedures for the Study Intervention/Investigational 
Product(s) 
Influenza A/Bethesda/MM2/H1N1 will be stored and shipped from the DMID contractor CMS to 
the Clinical Sites. Once received, Influenza A/Bethesda/MM2/H1N1 will be stored in and 
dispensed by the Investigational Pharmacy.  
After receipt of the study products, the site principal investigator is responsible for study product 
distribution and disposition and has ultimate responsibility for study product accountability. The 
site principal investigator may delegate to the participating site’s research pharmacist 
responsibility for study product accountability. The participating site’s research pharmacist will 
be responsible for maintaining complete records and documentation of study product receipt, 
accountability, dispensation, storage conditions, and final disposition of the study product(s). All 
study product(s), whether administered or not, must be documented on the appropriate study 
product accountability record or dispensing log. The sponsor’s monitoring staff will verify the 
participating site’s study product accountability records and dispensing logs per the site 
monitoring plan. 
Used and unused of study products will be retained until monitored and released for disposition, 
as applicable. This can occur on an ongoing basis for used study products.  
Used study products may be destroyed in accordance with site-specific SOPs following each 
monitoring visit where study product accountability is monitored, and resolution of any 
discrepancies. 

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Final disposition of the unused study products will be determined by DMID and communicated 
to the participating sites by the DMID Clinical Project Manager. 
The FDA requires accounting for the disposition of all investigational products. The Investigator 
is responsible for ensuring that a current record of product disposition is maintained, and product 
is dispensed only at an official study site by authorized personnel as required by applicable 
regulations and guidelines. Records of product disposition, as required by federal law, consist of 
the date received, date administered, quantity administered, and the subject number to whom the 
drug was administered.  
The Investigational Pharmacist will be responsible for maintaining accurate records of the 
shipment and dispensing of the investigational product. The pharmacy records must be available 
for inspection by the DMID monitoring contractors and is subject to inspection by a regulatory 
agency (e.g., FDA) or the Sponsor at any time. An assigned Study Monitor will review the 
pharmacy records. 

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SELECTION OF SUBJECTS AND STUDY 
ENROLLMENT AND WITHDRAWAL 
Subject Inclusion and Exclusion Criteria must be confirmed by a study clinician licensed to make 
medical diagnoses. No exemptions are granted on Subject Inclusion/Exclusion Criteria in DMID-
sponsored studies. Questions about eligibility will be directed toward the DMID Medical Officer. 
 
Eligibility Criteria 
6.1.1 
Subject 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 seven days after challenge, AND until they have no 
virus shedding,1 determined by qualitative RT-PCR for a minimum of two consecutive days 
post-challenge 
1For a minimum of seven days post-challenge (Study Day 8). 
4. Healthy2 males and non-pregnant, non-breastfeeding females3 aged ≥18 and ≤49 years of 
age, inclusive, at enrollment. 
2Good health is defined in inclusion criteria #10. 
3Females subjects of childbearing potential must agree to have a serum pregnancy test at 
screening, a urine pregnancy test before CXR performed (if more than 7 days have passed 
between CXR and serum pregnancy test), and a urine pregnancy test upon admission to the 
inpatient unit prior to CHI, and results must be negative.  
5. Women of childbearing potential4 must agree to use or have practiced true abstinence5 or use 
at least 1 acceptable primary form of contraception6,7  
These criteria are applicable to females in a heterosexual relationship and child-bearing 
potential (i.e., the criteria do not apply to subjects in a same sex relationship).  
4Not of child bearing 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). 

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5True 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). 
6Acceptable 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.  
7Must use at least one acceptable primary form of contraception for at least 30 days prior to 
admission and at least one acceptable primary form of contraception during the remainder 
of the study. 
6. Non-habitual smoker8 of tobacco, e-cigarettes or marijuana.  
8Non-habitual smokers are those who smoke no more than four cigarettes, other tobacco 
products, e-cigarettes or marijuana in a week for more than three months 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 or drug use within the last 30 days and 
agrees to abstain from alcohol and drugs9 for at least one week before admission and 
throughout the inpatient period. 
9 including forms of marijuana not included in criterion 6 
8. Negative drug urine toxicology result on screening (i.e., amphetamines, cocaine, and opiates) 
and on admission to the challenge unit (i.e., amphetamines, cocaine, opiates, and 
cannabinoids). 
9. Agree not to use the listed10 prescription or over-the-counter medications within 7 days prior 
to inpatient stay and through inpatient stay, unless approved by the investigator. 
10Oseltamivir, zanamivir, peramivir, baloxavir marboxil, amantadine (generic) and 
rimantadine (Flumadine and generic), aspirin, intranasal steroids, decongestants, 
antihistamines, and other non-steroidal anti-inflammatory drugs (NSAIDs). 
10. In good health11 and not have clinically significant medical, psychiatric, chronic or 
intermittent health conditions including those listed in Exclusion Criteria (Section 6.1.2). 
11Good 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 

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(Section 6.1.2). 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 6.1.2.) 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 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.  
11. Does not have an ongoing symptomatic condition12 for which subject has had or has ongoing 
medical investigations but has not yet received a diagnosis or treatment plan.  
12e.g., ongoing fatigue without a diagnosis for symptom. 
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 100.0°F.  
13. Eligibility laboratory values (WBC, Absolute Lymphocyte Count, Hgb, PLTs, ALT and Cr) 
are within acceptable parameters. (See Table 6) 
14. Body mass index (BMI) >18.5 and <35 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-investigator13 
13Designated 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 
blood draw. 
17. Negative respiratory virus panel by BIOFIRE® FILMARRAY® respiratory panel by 
bioMérieux or by Luminex xTAG® on Day -2, and Day -1. 
6.1.2 
Subject Exclusion Criteria 
Subjects eligible to participate in this study must not meet any of the following exclusion 
criteria: 
1. Female subject who has a positive pregnancy test on screening or admission, is breastfeeding 
or planning to become pregnant from 30 days prior to challenge through the end of the study. 

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2. Presence of self-reported or medically documented significant medical or psychiatric 
condition(s)14 
14Significant medical or psychiatric conditions include but are not limited to: 
a. Respiratory disease (e.g., chronic obstructive pulmonary disease [COPD], asthma) 
requiring daily medications15 currently or any treatment of respiratory disease 
exacerbations (e.g., asthma exacerbation) in the last 5 years 
15Asthma medications: inhaled, oral, or intravenous (IV) corticosteroids, leukotriene 
modifiers, long and short acting beta agonists, theophylline, ipratropium, biologics.  
b. Presence of any febrile illness or symptoms suggestive of a respiratory infection 
within two weeks prior to CHI. 
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, which is allowed. 
f. An autoimmune disease.  
g. An immunodeficiency of any cause. 
h.  A history of diabetes.  
3. Presence of immunosuppression or any medications that may be associated with impaired 
immune responsiveness16- 
16Including, 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 8.1). 
4. Known allergy or intolerance to treatments for influenza (including but not limited to 
oseltamivir, baloxavir marboxil, acetaminophen). 
5. Known allergy to two or more classes of antibiotics (e.g., penicillins, cephalosporins, 
fluoroquinolones, or glycopeptides).  
6. Known allergy to excipients in the challenge virus inoculum. 

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7. Receipt or planned receipt of any investigational drug/investigational vaccine/licensed 
vaccine within 30 days prior to the date of CHI.  
8. Prior enrollment in any influenza virus challenge study. 
9. Currently enrolled in any investigational study or intends to enroll in such a study within the 
ensuing study period. 
10. Receipt of any influenza vaccine six months prior to challenge or plans to receive influenza 
vaccine within 60 days post-challenge. 
11. History of a previous severe allergic reaction of any kind with generalized urticaria, 
angioedema, or anaphylaxis. 
12. Receipt of blood or blood products during the six months prior to the planned date of 
challenge. 
13. History of blood donation during the two months prior to the planned date of challenge and 
or plans to donate blood for the duration of the study. 
14. Any condition, to include medical and psychiatric conditions, that in the opinion of the 
Investigator, might interfere with the safety of the subject and/or study objectives.  
15. Known close contact with anyone known to have influenza 7 days prior to challenge. 
16. Acute medical condition with new prescription medication use in the last 30 days. 
17. Significant abnormality altering the anatomy of the nose/nasopharynx17 clinically significant 
nasal deviation, or nasal/sinus surgery within 180 days prior to challenge.  
17including significant nasal polyps. 
18. History in the last five years of chronic or frequent intermittent sinusitis. 
19. Recent history (180 days) of epistaxis or anatomic or neurologic abnormality impairing the 
gag reflex or contributing to aspiration. 
Table 6: 
Clinical Laboratory Normal Ranges 
Hematology 
Normal Range 
WBC x103/µL  
3.70 -11.00 
Absolute Lymphocyte count x103/µL  
 > 1.0 
Hgb g/dL (Female) 
> 11.0 
Hgb g/dL (Male) 
> 12.5 
Platelets cell x103/µL EDTA 
163 – 375 

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Platelets K/cu mm Citrate 
125 – 375 
Chemistry 
 
ALT IU/L (Female)  
< 43 
ALT IU/L (Male)  
< 60 
Creatinine mg/dL  
< 1.4 
 
Withdrawal from the Study, Discontinuation of Study Product, or 
Study Termination  
6.2.1 
Discontinuation of the Study Product  
Not applicable 
6.2.2 
Subject Replacement 
Subjects who withdraw, 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, are withdrawn from this study, or are lost to follow-up after signing the 
ICF but before administration of the study product may be replaced.  
6.2.3 
Study Termination  
If the study is prematurely terminated by the Sponsor, any regulatory authority, or the 
investigator for any reason, the investigator will promptly inform the study subjects and assure 
appropriate therapy or follow-up for the subjects, as necessary. The investigator will provide a 
detailed written explanation of the termination to the IRB/IEC. 
6.2.4 
Withdrawal during the Inpatient Period  
Subjects may withdraw voluntarily from participation in the study at any time. 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 inpatient unit before CHI. The investigator should be explicit regarding 
study follow-up (e.g., safety follow-up) that might be carried out once the subject undergoes CHI 
but decides to leave inpatient unit before discharge criteria are met.  
Subjects will be strongly discouraged from withdrawing from the study post-challenge. Any 
subjects who withdraws 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 (Appendix C), and that they will stay sequestered in their homes and avoid 
contact with others until they have two negative tests for influenza virus by qualitative RT-PCR. 

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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 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 inpatient 
unit prior to 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, 11 and phone 
call at Visit 12). 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 local 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).  
6.2.5 
Withdrawal during the Follow-up Period  
Subjects may withdraw voluntarily from participation in the study at any time. Subjects will be 
strongly discouraged from withdrawing from the study during the follow-up period. For the 
purposes of safety and serological testing, the subject will also be asked to come for all follow-
up visits. The subject will be encouraged to return for follow-up visits per outpatient schedule 
(i.e., Visits 9, 10, 11 and phone call at Visit 12). The investigator will inform the subject that 
already collected data will be retained and analyzed even if the subject withdraws from this 
study. As discussed in 5.2.6, 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).  
6.2.6 
Reasons for withdrawal might include, but are not limited to the following: 
• Subject no longer meets eligibility criteria (prior to challenge) 
• Subject meets individual halting criteria (Section 9.6.1) 
• Subject becomes noncompliant 
• 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 lost to follow-up 
• Subject becomes pregnant, if applicable 

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• Determined by a physician’s discretion to require additional therapy not indicated in the 
protocol to ensure subject’s health and well-being (or treatment failure, if applicable) 
• If the subject consents, every attempt will be made to follow all adverse events (AEs) 
through resolution or stabilization. 
• Clinical samples/data may be used for study analyses despite withdrawing early from the 
study. 

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STUDY PROCEDURES 
 
Screening Period  
7.1.1 
Study Visit 00A (Day -30 to -3) 
The screening sequence is designed to minimize subjects study visits and needle sticks, while 
identifying exclusionary criteria early and before more time-intensive and costly tests are 
performed and before the inpatient challenge stay begins. The screening may occur over more 
than one day and may encompass more than one visit. 
• Subjects will be provided with a description of the study (purpose and study procedures) 
and asked to read and sign the ICF. The ICF will be signed prior to performing any study 
procedure. 
• Demographic information will be obtained by interview of subjects. 
• Eligibility criteria will be reviewed with subjects. 
• Complete medical history will be obtained. 
• 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 CHI.  
• Documentation of receipt of 2018/2019 influenza vaccine 
• Subject receipt of non-seasonal influenza vaccine, including those that are experimental,  
product type (inactivated or live attenuated), vaccine virus strains (e.g., A/H3N2v, A/H5, 
A/H7, A/H9, etc.) and approximate date of vaccination will be recorded on the 
appropriate DCF, if known. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature) will be obtained.  
• Physical exam 
• A serum pregnancy test will be performed on all female subject of childbearing potential 
and result must be negative. 
• Height and weight will be collected. 
• Blood for safety labs (approximately 15 mL), including HIV, hepatitis B, and hepatitis C 
testing, and testing for white blood cells (WBCs), absolute lymphocyte count, 
hemoglobin, platelets, alanine transaminase (ALT) and creatinine (Cr) will be sent to a 
central lab for evaluation and uploaded and entered into Advantage eClinicalSM. These 

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values must be confirmed to meet the eligibility criteria as outlined in the Inclusion 
Criteria (Section 5.1.1) prior to admission to the inpatient unit. 
• Urine toxicology test (amphetamines, cocaine, and opiates, ) will be performed at the 
Central Lab. 
• 12-lead ECG will be performed and confirmed to be within normal limits or not clinically 
significant as determined by the PI or designated clinician licensed to make medical 
diagnoses and listed on Form FDA 1572 prior to admission to the inpatient unit. 
• CXR will be performed and confirmed to be within normal limits or not clinically 
significant as determined by the PI or designated clinician licensed to make medical 
diagnoses and listed on Form FDA 1572 prior to admission to the inpatient unit. Among 
female subjects 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. 
• Eligibility criteria will be reviewed with subjects in the week prior to admission to the 
inpatient unit to confirm no new exclusion criteria have been met since the review at 
initial enrollment. 
 
Inpatient Period 
7.2.1 
Study Visit 00B (Day -2) 
• The subjects will be admitted to inpatient unit.  
• Eligibility criteria will be reviewed with subjects. 
• Subject’s willingness to participate will be reconfirmed and documented in the study 
records prior to performing any further study procedures. The subject will have 
approximately a 48-hour window to decide if they would like to drop out of the study and 
leave the inpatient unit before virus is administered. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature) will be obtained. 
• Weight will be collected for calculation of BMI (with height collected on Day -30 to -3).  
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• Medical history will be obtained. 
• All concomitant medications will be reviewed with subjects for accuracy and 
completeness. Any new concomitant medications taken since the screening visit will be 
reviewed with subjects and assessed for continued eligibility.  

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• A urine pregnancy test will be performed locally on all female subjects of childbearing 
potential and a positive result will exclude the subject from receipt of study product.  
• Blood (approximately 20 mL) for antibody and cytokine assays  
• From at least a subset of participants: Blood (approximately 8 mL) for innate, T and B 
phenotyping (cryopreserved PBMC) 
• Blood (approximately 48 mL) for T and B immunology (cryopreserved PMBC) 
• Blood (approximately 2.5 mL) for transcriptomics 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed to assure that no subject has a respiratory viral infection (positive results will 
exclude the subject from enrollment, and additional subjects may be admitted as 
replacements if any subjects are excluded prior to challenge)..  
• NLF will be collected for baseline immunology assays. 
• Urine toxicology test (amphetamines, cocaine, opiates, and cannabinoids) will be 
performed locally. Results must be negative for subject to continue participation in the 
study.  
7.2.2 
Study Visit 00C (Day -1) 
• The subjects will remain in the inpatient unit.  
• Eligibility criteria will be reviewed with subjects. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature) will be obtained. 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• Any new concomitant medications taken since the screening visit will be reviewed with 
subjects and assessed for continued eligibility.  
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed to assure that no subject has a respiratory viral infection (positive results will 
exclude the subject from enrollment, and additional subjects may be admitted as 
replacements if any subjects are excluded prior to challenge). Quantitative RT-PCR and 
culture for influenza will be performed on the same NP swab specimen.  
• NLF will be collected for baseline immunology assays if not collected at Day -2. 

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• Subjects will be trained on Flu-PRO Survey Instrument and Validation Diary. 
7.2.3 
Study Visit 01 (Day 1) Before Challenge 
• Subjects will remain in the inpatient unit. 
• Eligibility criteria will be reviewed with subjects. 
• Informed consent will be reviewed with subjects. 
• Baseline local and systemic symptoms will be assessed using daily Flu-PRO Survey 
Instrument and Validation Diary prior to challenge. All subjects will perform self-
assessment using the Flu-PRO Survey Instrument Validation Diary daily during the 
inpatient stay to generate a symptom severity.  
• Vital signs (including SpO2*, HR, RR, and BP and oral temperature) will be obtained. 
(*For data analysis, this measurement of SpO2 will be considered baseline.)  
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• Any new concomitant medications taken since the screening visit will be reviewed with 
subjects and assessed for continued eligibility. 
• Medical history will be obtained. 
• Blood (approximately 2.5 mL) for transcriptomics. 
• Subjects will receive study influenza challenge virus. The challenge virus 
(A/Bethesda/MM2/H1N1) will be administered intranasally at a dose of 2 mL of 
approximately 5x106 TCID50. Intranasal challenge will be carried out using the MAD 
Nasal sprayer device (Wolfe-Tory Medical, Salt Lake City, Utah) attached to a 1 mL 
syringe. One mL of virus will be delivered in each nostril of a recumbent subject. 
7.2.4 
Study Visit 01 (Day 1) Post-challenge 
• Subjects will remain in the inpatient unit. 
• Post-challenge vital signs (including SpO2, HR, RR, BP and oral temperature will be 
obtained approximately within each 8-hour period, or three times per day while subject is 
awake, and as clinically indicated). 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary at approximately 5 PM (see MOP for details) 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 

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• All AEs and SAEs, will be recorded on the appropriate DCF.  
7.2.5 
Study Visit 02 (Day 2) 
• Subjects will remain in the inpatient unit. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary at approximately 5 PM (see MOP for details) 
• All concomitant medication will be reviewed. 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• NLF will be collected for immunology assays. 
• Blood for safety labs (approximately 10 mL), including white blood cells (WBCs), 
absolute lymphocyte count, hemoglobin, platelets, and alanine transaminase (ALT), 
creatinine (Cr) will be sent to a central lab for evaluation and uploaded and entered into 
Advantage eClinicalSM.  
• Blood (approximately 5 mL) for cytokine assays  
• Blood (approximately 2.5 mL) for transcriptomics 
• All AEs and SAEs, will be recorded on the appropriate DCF.  
7.2.6 
Study Visit 03 (Day 3) 
• Subjects will remain in inpatient unit. 
• All concomitant medication will be reviewed. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary at approximately 5 PM (see MOP for details) 

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• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• All AEs and SAEs will be recorded on the appropriate DCF.  
7.2.7 
Study Visit 04 (Day 4) 
• Subjects will remain in inpatient unit. 
• All concomitant medication will be reviewed. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary at approximately 5 PM (see MOP for details) 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• Blood for safety labs (approximately 10 mL), including white blood cells (WBCs), 
absolute lymphocyte count, hemoglobin, platelets, and alanine transaminase (ALT), 
creatinine (Cr) will be sent to a central lab for evaluation and uploaded and entered into 
Advantage eClinicalSM.  
• Blood (approximately 5 mL) for cytokine assays  
• From at least a subset of participants: Blood (approximately 15 mL) for ASC (fresh 
PBMC) 
• From at least a subset of participants: Blood (approximately 8 mL) for innate, T and B 
cell phenotyping (cryopreserved PBMC) 
• Blood (approximately 32 mL) for T and B cell immunology (cryopreserved PMBC) 
• Blood (approximately 2.5 mL) for transcriptomics 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• NLF will be collected for immunology assays. 

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• All AEs and SAEs will be recorded on the appropriate DCF.  
7.2.8 
Study Visit 05 (Day 5) 
• Subjects will remain in inpatient unit. 
• All concomitant medication will be reviewed. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary at approximately 5 PM (see MOP for details) 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• All AEs and SAEs will be recorded on the appropriate DCF.  
7.2.9 
Study Visit 06 (Day 6) 
• Subjects will remain in inpatient unit. 
• All concomitant medication will be reviewed. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary at approximately 5 PM (see MOP for details) 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• NLF will be collected for immunology assays. 
• Blood (approximately 5 mL) for cytokine assays  

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• From at least a subset of participants: Blood (approximately 15 mL) for ASC (fresh 
PBMC) 
• From at least a subset of participants: Blood (approximately 8 mL) for innate, T and B 
phenotyping (cryopreserved PBMC) 
• Blood (approximately 32 mL) for T and B immunology (cryopreserved PMBC) 
• ECG will be performed. 
• All AEs and SAEs will be recorded on the appropriate DCF.  
7.2.10 
Study Visit 07 (Day 7) 
• Subjects will remain in inpatient unit. 
• All concomitant medication will be reviewed. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary at approximately 5 PM (see MOP for details) 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• All AEs and SAEs, will be recorded on the appropriate DCF.  
7.2.11 
Study Visit 08 (Day 8) 
• Subjects begin the day in the inpatient unit and will remain if discharge criteria are not 
met. 
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All concomitant medication will be reviewed. 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary. On this day, subjects may complete the self-assessment earlier than 
previous days if they are being discharged to home. New or changes in symptoms 

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between the time of self-assessment on this day and 5 PM (the usual time for self-
assessment) will be captured in the Flu-PRO Survey Instrument and Validation Diary on 
Day 9 post-challenge (see MOP for details). 
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• NLF will be collected for immunology assays. 
• Blood for safety labs (approximately 10 mL), including white blood cells (WBCs), 
absolute lymphocyte count, hemoglobin, platelets, and alanine transaminase (ALT), 
creatinine (Cr) will be sent to a central lab for evaluation and uploaded and entered into 
Advantage eClinicalSM.  
• Blood (approximately 20 mL) for antibody and cytokine assays  
• From at least a subset of participants: Blood (approximately 15 mL) for ASC (fresh 
PBMC) 
• From at least a subset of participants: Blood (approximately 8 mL) for innate, T and B 
phenotyping (cryopreserved PBMC) 
• Blood (approximately 32 mL) for T and B immunology (cryopreserved PMBC) 
• Blood (approximately 2.5 mL) for transcriptomics 
• Subjects will 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):  
o Have two consecutive qualitative RT-PCR tests on NP swabs (that are performed on 
Day 7 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®).  
o Are afebrile (< 100.4°F/38.0°C). 
o SpO2≥95% on room air 
o Show no moderate or severe influenza signs or symptoms by clinical evaluation. 
o Are clinically and hemodynamically stable for at least 48 hours (per the evaluation of 
the study physician). 

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• A single dose of baloxavir marboxil will be offered to all subjects that have not had two 
consecutive qualitative RT-PCR tests negative for influenza A. 
• All AEs and SAEs, will be recorded on the appropriate DCF.  
7.2.11.1 
Unscheduled Inpatient Days After Day 8 
• Subjects who are still shedding on Day 8 will remain in the inpatient unit and will 
continue to be tested until they have qualitative NP swabs that are negative for influenza 
A on two consecutive days  
• Vital signs (including SpO2, HR, RR, BP and oral temperature will be obtained 
approximately within each 8-hour period, or three times per day while subject is awake, 
and as clinically indicated). 
• All concomitant medication will be reviewed. 
• All subjects will perform self-assessment using the Flu-PRO Survey Instrument and 
Validation Diary once daily at approximately 5 PM through day 14 post challenge (see 
MOP for details).  
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• A qualitative multiplex respiratory virus assay on NP swab specimen (BIOFIRE® 
FILMARRAY® respiratory panel by bioMérieux or Luminex xTAG®) will be 
performed.  
• Subjects will 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):  
o Have two consecutive qualitative RT-PCR tests on NP swabs (that are performed on 
Day 7 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®).  
o Are afebrile (< 100.4°F/38.0°C). 
o SpO2 ≥95% on room air 
o Show no moderate or severe influenza signs or symptoms by clinical evaluation. 
o Are clinically and hemodynamically stable for at least 48 hours (per the evaluation of 
the study physician). 
o All AEs and SAEs, will be recorded on the appropriate DCF.  

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• A single dose of baloxavir marboxil will be offered to all subjects that have not 
previously received a dose and have not had two consecutive qualitative RT-PCR tests 
negative for influenza A. 
 
Follow-up Period 
7.3.1.1 
Post-discharge (Approximately Day 9 to Day 14) 
• After discharge and through Day 14 post-challenge, subjects continue to perform daily 
self-assessment using the Flu-PRO Survey Instrument and Validation Diary at 
approximately 5 PM through day 14 post challenge.  
7.3.1.2 
Study Visit 09 (Day 15 ± 3 Days)3Days 
• Subjects will visit the study clinic. 
• Study personnel will collect the Flu-PRO Survey Instrument and Validation Diary from 
subjects including symptoms experienced from Days 9 to 14 post challenge.  
• All concomitant medications will be reviewed. 
• Vital signs including HR, RR, BP and oral temperature will be obtained.  
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• Blood (approximately 5 mL) for cytokine assays  
• From at least a subset of participants: Blood (approximately 8 mL) for innate, T and B 
phenotyping (cryopreserved PBMC) 
• Blood (approximately 40 mL) for T and B immunology (cryopreserved PMBC) 
• Blood (approximately 2.5 mL) for transcriptomics 
• NLF for immunology assays. 
• All AEs and SAEs will be recorded on the appropriate DCF.  
7.3.1.3 
Study Visit 10 (Day 29 ± 3 Days) 
• Subjects will visit the study clinic. 
• All concomitant medication will be reviewed. 
• Vital signs including HR, RR, BP and oral temperature will be obtained.  

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• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• Blood (approximately 20 mL) for antibody assays  
• From at least a subset of participants: Blood (approximately 8 mL) for innate, T and B 
phenotyping (cryopreserved PBMC) 
• Blood (approximately 40mL) for T and B immunology (cryopreserved PMBC) 
• NLF for immunology assays. 
• All unsolicited AEs and SAEs will be recorded on the appropriate DCF.  
7.3.1.4 
Study Visit 11 (Day 61 ± 5 Days) 
• Subjects will visit the study clinic. 
• All concomitant medication will be reviewed. 
• Vital signs including HR, RR, BP and oral temperature will be obtained.  
• Targeted physical exam (lung, heart, oral/pharyngeal, and neck exams) will be 
performed; additional clinical evaluation will be performed if clinically indicated. 
• Blood (approximately 20 mL) for antibody assays  
• From at least a subset of participants: Blood (approximately 8 mL) for innate, T and B 
phenotyping (cryopreserved PBMC) 
• Blood (40mL) for T and B immunology (cryopreserved PMBC) 
• NLF for immunology assays. 
• All SAEs will be recorded on the appropriate DCF.  
7.3.2 
Final Study Visit -- Telephone Follow-up (Day 91 ± 7 Days) 
• A phone call for safety follow up will be performed.  
• All SAEs will be recorded on the appropriate DCF.  
 
Early Termination Visit 
Any of the following activities may be performed at the Early Termination Visit on subjects who 
withdraw or are withdrawn or terminated from this study. 
• All concomitant medications will be recorded on the appropriate DCF.  

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• Subject self-assessment using the Flu-PRO Survey Instrument and Validation Diary will 
be reviewed (if within 14 days post-challenge). 
• Vital signs, including oral temperature, pulse, RR, and BP may be obtained. 
• A targeted physical examination, may be performed 
• Blood or nasal specimens for safety or research purposes may be collected depending on 
when this visit occurs. 
• All AE/SAEs will be recorded on the appropriate DCF per Section 9. 
 
Unscheduled Study Visits  
An Unscheduled Visit may occur at any time during this study. Any of the following activities 
may be performed: 
• All concomitant medications will be recorded on the appropriate DCF.  
• Subject self-assessment using the Flu-PRO Survey Instrument and Validation Diary will 
be reviewed (if within 14 days post-challenge). 
• Vital signs, including oral temperature, pulse, RR, and BP may be obtained if indicated. 
• A targeted physical examination, may be performed 
• Blood or nasal specimens for safety or research purposes may be collected depending on 
when this visit occurs. 
• All AE/SAEs will be recorded on the appropriate DCF. 
 
 
Protocol Deviations 
A protocol deviation is any noncompliance with the study protocol, GCP, or protocol-specific 
MOP requirements. The noncompliance may be either on the part of the subject, the site PI, or 
the site personnel. As a result of deviations, corrective actions are to be developed by the site and 
implemented promptly. 
These practices are consistent with ICH E6: 
4.5 
Compliance with Protocol, Sections 4.5.1, 4.5.2, and 4.5.3 
5.1 
Quality Assurance and Quality Control, Section 5.1.1 
5.20 
Noncompliance, Sections 5.20.1, and 5.20.2. 

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It is the responsibility of the site PI and personnel to use continuous vigilance to identify and 
report deviations within five working days of identification of the protocol deviation, or within 
five working days of the scheduled protocol-required activity. All deviations must be promptly 
reported to DMID per the data coordinating center (DCC) protocol deviation reporting 
procedures. 
All protocol deviations, as defined above, must be addressed in study subject 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. Protocol deviations must be sent to the local 
IRB/IEC per their guidelines. The site PI and personnel are responsible for knowing and 
adhering to their IRB requirements. 

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DESCRIPTION OF CLINICAL AND LABORATORY 
EVALUATIONS 
 
Clinical Evaluations 
Specifics of clinical and laboratory evaluations are in Table 11(Appendix A). 
Screening for eligibility will occur from Days -30 to -3 to determine health status of potential 
subjects. This will include the following clinical evaluations: 
• A complete medical and medication history 
• Review of concomitant medications 
• Review of receipt of 2018/2019 influenza vaccine 
• Subject receipt of non-seasonal influenza vaccine, including those that are experimental, 
product type, vaccine virus strains and approximate date of vaccination (Section 7.1.1) 
• Height and weight will be collected. 
• Vital signs (including SpO2, RR, HR, BP, oral temperature)  
• Physical exam 
• Collection of blood for screening and baseline safety laboratory testing 
• Urine for toxicology 
• ECG 
• Serum pregnancy test 
• Baseline 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.) 
After enrollment, the subjects will be admitted to the inpatient unit (Day -2). Baseline 
assessments and testing will be performed after admission and the subjects will have 
approximately a 48-hour window to decide if they would like to drop out of the study and leave 
the inpatient unit before challenge virus is administered. The following clinical evaluations will 
be performed in the 48 hours before challenge (Days -2 and Day -1): 
• Vital signs (including SpO2, RR, HR, BP, oral temperature)  
• Targeted physical exam (including lung, heart, oral/pharyngeal, and neck exams)  
• Weight will be collected. 
• Complete medical and medication history  
• Review of concomitant medications 
• Urine for toxicology; results must be negative for subject to be eligible for challenge on 
day 1. 
• A urine pregnancy test will be performed locally on all female subjects of childbearing 
potential and a positive result will exclude the subject from receipt of study product.  

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• NLF will be collected for immunology assays on Day -2. If not collected on Day-2, NLF 
may be collected on Day -1, but not both days.  
• NP swab will be collected for testing for qualitative evaluation of viral respiratory 
infection 
On Day 1, the challenge virus will be administered. Baseline evaluations before CHI will include 
the following: 
• Local and systemic symptom assessment using Flu-PRO Survey Instrument and 
Validation Diary 
• Targeted physical exam (including lung, heart, oral/pharyngeal, and neck exams)  
• Complete medical and medication history 
• Review of concomitant medications 
• Vital signs including SpO2, HR, RR, BP, oral temperature 
Subjects will be followed for a minimum of 7 days post-challenge (through Study Day 8) in the 
inpatient unit. The following clinical evaluations will be performed during this post-challenge, 
inpatient period: 
• Local and systemic symptom assessment using Flu-PRO Survey Instrument and 
Validation Diary 
• Targeted physical exam including height, weight, clinical evaluation (lung, heart, 
oral/pharyngeal, and neck exams) 
• Complete medical and medication history 
• Review of concomitant medications 
• Vital signs including SpO2, HR, RR, BP, oral temperature 
• ECG will be performed 
• NP Swab will be collected for testing for qualitative evaluation of viral respiratory 
infection 
• Collection of blood for clinical laboratory tests  
 
8.1.1 
Research Procedures  
• Flu-PRO Survey Instrument and Validation Diary to generate a symptom severity score 
• Collection of blood for antibody and cytokine assays  
• Collection of blood for transcriptomics  
• From at least a subset of participants: Collection of blood for ASC (fresh PBMC) 
• From at least a subset of participants: Collection of blood for innate, T and B 
phenotyping (cryopreserved PBMC) 
• Collection of blood for T and B immunology (cryopreserved PMBC) 
• Collection of NLF for immunology assays 
• Collection of NP swab for viral shedding and viral load assessment.  
 

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8.1.2 
Assessment of Concomitant Medications/Treatments other than Study Product 
Concomitant medications will be reviewed at every study visit through the end of the study. 
Women of child bearing 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 2018/2019 influenza vaccine season 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 
vaccination (Section 7.1.1) 
The following prescription or over-the-counter medications cannot be used within 7 days prior to 
admission to the inpatient unit and through the inpatient stay, 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 CHI.  
Subjects should not have received influenza vaccine six months prior to challenge and must not 
plan to receive an influenza vaccine 60 days post challenge. 
Subjects should not have received blood or blood products during the six months prior to the 
planned date of CHI. 
8.1.3 
Assessment of Subject Compliance with Study Intervention/Investigational 
Product/Investigational Device  
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 and 
entered into the eCRF. 

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8.1.4 
Non-Research Standard of Care  
Baloxavir marboxil (Xofluza)may be given to each subject who has not had two consecutive 
days of negative qualitative RT-PCR tests for influenza virus on Study Day 8. Baloxavir 
marboxil is a polymerase acidic endonuclease inhibitor indicated for the treatment of acute 
uncomplicated influenza in patients 12 years of age and older [40]. Baloxavir marboxil is 
licensed as a single-dose drug.  
8.1.5 
Non-Research Standard of Care  
Subjects who continue to shed virus beyond Day 8 will undergo routine daily evaluations as 
described in Sections 6 and 7. If the subject is considered by the study physician to be clinically 
well, no additional changes to study procedures will be made. The study physician may 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. 
 
Laboratory Evaluations 
8.2.1 
Clinical Laboratory Evaluations 
• Hematology at Screening: white blood cells (WBCs), absolute lymphocyte count, 
hemoglobin, and platelets (if platelets in the EDTA tube are clumped and the result is not 
available, platelets may be repeated in a citrate tube) 
• Hematology at Follow-up (at Days 2, 4, and 8): white blood cells (WBCs), absolute 
lymphocyte count, hemoglobin, and platelets (if platelets in the EDTA tube are clumped 
and the result is not available, platelets may be repeated in a citrate tube) 
• Biochemistry at Screening: alanine transaminase (ALT) and creatinine (Cr) 
• Biochemistry at Follow-up (at Days 2, 4, and 8): alanine transaminase (ALT) and 
creatinine (Cr) 
• Hepatitis B and Hepatitis C, HIV at Screening. 
• A urine toxicology test (amphetamines, cocaine, and opiates,) at screening (to be 
performed at the central laboratory) and on the day of admission to the inpatient unit 
(amphetamines, cocaine, opiates, and cannabinoids) (to be performed locally). 
• Serum HCG pregnancy test (for female subjects of childbearing potential) at screening to 
be performed at the Central Laboratory 
• 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 the day of admission to the inpatient unit, to be performed locally. 

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• Qualitative multiplex respiratory virus assay (BIOFIRE® FILMARRAY® respiratory 
panel by bioMérieux or Luminex xTAG®) on NP swab 
8.2.2 
Research Assays  
• HAI, MN, NAI, anti-HA-stalk antibody titers from serum 
• sIgA titers from NLF 
• Human leukocyte antigen (HLA) class I and II alleles from blood 
• Systemic cytokines and chemokines from serum 
• Mucosal cytokines and chemokines from NLF 
• Transcriptional responses from whole blood 
• Plasmablasts (ASC) in circulation (from PBMC) 
• Influenza-specific B and T cell immune responses from PBMCs 
• Immunophenotyping of innate, B and T cell subsets in NLF  
• Quantitative RT-PCR for influenza from NP swab specimen. 
8.2.2.1 
Laboratory Specimen Preparation, Handling, and Storage 
Instructions for specimen preparation, handling, and storage are included in the central (clinical) 
laboratory manual and protocol-specific MOP as appropriate. 
8.2.2.2 
Laboratory Specimen Shipping 
Specimen shipment 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 central (clinical) laboratory manual and 
protocol-specific MOP as appropriate. 
Specimens for clinical screening and safety laboratory evaluations will be transported from the 
participating sites to the central (clinical) laboratory. Urine toxicology on the day of admission 
will be performed at the local laboratory. 
Specimens for HAI, MN, NAI and anti-HA-stalk antibody assays, transcriptomics and 
Plasma/PBMC-based and NLF-based assays will be shipped from the participating sites to 
DMID Clinical Materials Services (CMS) and then provided by the CMS to the appropriate 
laboratory in a blinded manner. 
Further instructions for specimen shipment are included in the central (clinical) laboratory 
manual and protocol-specific MOP, as appropriate. 

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ASSESSMENT OF SAFETY 
 
Assessing and Recording Safety Parameters 
Signs, symptoms, laboratory findings that are part of MMID from the time of CHI through 14 
days post-challenge will not be considered adverse events. These signs, symptoms and laboratory 
findings (mild and moderate) which define MMID include the following: arthralgia, chest 
tightness, chills, conjunctivitis, nasal congestion, sinus congestion, coryza, decreased appetite, 
diarrhea, cough, dyspnea/shortness of breath, fatigue/tiredness, fever (>38.0°C), headache, 
lymphopenia (<1000 cells/mL), myalgia, nausea, oxygen saturation decrease by ≥3% from 
baseline, rhinorrhea, sore throat, and sweats.  
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 3 months post-challenge). 
2. Clinical safety laboratory adverse events occurring from the time of CHI through study 
Day 8. Parameters to be evaluated include: white blood cells (WBCs), absolute 
lymphocyte count (except as part of MMID), hemoglobin, platelets, and alanine 
transaminase (ALT), and creatinine (Cr). 
3. Adverse Events – non-serious adverse events occurring from the time of CHI through 
approximately 30 days post-challenge. See below for how adverse events are defined. 
 
9.1.1 
Adverse Events (AEs) 
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. 
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 MMID will be collected from the time of CHI through 14 
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 MMID will 

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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. 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 6.1) 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. 
9.1.1.1 
Adverse Events Grading 
AEs must be assessed for severity and relationship to study product (see definitions below). 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 DCF and entered into 
the eCRF. 
Severity of Event: AEs will be assessed by a licensed study physician listed on the Form FDA 
1572 as the site PI or sub-investigator using a protocol-defined grading system (described below 
in this Section). For events not included in the protocol-defined grading system, the following 
guidelines will be used to quantify severity: 
• 
Mild (Grade 1): Events require minimal or no treatment and do not interfere with the 
subject’s daily activities.  
• 
Moderate (Grade 2): Events result in a low level of inconvenience or concern with 
therapeutic measures. Moderate events may cause some interference with functioning and 
daily activities.  
• Severe (Grade 3): Events interrupt the subject’s daily activities and may require systemic 
drug therapy or other treatment. Severe events are usually incapacitating. 
Relationship to Study Product: The licensed study physician’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 CHI, the study product must always 
be suspect. To help assess, the following guidelines are used: 

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• Related – There is a reasonable possibility that the study product caused the AE. Reasonable 
possibility means that there is evidence to suggest a causal relationship between the study 
product and the AE. 
• Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event. 
Adverse Events Grading: All AEs and laboratory findings (that are not part of MMID) will be 
graded for severity and assessed for relationship to study product (see definitions). 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.  
Oral temperature will be graded as follows: 
Table 7: 
Severity Grading for Oral Temperature 
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, respiratory rate, and SpO2 will be graded as follows: 
Table 8: 
Pulse, BP, RR and SpO2 Adverse Event Grading 
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 

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Physiologic Parameter 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
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 
Clinical safety laboratory values# will be graded as follows: 
Table 9: 
Clinical Safety and Laboratory AEs Grading 
Hematology 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
WBC x103/µL (Decrease) 
2.50 – 3.69 
1.50 – 2.49 
<1.50 
WBC x103/µL (Increase) 
11.01 – 15.09 
15.10 – 20.00 
>20.00 
Absolute Lymphocyte count x 
103/µL (Decrease) 
0.75 - 0.99 
0.50 - 0.74 
< 0.50  
Hgb g/dL (Decrease) (Female) 
10.1 – 10.9 
8.5 – 10.0 
<8.5 
Hgb g/dL (Decrease) (Male) 
11.0 – 12.4 
9.5 – 10.9 
<9.5 
Platelets cell x 103/µL (Decrease) 
EDTA 
140 – 162 
100 – 139 
<100 
Platelets x 103/µL (Increase) 
EDTA 
376 – 550 
551– 750 
>750 
Platelets K/cu mm (Decrease) 
Citrate 
115-124 
100-114 
<100 
Platelets K/cu mm (Increase) 
Citrate 
376 - 550 
551-750 
>750 
Chemistry 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
ALT IU/L (Increase) (Female)  
44 – 100  
101 – 200 
>200 
ALT IU/L (Increase) (Male)  
61- 138 
139 – 275 
>275 

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Creatinine mg/dL (Increase)  
1.5 – 1.8 
1.9 – 2.2 
> 2.2 
 
9.1.2 
Serious Adverse Events (SAEs) 
An AE or suspected adverse reaction is considered a SAE if, in the view of either the site 
principal investigator or Sponsor, it results in any of the following outcomes:  
• 
Death, 
• 
a life-threatening adverse event1,  
• 
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 
hospitalizations 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 ER or at home, blood 
dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of 
drug dependency or drug abuse. 
1 Life-threatening adverse event. An AE is considered “life-threatening” if, in the view of either 
the site principal investigator or Sponsor, its occurrence places the patient or subject at 
immediate risk of death. It does not include an AE that, had it occurred in a more severe form, 
might have caused death. 
SAEs will be: 
• 
Assessed for severity and relationship to study product and alternate etiology (if not related 
to study product) by a licensed study physician 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. 
• 
Followed through resolution by a licensed study physician (for investigational new drug 
application [IND] studies, a physician 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. 

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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 6.1and the 
protocol-specific MOP. 
 
Specification of Safety Parameters 
Safety will be assessed by the frequency and severity of: 
Solicited Symptoms: Influenza symptoms elicited by the Flu-PRO Survey Instrument and 
Validation Diary (Appendix D).  
Unsolicited Events: Unsolicited events are any non-serious adverse events occurring from the 
time of CHI through approximately 30 days post-challenge. 
 
Reporting Procedures 
9.4.1 
Reporting Serious Adverse Events 
Any AE that meets a protocol-defined serious criterion must be submitted immediately (within 
24 hours of site awareness) on an SAE form to the DMID Pharmacovigilance Group, at the 
following address: 
 
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX Number: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
In addition to the SAE form, select SAE data fields must also be entered into the DCC system. 
Please see the protocol-specific MOP for details regarding this procedure.  

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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 and DMID 
Clinical Project Manager will be notified of the SAE by 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 designated clinician 
licensed to make medical diagnoses and listed on Form FDA 1572 becomes aware of an SAE 
that is suspected to be related to study product, the site principal investigator or designated 
clinician licensed to make medical diagnoses and listed on Form FDA 1572 will report the event 
to the DMID Pharmacovigilance Group. 
9.4.2 
Regulatory Reporting for Studies Conducted Under DMID Sponsored- IND 
Following notification from the site PI or designated clinician licensed to make medical 
diagnoses and listed on Form FDA 15721572, DMID, as the IND Sponsor, will report any 
suspected unexpected SAE. DMID will report an AE as a suspected unexpected AE only if there 
is evidence to suggest a causal relationship between the study intervention and the AE. DMID 
will submit an IND safety report to the FDA and will notify all participating site PIs (i.e., all PIs 
to whom the Sponsor is providing drug under its IND(s) or under any PI’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 seven 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.  
All SAEs designated as “not related” to study product(s), will be reported to the FDA at least 
annually in a summary format. 
9.4.3 
Reporting of Pregnancy 
Pregnancies occurring in study subjects will be reported via Advantage electronic data capture 
system (Advantage eClinical) on the Pregnancy Report form. No further study product will be 
administered to pregnant subjects, but 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 

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for safety for the duration of this study. Efforts will be made to follow all pregnancies reported 
during the course of this study to pregnancy outcome pending the subject’s permission. 
 
Type and Duration of Follow-up of Subjects after Adverse Events 
AEs will be assessed (per Section 9.1.1) and followed from initial recognition of the AE through 
end of the protocol defined follow-up period.  
SAEs will be followed up through resolution even if duration of follow-up goes beyond the 
protocol-defined the follow-up period. 
Resolution of an AE is defined as the return to pre-treatment status or stabilization of the 
condition with the expectation that it will remain chronic. 
 
Halting Rules 
9.6.1 
Study Halting Criteria 
Additional enrollment and study interventions/administration of study products in this study will 
be halted for Safety Monitoring Committee (SMC) review/recommendation if any of the 
following are reported: 
1. Any subject experiences laryngospasm, bronchospasm, or anaphylaxis within one day 
after challenge. 
2. Four or more subjects (approximately ≥5% of the anticipated sample size) experience the 
same related grade 3 AE of any kind after challenge through the inpatient unit period and 
related to study product. 
3. Two or more subjects experience generalized urticaria (defined as occurring at more than 
two body parts) within three days after challenge. 
4. Any subject experiences a related SAE during the inpatient unit period.  
5. Any subject develops severe influenza (Section 2.3.2) during the inpatient unit period and 
requires admission to an Intensive Care Unit (ICU), and/or receipt of ICU level of care. 
6. Any medically significant safety issue that the PI determines should halt the study. 
 
Safety Oversight 
9.7.1 
Safety Monitoring Committee (SMC) 
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 

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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.  

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HUMAN SUBJECTS PROTECTION 
 
Institutional Review Board/Independent Ethics Committee 
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. The 
IRB must be registered with OHRP [OHRP-only or OHRP/FDA] as applicable to the research. 
The IRB FWA number will be provided to DMID. 
 
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. 
 
Informed Consent Process 
Informed consent is a process that is initiated prior to an individual agreeing to participate in a 
study and continuing throughout the individual’s study participation. During recruitment, 
investigator or designee may obtain information regarding eligibility through oral or written 
communication with the prospective subject. The screening 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.  
At the first screening visit before any study procedures are performed, informed consent will be 
obtained and documented. 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. 
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, 

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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 specifically to genetic testing 
(HLA testing and transcriptomics) planned for this study. 
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 study, as well as any anticipated prorated payments, if any, to the subject 
for participating in the study. 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 study 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 extent of the confidentiality of the subjects’ records will be defined, and subjects will be 
informed that applicable data protection legislation will be followed. 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 study 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 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 study 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 allowed sufficient time to consider participation in this research study and have 
the opportunity to discuss this study with their family, friends or legally authorized 
representative, or think about it prior to agreeing to participate. 

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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 study. 
Once signed, a copy of the informed consent form will be given to the subject(s) for their 
records. The subject(s) may withdraw consent at any time throughout the course of the study. 
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. 
Study personnel may employ recruitment efforts prior to obtaining study consent if a patient-
specific screening consent is on record or if the IRB has agreed that chart review is allowed 
without a fully executed screening consent. In cases where there is not a patient-specific 
screening consent on record, site clinical staff may pre-screen via chart review and refer potential 
subjects to the Research staff. Research staff would obtain written consent per the standard 
informed consent process before conducting protocol-specific activities. 
New information will be communicated by the site principal investigator to subjects who consent 
to participate in this study in accordance with IRB requirements. The informed consent 
document will be updated, and subjects will be re-consented per IRB requirements, if necessary. 
Subjects will be given a copy of all informed consent forms that they sign. 
 
Consent for Future Use of Stored Specimens and Data 
Subjects who agree to participate in this study will have venous blood collected for clinical 
safety laboratory evaluations, serological and cellular immunology assays, as well as for 
transcriptomic profiling assessments. The amount of venous blood collected for these assays may 
exceed the amount required to perform per protocol (PP) defined assays, and this excess/residual 
blood and corresponding serum, plasma and PBMCs will be designated for future research use 
and stored indefinitely at a central storage facility. Similarly, subjects will also have NP swabs 
and NLF collected for clinical laboratory evaluations and immunology assays. The NP swab 
supernatant can be stored after it is used to test for respiratory viruses. The NLF collected for 
immunology assays exceeds the amount required to perform per protocol (PP) defined assays. 
The NP swabs and NLF excess/residual fluid and corresponding isolated cells will be designated 
for future research use and stored indefinitely at a central storage facility. 
Samples designated for future research use may be used to determine additional immunological 
assessments that may include but are not limited to antibody epitope mapping, B and T cell 
repertoire determination, nontraditional immune assay development, determination of innate 
immune factors and the ability of challenge virus-induced antibodies to cross-react to different 
influenza proteins and virus strains. These blood samples might be used in new or different 
immunological laboratory tests, to provide information for the development of new vaccines, or 
for the studies of influenza or other infections. Subjects will be asked to consent specifically to 
future genetic testing, including whole genome sequencing An IRB will review future research 

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prior to the use of identifiable specimens or data. Future research use specimens, upon written 
request and approval from DMID, may be shared with investigators at the participating VTEU 
sites, with other investigators or company designated research laboratories for purposes of 
conducting additional immunological assessments other than PP analysis. DMID would 
authorize shipment from the DMID CMS. There are no benefits to subjects in the collection, 
storage and future research use of their samples/specimens. Future research use 
samples/specimens will not be sold or used directly for production of any commercial product. 
Each sample/specimen will be encoded (labeled) only with a barcode and a unique tracking 
number to protect subject confidentiality. Reports from future research studies performed using 
subjects’ samples/specimens will NOT be kept in their health records. 
Subjects will be asked to consent to the future research use of excess/residual specimens at the 
time of enrollment. Subjects will be asked to consent to sharing genetic information and samples. 
Subjects may withdraw permission to use samples for future use at any time. They will need to 
contact the study site and the samples will be removed from the study repository after this study 
is completed and documentation will be completed that outlines the reason for withdrawal of 
permission for future use of samples.  
 
Exclusion of Women, Minorities, and Children (Special Populations) 
This study will be inclusive of all healthy adults who meet the inclusion/exclusion criteria, 
regardless of religion, sex, or ethnic background.  
 
Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and 
the Sponsor(s) and their agents. This confidentiality includes documentation, investigation data, 
subject’s clinical information, and all other information generated during participation in the 
study. No information concerning the study or the data generated from the study will be released 
to any unauthorized third party without prior written approval of the DMID and the subject. 
Subject confidentiality will be maintained when study results are published or discussed in 
conferences. The study monitor or other authorized representatives of the Sponsor or 
governmental 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 subjects in this study. The clinical study site will permit 
access to such records. 
All records will be kept locked and all computer entry and networking programs will be carried 
out with coded numbers only and with password protected systems. All non-clinical specimens, 
evaluation forms, reports, and other records that leave the site will be identified only by a coded 
number. 

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Certificate of Confidentiality 
To protect privacy, we have received a Certificate of Confidentiality. With this Certificate, the 
researchers cannot be forced to release information that may identify the research subject, even 
by a court subpoena, in any federal, state, or local civil, criminal, administrative, legislative, or 
other proceedings. The researchers will use the Certificate to resist any demands for information 
that would identify the subject, except as explained below. 
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 subject 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 is in compliance with applicable Federal 
regulations governing the protection of human subjects in research. 
 
Costs, Subject Compensation, and Research Related Injuries 
There is no cost to subjects for the research tests, procedures, and study product while taking part 
in this study. Procedures and treatment for clinical care may be billed to the subject, subject’s 
insurance or third party. Subjects may be compensated for their participation in this study. 
Compensation will be in accordance with the local IRB’s policies and procedures, and subject to 
IRB approval. 
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 to the subject by the NIAID, NIH 
to the subject, or by the participating site for any injury suffered due to participation in this 
study. 

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STATISTICAL CONSIDERATIONS 
 
Introduction 
The goal of this study is to assess the impact of pre-existing immunity to 
A/California/04/2009/H1N1 Variant Influenza A virus on clinical responses and immune 
responses after subjects are challenged intranasally with 2 mL of approximately 5x106 tissue 
culture infective dose (TCID50) of the virus. The study will evaluate and further refine the H1N1 
CHI model across multiple sites, which will help develop the model for future vaccine studies. 
 
Study Hypotheses 
This study is not designed to achieve pre-set levels of power and precision to address the 
primary, secondary, or exploratory objectives. The power and precision allowed by the study’s 
sample size are discussed in Section 11.3, and the general methodology planned to address study 
objectives is described in Section 11.5. Specific null and alternative hypotheses and details about 
planned tables, figures, and data listings will be defined in a separate statistical analysis plan. 
 
Sample Size Considerations 
11.3.1 
Study Design 
This is a CHI study of A/Bethesda/MM2/H1N1 virus infection to assess clinical response, 
immunological response, and safety. A total of up to 80 participants will receive controlled 
A/Bethesda/MM2/H1N1 virus inoculation. 
Influenza A/Bethesda/MM2/H1N1 challenge will be performed using a 2 mL dose of 
approximately 5x106 TCID50/mL. The primary objective of the study will be to evaluate the 
association of symptomatic RT-PCR-positive influenza virus infection post-challenge and pre-
existing HAI antibody titers in healthy subjects. 
Details of the study design are presented in Section 4, and the schedule of events is given in 
Appendix A (Table 11). 
11.3.2 
Sample Size 
The study is planned to enroll up to 80 subjects, all of whom will receive the study influenza 
challenge virus. While the study is not designed to test any specific null hypothesis or reach a 
pre-determined level of estimation precision, the following gives the precision and power for 
estimates and hypothesis tests of interest, respectively, 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 will be described in the statistical analysis plan. 

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The primary objective is to evaluate the association between pre-existing (baseline) HAI 
antibody titers and MMID post-challenge. Table 9 below gives the power to detect varying 
decreases in the odds of a binary endpoint such as MMID with a one standard deviation (SD) 
increase in a continuous covariate (assumed to be normally distributed) among all subjects 
(n=80), via a Wald test from a logistic regression model at the α=0.05-level. This would roughly 
correspond to testing for an association between log-transformed baseline HAI titer and MMID. 
Table 10: 
Power to detect odds ratios (OR) for a one SD increase in a continuous, 
normally distributed covariate in a logistic regression model at the α=0.05-
level 
 
Power (%) 
Prevalence* 
OR=0.4 
OR=0.5 
OR=0.6 
OR=0.7 
0.5 
96.1 
82.8 
59.1 
34.2 
0.6 
95.5 
81.3 
57.3 
32.9 
0.7 
94.9 
79.6 
55.1 
31.3 
*The probability of a binary outcome such as MMID within the study population 
Table 10 indicates the probability of observing one or more safety events, such as a solicited 
symptom or an SAE of a particular classification in all subjects (n=80), given underlying event 
probabilities. 
Table 11: 
Probability (%) of observing at least one safety event, given varying 
underlying event probabilities and group sizes 
True Event Probability 
All Subjects 
(N=80) 
10% (very common) 
>99.9 
1% (common) 
55.2 
0.1% (uncommon) 
7.7 
0.01% (rare) 
0.8 
 
Binomial confidence intervals (CI) are widest (have the least precision) when the response rate 
is 50%. With this event rate among all subjects (40 events among n=80 subjects), the 
corresponding exact Clopper-Pearson CI would be (38.6%, 61.4%). 
 
Planned Interim Analyses 
Interim analyses would only be used to terminate this trial in the event that unanticipated safety 
events were determined to be of sufficient concern to require such action by the sponsor. These 
assessments will not be made on the basis of testing a formal statistical hypothesis. As described 
in Section 9.7, an SMC will be convened by DMID to review participant, clinical, and safety 
data. 

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11.4.1 
Interim Safety Review  
An interim safety review may include enrollment and demographic information, clinical 
laboratory tests, reported influenza symptoms, and AE/SAEs. Additional data may be requested 
by the SMC, and interim statistical reports may be generated as deemed necessary and 
appropriate by DMID. The SMC will meet and review these data at scheduled time points or ad 
hoc as needed during this trial, as delineated in the SMC charter. 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 11.4.1are met, and if so, the 
SMC will be convened to review. 
 
Final Analysis Plan 
Clinical, safety, and immunology data through the last follow-up at Day 91 will comprise the 
primary clinical database for this study. Once the last subject has completed the final telephone 
follow-up, the primary clinical database will be cleaned, monitored, and locked. Analyses of 
safety, clinical, and available immunological data will be performed by the SDCC after the 
primary clinical database is locked and the detailed statistical analysis plan is 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 may be included in an addendum to the CSR, 
publication of manuscript(s), or other report. 
A formal statistical analysis plan (SAP) that elaborates on the analyses indicated here will be 
developed and finalized prior to performing the final analysis. 
11.5.1 
Analysis Populations 
The Safety Analysis (SA) population will include all subjects who received the study influenza 
challenge. 
The modified Intent-to-Treat (mITT) population will consist of all subjects who received the 
study influenza challenge and were followed for the entire challenge period of the study. 
The Per-Protocol (PP) population will include all subjects in the mITT population with the 
following exclusions: 
• Data from all available visits for subjects found to be ineligible at baseline 
• Data from any visit that occurs substantially out of window 
• Data from all visits subsequent to: 
o Major protocol deviations 
o Receipt of immunosuppression or any medications that may be associated with 
impaired responsiveness 
o Receipt of any investigational drug/investigational vaccine/licensed vaccine 

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o Receipt of blood or blood products, or blood donation 
The Infected (INF) population will include only the infected subjects (i.e. those with detectable 
influenza virus) from the PP population. 
The Symptomatic (SYMP) population will include only the subjects from the PP population who 
report symptoms throughout the challenge period. 
11.5.2 
Safety and Clinical Data 
Summaries of safety and clinical data will be presented for the SA population. Analysis 
involving endpoints calculated over the course of the entire challenge period (e.g. MMID) will 
be done within the mITT population, or the PP, INF, or SYMP populations as appropriate. 
Binary safety and clinical outcomes such as influenza infection, MMID, solicited symptoms, and 
SAEs will be presented as frequencies and percentages. Results will be presented overall and by 
infection status, and separately by study day where applicable. Associated confidence intervals 
and comparisons between infected and uninfected subjects via exact binomial tests will also be 
included, as appropriate.  
Viral shedding will be summarized over the challenge period with the following: a binary yes/no 
variable separately by inpatient study day (Day 1 through Day 8) and over the entire challenge 
period; peak (quantitative) shedding and total shedding, as measured by the area under the curve 
(AUC) over the challenge period; the total number of days of shedding. 
Solicited symptoms of influenza recorded via the Flu-PRO survey instrument will be 
summarized by extent (not at all, a little bit, somewhat, quite a bit, very much) for each study 
day, the maximum extent over the first 8 days, and the maximum extent over the entire study. 
Symptoms will also be analyzed by treating the extent as an ordinal variable (0=not at all, 1=a 
little bit, etc.) and calculating the mean scores in each domain (e.g. nose, throat, etc.) and across 
all collected symptoms. Symptoms will also be analyzed by taking the most extensive response 
over the follow-up period and dichotomizing into a binary variable (not at all versus at least a 
little bit) and using standard techniques, such as exact confidence intervals, to summarize the 
proportion of subjects reporting each symptom. Analysis of reported symptoms will be presented 
across all subjects and by infection status (RT-PCR-confirmed symptomatic, RT-PCR-confirmed 
asymptomatic, RT-PCR-negative symptomatic, RT-PCR-negative asymptomatic), and subjects 
may be compared by infection status using appropriate hypothesis tests. The Flu-PRO Survey 
Instrument will be assessed for its accuracy in predicting infection status in an exploratory 
analysis, by randomly splitting the data into training and testing data. 
Unsolicited AEs will be coded by Medical Dictionary for Regulatory Activities (MedDRA) for 
preferred term (PT) and system organ class (SOC). SAEs will be reported by detailed listings 
showing the event description, MedDRA preferred term and SOC, relevant dates (challenge 
dates and AE onset dates), severity, relatedness, and outcome for each event. Non-serious 
unsolicited AEs will be summarized as number and percentage of subjects reporting at least one 

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event in each MedDRA SOC and PT, cross tabulated by severity and relationship to the 
influenza challenge. Additionally, the proportion of subjects and exact 95% confidence intervals 
of AEs in aggregate and by MedDRA categories will be computed. SAEs will be described by 
visit, over the challenge period and over the entire study. 
Clinical laboratory data will be summarized by severity for each visit and as the maximum over 
all visits. Graphical presentations may include box plots and plots of changes from pre-infection 
measurements. 
11.5.3 
Immunological Data 
Analysis of the primary objective will be done in both the mITT and PP populations, the latter as 
a sensitivity analysis. Analysis of secondary and exploratory immunological objectives will be 
done in the PP, INF, and/or SYMP populations, as appropriate. 
The associations between MMID and variable baseline HAI titers (the primary analysis) will be 
assessed in the PP population via logistic regression, treating baseline titer as a continuous 
variable after appropriate transformation. Associations between other infection-related endpoints 
(secondary outcomes: asymptomatic infection; symptoms without detected influenza virus) and 
baseline HAI, NAI, or MN antibody titers will be assessed similarly. For asymptomatic 
infection, the analysis will be performed in the INF population to evaluate the likelihood of 
symptoms given influenza infection; for symptoms without detected influenza virus, the analysis 
will be performed in the SYMP population to evaluate the likelihood of infection given reported 
symptoms. 
Study strain-specific immune responses will be summarized at each relevant study visit, overall 
and by infection status. Analyses for antibody titers will include the number and proportion of 
subjects with a titer ≥1:40, number and proportion of subjects achieving seroconversion (defined 
as either a pre-challenge titer <1:10 and a post-challenge titer ≥1:40 or a pre-challenge titer 
≥1:10 and a minimum four-fold rise in post-challenge antibody titer), and 
Geometric Mean Titers (GMTs) along with corresponding 95% confidence intervals. Descriptive 
summary statistics will be provided for all assays and time points. The correlation between HAI, 
MN, and NAI antibody titers will be evaluated. Plots such as reverse cumulative distributions or 
longitudinal presentations of GMTs will be presented. 
Exploratory objectives also include evaluating the associations between MMID and baseline 
HAI titers, as well as potential confounders age, sex, and receipt of seasonal influenza vaccine in 
the previous year. This will be assessed via multivariable logistic regression in the PP 
population. 
Assays measured on continuous scales will be summarized by the mean, median, standard 
deviation, and range. 

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11.5.1 
Transcriptomic Data 
For the transcriptomics exploratory analysis, 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 [44]. Gene expression quantification will 
be carried out by using the Subread software [45] using the latest Ensembl [46] gene model 
annotations as a reference. Systematic differences in sequence coverage between samples will be 
accounted for using the TMM method [47] as implemented in the edgeR [48] R package. 
Principal component analysis, non-metric multidimensional scaling, and hierarchical clustering 
analysis will be used to identify potential global outliers and systematic batch effect. Negative 
binomial models as implemented in edgeR [48] 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 [49] 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 [50]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 [51] and KEGG [52]databases as well 
as Blood Transcription Modules [53] accounting for gene length bias will be carried out using 
the implementation provided by the goseq [54] 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. 
11.5.2 
Genomics Data 
To assess the association of subject HLA class I and II alleles with development of MMID post-
challenge through Day 8, pre-existing immune status, and magnitude and breadth of the elicited 
immune responses post-challenge, regularized logistic regression (for binary outcomes) and 
regularized linear regression (for continuous outcomes) using HLA alleles as predictors as 
implemented in the glmnet [55] R packages will be utilized. Cross-validation will be used to 
select optimal regularization parameters. Additional details/analyses will be described in the 
SAP. 
11.5.3 
Missing Values and Outliers 
All attempts will be made to collect all data per protocol. If all Flu-PRO Survey Instrument data 
for one study day are missing, the missingness will be indicated in related summaries, but no 
imputation is planned for endpoints derived from these data (e.g. MMID). No imputation for 
missing immunological or transcriptomic/genomic data is planned either. 
Any data point that appears to be erroneous or inexplicable based on clinical judgment will be 
investigated as a possible outlier. If data points are identified as outliers, sensitivity analyses will 
be performed to examine the impact of including or excluding the outliers. Any substantive 
differences in these analyses will be reported. 

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SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
Each participating site will maintain appropriate medical and research records in compliance 
with ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of subjects. Each site will permit authorized representatives of the DMID, its 
designees, and appropriate regulatory agencies to examine (and when required by applicable law, 
to copy) clinical records for the purposes of quality assurance reviews, audits, and evaluation of 
the study safety and progress. These representatives will be permitted access to all source data 
and source documents, which include, but are not limited to, hospital records, clinical and office 
charts, laboratory notes, memoranda, subjects’ memory aids or evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions certified 
after verification as being accurate and complete, microfiches, photographic negatives, microfilm 
or magnetic media, x-rays, and subject files and records kept at the pharmacy, at the laboratories, 
and medico-technical departments involved in the study.  

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QUALITY CONTROL AND QUALITY ASSURANCE 
Following a written DMID-accepted site quality management plan, each participating site(s) and 
its subcontractors are responsible for conducting routine quality assurance (QA) and quality 
control (QC) activities to internally monitor study progress and protocol compliance. The site 
principal investigator will provide direct access to all study-related sites, source data/data 
collection forms, and reports for the purpose of monitoring and auditing by the Sponsor, and 
inspection by local and regulatory authorities. The site principal investigator will ensure all study 
personnel are appropriately trained and current documentations are maintained on site. 
The DCC 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 participating site(s) for clarification and resolution. 

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DATA HANDLING AND RECORD KEEPING 
 
Data Management Responsibilities 
The investigator is responsible to ensure the accuracy, completeness, legibility, and timeliness of 
the data reported. All source documents should be completed in a neat, legible manner to ensure 
accurate interpretation of data. Black or blue permanent ink is required to ensure clarity of 
reproduced copies. When making changes or corrections, cross out the original entry with a 
single line, and initial and date the change. DO NOT ERASE, OVERWRITE, OR USE 
CORRECTION FLUID OR TAPE ON THE ORIGINAL. 
Copies of the eCRF will be provided for use as source data collection forms and maintained for 
recording data for each subject enrolled in the study. Data reported in the eCRF derived from 
source data collection forms should be consistent or the discrepancies should be explained. 
The Sponsor and/or its designee will provide guidance to the site principal investigators and 
other study personnel on making corrections to the data collection forms and eCRF. 
 
Data Coordinating Center/Biostatistician Responsibilities 
Data collection is the responsibility of the study personnel at the participating clinical study site 
under the supervision of the site principal investigator. During the study, the site principal 
investigator must maintain complete and accurate documentation for the study. 
The data coordinating center for this study will be responsible for data management, quality 
review, analysis, and reporting of the study data. 
 
Data Capture Methods 
Clinical information (including, but not limited to, AE/SAEs, concomitant medications, receipt 
of 2018/2019 influenza vaccine, receipt of non-seasonal influenza vaccine, including those that 
are experimental, product type, vaccine virus strains and approximate date of vaccination 
(Section 7.1.1) will be recorded on the appropriate DCF, medical history, physical assessments, 
and clinical laboratory values) will be collected on data collection forms 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.  

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Types of Data 
Data for this study will include clinical, safety, and outcome measures (e.g., clinical laboratory 
values, and immunological data). 
 
Study Records Retention 
Study records and reports including, but not limited to, eCRFs, source documents, ICFs, 
laboratory test results, and study drug disposition records will be retained for 2 years after a 
marketing application is approved for the study 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 the study 
product, until 2 years after the investigation is discontinued and the FDA has been notified. 
These documents will be retained for a longer period, however, if required by local regulations. 
ICFs for future use will be maintained as long as the sample/specimen exists. 
No records will be destroyed without the written consent of the Sponsor. It is the responsibility 
of the Sponsor to inform the site principal investigator when these documents no longer need to 
be retained. The participating VTEU sites must contact DMID for authorization prior to the 
destruction of any study records. 

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CLINICAL MONITORING 
Site monitoring is conducted to ensure that the human subjects’ protections, study and laboratory 
procedures, study intervention administration, and data collection processes are of high quality 
and meet Sponsor, ICH/GCP guidelines and applicable regulations, and that this study is 
conducted in accordance with the protocol, protocol-specific MOP and applicable Sponsor 
standard operating procedures. DMID, the sponsoring agency, or its designee will conduct site-
monitoring visits as detailed in the clinical monitoring plan. 
Site visits will be made at standard intervals as defined by DMID and may be made more 
frequently as directed by DMID. Monitoring visits will include, but are not limited to, review of 
regulatory files, accountability records, eCRFs, informed consent forms, medical and laboratory 
reports, and protocol and GCP compliance. Site monitors will have access to each participating 
site, study personnel, and all study documentation according to the DMID-approved site 
monitoring plan. Study monitors will meet with site principal investigators to discuss any 
problems and actions to be taken and will document site visit findings and discussions. 

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PUBLICATION POLICY 
Following completion of the study, the lead Principal Investigator is expected to publish the 
results of this research in a scientific journal. All investigators funded by the NIH must submit or 
have submitted for them to the National Library of Medicine’s PubMed Central 
(http://www.ncbi.nlm.nih.gov/pmc/) an electronic version of their final, peer-reviewed 
manuscripts upon acceptance for publication, to be made publicly available no later than 12 
months after the official date of publication. The NIH Public Access Policy ensures the public 
has access to the published results of NIH funded research. It requires investigators to submit 
final peer-reviewed journal manuscripts that arise from NIH funds to the digital archive PubMed 
Central upon acceptance for publication. Further, the policy stipulates that these papers must be 
accessible to the public on PubMed Central no later than 12 months after publication. 
Refer to: 
• NIH Public Access Policy, http://publicaccess.nih.gov/ 
• NIH Office of Extramural Research (OER) Grants and Funding, 
http://grants.nih.gov/grants/oer.htm 
As of January 2018, all clinical trials supported by the NIH must be registered on 
ClinicalTrials.gov, no later than 21 days after the enrollment of the first subject. Results of all 
clinical trials supported by the NIH, generally, need to be submitted no later than 12 months 
following the primary completion date. A delay of up to 2 years is available for trials that meet 
certain criteria and have applied for certification of delayed posting. 
As part of the result posting a copy of this protocol (and its amendments) and a copy of the 
Statistical Analysis Plan will be posted on ClincialTrials.gov. 
For this study the responsible party is NIAID/DMID which will register the study and post 
results.  
The responsible party does not plan to request certification of delayed posting. 
Refer to: 
• Public Law 110-85, Section 801, Clinical Trial Databases 
• 42CFR11 
• NIH NOT-OD-16-149 

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17. Nicholson KG, Webster RG, Hay AJ. Textbook of influenza: Blackwell Science Ltd, 1998. 

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18. Wilkinson TM, Li CK, Chui CS, et al. Preexisting influenza-specific CD4+ T cells correlate 
with disease protection against influenza challenge in humans. Nat Med 2012; 18:274-80. 
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challenge model H1N1pdMIST: an A(H1N1)pdm09 dose-finding investigational new drug 
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20. Memoli MJ, Shaw PA, Han A, et al. Evaluation of Antihemagglutinin and 
Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy 
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Antibody as a Correlate of Protection in a Healthy Volunteer Challenge with Influenza 
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22. Memoli MJ, Han A, Walters KA, et al. Influenza A Reinfection in Sequential Human 
Challenge: Implications for Protective Immunity and "Universal" Vaccine Development. Clin 
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23. Powers JH, 3rd, Bacci ED, Guerrero ML, et al. Reliability, Validity, and Responsiveness of 
InFLUenza Patient-Reported Outcome (FLU-PRO(c)) Scores in Influenza-Positive Patients. 
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24. Treanor JJ, Betts RF, Erb SM, Roth FK, Dolin R. Intranasally administered interferon as 
prophylaxis against experimentally induced influenza A virus infection in humans. J Infect Dis 
1987; 156:379-83. 
25. Treanor JJ, Roth FK, Betts RF. Use of live cold-adapted influenza A H1N1 and H3N2 virus 
vaccines in seropositive adults. J Clin Microbiol 1990; 28:596-9. 
26. Clements ML, Murphy BR. Development and persistence of local and systemic antibody 
responses in adults given live attenuated or inactivated influenza A virus vaccine. J Clin 
Microbiol 1986; 23:66-72. 
27. Betts RF, Douglas RG, Jr., Murphy BR. Resistance to challenge with influenza A/Hong 
Kong/123/77 (H1N1) wild-type virus induced by live attenuated A/Hong Kong/123/77 (H1N1) 
cold-adapted reassortant virus. J Infect Dis 1985; 151:744-5. 
28. Youngner JS, Treanor JJ, Betts RF, Whitaker-Dowling P. Effect of simultaneous 
administration of cold-adapted and wild-type influenza A viruses on experimental wild-type 
influenza infection in humans. J Clin Microbiol 1994; 32:750-4. 
29. Hayden FG, Tunkel AR, Treanor JJ, Betts RF, Allerheiligen S, Harris J. Oral LY217896 for 
prevention of experimental influenza A virus infection and illness in humans. Antimicrob Agents 
Chemother 1994; 38:1178-81. 
30. Sears SD, Clements ML, Betts RF, Maassab HF, Murphy BR, Snyder MH. Comparison of 
live, attenuated H1N1 and H3N2 cold-adapted and avian-human influenza A reassortant viruses 
and inactivated virus vaccine in adults. J Infect Dis 1988; 158:1209-19. 
31. Sears SD, Clements ML. Protective efficacy of low-dose amantadine in adults challenged 
with wild-type influenza A virus. Antimicrob Agents Chemother 1987; 31:1470-3. 
32. Hayden FG, Treanor JJ, Betts RF, Lobo M, Esinhart JD, Hussey EK. Safety and efficacy of 
the neuraminidase inhibitor GG167 in experimental human influenza. Jama 1996; 275:295-9. 
33. Reuman PD, Bernstein DI, Keely SP, Young EC, Sherwood JR, Schiff GM. Differential 
effect of amantadine hydrochloride on the systemic and local immune response to influenza A. J 
Med Virol 1989; 27:137-41. 

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34. Al-Nakib W, Higgins PG, Willman J, et al. Prevention and treatment of experimental 
influenza A virus infection in volunteers with a new antiviral ICI 130,685. J Antimicrob 
Chemother 1986; 18:119-29. 
35. Snyder MH, Betts RF, DeBorde D, et al. Four viral genes independently contribute to 
attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines. 
J Virol 1988; 62:488-95. 
36. Clements ML, Subbarao EK, Fries LF, Karron RA, London WT, Murphy BR. Use of single-
gene reassortant viruses to study the role of avian influenza A virus genes in attenuation of wild-
type human influenza A virus for squirrel monkeys and adult human volunteers. J Clin Microbiol 
1992; 30:655-62. 
37. Clements ML, Snyder MH, Sears SD, Maassab HF, Murphy BR. Evaluation of the 
infectivity, immunogenicity, and efficacy of live cold-adapted influenza B/Ann Arbor/1/86 
reassortant virus vaccine in adult volunteers. J Infect Dis 1990; 161:869-77. 
38. Keitel WA, Couch RB, Quarles JM, Cate TR, Baxter B, Maassab HF. Trivalent attenuated 
cold-adapted influenza virus vaccine: reduced viral shedding and serum antibody responses in 
susceptible adults. J Infect Dis 1993; 167:305-11. 
39. Treanor JJ, Betts RF. Evaluation of live attenuated cold-adapted influenza B/Yamagata/16/88 
reassortant virus vaccine in healthy adults. J Infect Dis 1993; 168:455-9. 
40. Food and Drug Administration. Highlights of Prescribing Information. Available at: 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/210854s000lbl.pdf. Accessed 7 June 
2019. 
41. Powers JH, Guerrero ML, Leidy NK, et al. Development of the Flu-PRO: a patient-reported 
outcome (PRO) instrument to evaluate symptoms of influenza. BMC Infect Dis 2016; 16:1. 
42. Takashita E, Morita H, Ogawa R, et al. Susceptibility of Influenza Viruses to the Novel Cap-
Dependent Endonuclease Inhibitor Baloxavir Marboxil. Frontiers in Microbiology 2018; 9. 
43. Hussain M, Galvin HD, Haw TY, Nutsford AN, Husain M. Drug resistance in influenza A 
virus: the epidemiology and management. Infect Drug Resist 2017; 10:121-34. 
44. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory 
requirements. Nat Methods 2015; 12:357-60. 
45. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning 
sequence reads to genomic features. Bioinformatics 2014; 30:923-30. 
46. Aken BL, Achuthan P, Akanni W, et al. Ensembl 2017. Nucleic Acids Res 2017; 45:D635-
d42. 
47. Robinson MD, Oshlack A. A scaling normalization method for differential expression 
analysis of RNA-seq data. Genome Biol 2010; 11:R25. 
48. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential 
expression analysis of digital gene expression data. Bioinformatics 2010; 26:139-40. 
49. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful 
approach to multiple testing. Journal of the Royal statistical society: series B (Methodological) 
1995; 57:289-300. 
50. Suzuki R, Shimodaira H. Pvclust: an R package for assessing the uncertainty in hierarchical 
clustering. Bioinformatics 2006; 22:1540-2. 
51. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo P, Mesirov JP. 
Molecular signatures database (MSigDB) 3.0. Bioinformatics 2011; 27:1739-40. 

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52. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 
2000; 28:27-30. 
53. Li S, Rouphael N, Duraisingham S, et al. Molecular signatures of antibody responses derived 
from a systems biology study of five human vaccines. Nat Immunol 2014; 15:195-204. 
54. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: 
accounting for selection bias. Genome Biol 2010; 11:R14. 
55. Friedman J, Hastie T, Tibshirani R. Regularization Paths for Generalized Linear Models via 
Coordinate Descent. J Stat Softw 2010; 33:1-22. 
56. Centers for Disease Control and Prevention. People at High Risk For Flu Complications. 
Available at: https://www.cdc.gov/flu/highrisk/index.htm. Accessed 10 June 2019. 
 

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APPENDICES 

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Schedule of Events  
Table 12: 
Schedule of Events 
Evaluation  
Enrollment/Screening 
Study Visit 00A Day -30 to -3 
Inpatient Study Visit 00B Day -2 
Inpatient Study Visit 00C Day -1 
Inpatient Study Visit 1 Day 1 
Inpatient Study Visit 2 Day 2 
Inpatient Study Visit 3 Day 3 
Inpatient Study Visit 4 Day 4 
Inpatient Study Visit 5 Day 5 
Inpatient Study Visit 6 Day 6 
Inpatient Study Visit 7 Day 7 
Inpatient Study Visit 8 Day 8 
Follow-up Study Visit 9 Day 15±3 
Follow-up Study Visit 10 Day 29±3 
Follow-up Study Visit 11 Day 61±5 
Final Study Visit 12 Day 91 ± 7 
Early Termination Visit 
Unscheduled Visit 
Visit type1 
c 
i 
i 
i 
i 
i 
i 
i 
i 
i 
i 
c 
c 
c 
p 
c 
c 
Screening for eligibility 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signed informed consent  
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review/confirm eligibility criteria 
x2 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review/confirm informed consent  
 
x3 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inpatient period 
 
x4 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
Discharge from inpatient  
 
 
 
 
 
 
 
 
 
 
x5 
 
 
 
 
 
 
Treatment with antiviral 
 
 
 
 
 
 
 
 
 
 
x6 
 
 
 
 
 
 
Demographics 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
x 
x 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height measurements 
x7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight measurements 
x7 
x7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant medications 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x 
X 
Physical Exam 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Targeted physical exam8 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x22
x22 

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Evaluation  
Enrollment/Screening 
Study Visit 00A Day -30 to -3 
Inpatient Study Visit 00B Day -2 
Inpatient Study Visit 00C Day -1 
Inpatient Study Visit 1 Day 1 
Inpatient Study Visit 2 Day 2 
Inpatient Study Visit 3 Day 3 
Inpatient Study Visit 4 Day 4 
Inpatient Study Visit 5 Day 5 
Inpatient Study Visit 6 Day 6 
Inpatient Study Visit 7 Day 7 
Inpatient Study Visit 8 Day 8 
Follow-up Study Visit 9 Day 15±3 
Follow-up Study Visit 10 Day 29±3 
Follow-up Study Visit 11 Day 61±5 
Final Study Visit 12 Day 91 ± 7 
Early Termination Visit 
Unscheduled Visit 
Vital signs9 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
x22
x22 
SpO210 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
Influenza Challenge 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Flu-PRO Survey Instrument 
 
 
x11 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
x11 
x11 
Collect Flu-PRO Survey Instrument 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
Adverse event review12 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
x22 
x22 
SAE review 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
X 
ECG (12 lead) 
x13 
 
 
 
 
 
 
 
x13 
 
 
 
 
 
 
 
 
CXR (PA and lateral) 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for HIV, HBV, HCV 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum HCG 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine HCG14 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine toxicology15 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for Safety Lab Evaluations (Screen)16 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for Safety Lab Evaluations (Follow-up)17 
 
 
 
 
x 
 
x 
 
 
 
x 
 
 
 
 
x22 
 
Sera for cytokine and/or antibody assays18 
 
x 
 
 
x 
 
x 
 
x 
 
x 
x 
x 
x 
 
 
 
Blood for ASC (ELISpot; fresh PBMC)19 
 
 
 
 
 
 
x 
 
x 
 
x 
 
 
 
 
 
 
Blood for innate and/or T and B immunophenotyping 
(cryopreserved PBMC)19 
 
x 
 
 
 
 
x 
 
x 
 
x 
x 
x 
x 
 
 
 

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Evaluation  
Enrollment/Screening 
Study Visit 00A Day -30 to -3 
Inpatient Study Visit 00B Day -2 
Inpatient Study Visit 00C Day -1 
Inpatient Study Visit 1 Day 1 
Inpatient Study Visit 2 Day 2 
Inpatient Study Visit 3 Day 3 
Inpatient Study Visit 4 Day 4 
Inpatient Study Visit 5 Day 5 
Inpatient Study Visit 6 Day 6 
Inpatient Study Visit 7 Day 7 
Inpatient Study Visit 8 Day 8 
Follow-up Study Visit 9 Day 15±3 
Follow-up Study Visit 10 Day 29±3 
Follow-up Study Visit 11 Day 61±5 
Final Study Visit 12 Day 91 ± 7 
Early Termination Visit 
Unscheduled Visit 
Blood for T and B immunology (cryopreserved 
PBMC)  
 
x 
 
 
 
 
x 
 
x 
 
x 
x 
x 
x 
 
x22 
x22 
Blood for transcriptomics (whole blood) 
 
x 
 
x20 
x 
 
x 
 
 
 
x 
x 
 
 
 
x22 
x22 
NP swab for qualitative & quantitative respiratory 
21
 
x 
x 
 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
NLF for cytokine and/or antibody assays 
 
x 
 
 
x 
 
x 
 
x 
 
x 
x 
x 
x 
 
x22 
x22 
NLF for other immunology (immunophenotyping) 
 
x 
 
 
x 
 
x 
 
x 
 
x 
 
 
 
 
x22 
x22 
HLA Typing 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
x22 
x22 
 
1. 
Visit type: c=clinic, i=inpatient unit, p=phone call  
2. 
Eligibility criteria may change from the day of enrollment to Day -3. To avoid admitting subjects with new exclusion criteria into the inpatient unit, the eligibility criteria 
will be reviewed and confirmed before the inpatient stay. 
3. 
Subjects will have approximately a 48-hour window to decide if they would like to drop out of the study and leave the inpatient unit before challenge virus is 
administered. 
4. 
Eligible subjects will be admitted to the inpatient unit on Day -2 (which is two days prior to the planned challenge). 
5. 
Subjects will remain in the inpatient 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 (that are performed on Day 7 or thereafter) for influenza A by qualitative RT-PCR performed by the local clinical 
laboratory, are afebrile, 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 Day 8 will remain in the inpatient unit until the criteria are met. Study procedures 
for the inpatient unit will be the same as on Day 8, except for no additional blood draws unless clinically indicated. 
6. 
A single dose of baloxavir marboxil will be offered to all subjects who do not have two consecutive negative NP swabs (on Day 7 or thereafter) for influenza A by 
qualitative RT-PCR.  

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7. 
Height and weight will be measured at initial Screening. Weight will be documented a second time on admission to the inpatient unit. BMI calculation for the purpose of 
Eligibility will be based on the first measured weight. BMI for the purpose of analyses will be calculated with the second measured weight. 
8. 
Clinical evaluation (including lung, heart, oral/pharyngeal, and neck exams), and symptom evaluation daily while in the inpatient unit. 
9. 
Oral temperature, blood pressure, pulse, and respiratory rate will be assessed three times per day while the subjects are awake, and as clinically indicated. 
10. Peripheral mixed venous oxygen saturation (SpO2) on room air at screening, on admission, and when oral temperature, blood pressure, pulse, and respiratory rate are 
assessed through the inpatient stay. 
11. Training on Flu-PRO Survey Instrument and Validation Diary for evaluation of severity score. Self-assessment using Flu-PRO and diary to be performed in the morning 
pre-challenge on Day 1, and at approximately 5 PM through day 14 during the inpatient stay and following discharge to home.  
12. Unsolicited AEs will be monitored for approximately 30 days post challenge. Review of Flu-PRO for indicators of possible severe influenza associated illness (signs, 
symptoms, or lab findings) not consistent with MMID. 
13. ECG to be performed at screening, on Day 6, and as clinically indicated. VTEU sites will consult a cardiologist if ECG is abnormal to determine clinically necessary 
work up.  
14. 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.  
15. Negative drug urine toxicology result (amphetamines, cocaine, and opiates, ) required on screening performed at the Central Lab, and (amphetamines, cocaine, opiates, 
and cannabinoids) on admission to the challenge unit performed locally. 
16. Screening Safety Labs: white blood cells (WBCs), absolute lymphocyte count, hemoglobin, platelets, alanine transaminase (ALT), and creatinine (Cr). 
17. Follow-Up Safety Labs: 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). 
18. Including HAI, MN and NAI antibody titers at admission to the inpatient unit (Day -2) and Visits 8,10 and 11. 
19. Will be drawn from at least a subset of participants for local testing 
20. Blood draw to occur prior to challenge 
21. Multiplex respiratory virus assay to be performed at each site. 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. 
22. If indicated. 
 
 

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Venipuncture Volumes (mL) 
Table 13: 
Venipuncture Volumes (all volumes are approximate) 
Evaluation  
Enrollment/Screening 
Study Visit 00A Day -30 to -3 
Inpatient Study Visit 00B Day -2 
Inpatient Study Visit 00C Day -1 
Inpatient Study Visit 1 Day 1 
Inpatient Study Visit 2 Day 2 
Inpatient Study Visit 3 Day 3 
Inpatient Study Visit 4 Day 4 
Inpatient Study Visit 5 Day 5 
Inpatient Study Visit 6 Day 6 
Inpatient Study Visit 7 Day 7 
Inpatient Study Visit 8 Day 8 
Follow-up Study Visit 9 Day 15±3 
Follow-up Study Visit 10 Day 29±3 
Follow-up Study Visit 11 Day 61±5 
Final Study Visit 12 Day 91 ± 7 
Total Volumes 
Blood for Safety Lab Evaluations (Screen) 
151 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
15 
Blood for Safety Lab Evaluations (Follow-up) 
 
 
 
 
102 
 
102 
 
 
 
102 
 
 
 
 
30 
Blood for HLA typing 
 
 
 
 
 
 
 
 
 
 
 
 
 
03 
 
0 
Sera for antibody and cytokine assays 
 
204,5 
 
 
55 
 
55 
 
55 
 
204,5 
55 
204 
204 
 
100 
Blood for ASC (ELISpot; fresh PBMC)6 
 
 
 
 
 
 
157 
 
157 
 
157 
 
 
 
 
45 
Blood for innate and/or T and B 
immunophenotyping (cryopreserved PBMC) 6 
 
88,9 
 
 
 
 
88,9 
 
88,9 
 
88,9 
88,9 
89 
89 
 
56 
Blood for T and B immunology (cryopreserved 
PBMC)  
 
4810 
 
 
 
 
3210 
 
3210 
 
3210 
4010 
4010 
4010 
 
264 
Blood for transcriptomics (whole blood) 
 
2.5 
 
2.511 
2.5 
 
2.5 
 
 
 
2.5 
2.5 
 
 
 
15 
Total (fresh PBMC subset) 
15 
78.5 
0 
2.5 
17.5 
0 
72.5 
0 
60 
0 
87.5 
55.5 
68 
68 
0 
525 
Total (subset without fresh PBMC) 
15 
78.5 
0 
2.5 
17.5 
0 
57.5 
0 
45 
0 
72.5 
55.5 
68 
68 
0 
480 
 
 

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1. 
Safety Lab Evaluations (Screen): Hepatitis B surface antigen (HBsAg), hepatitis C antibodies (anti-HCV), human immunodeficiency virus (HIV) testing, and serum 
HCG (for women subjects of childbearing potential), white blood cells (WBCs), absolute lymphocyte count, hemoglobin, platelets, and alanine transaminase (ALT), and 
creatinine (Cr).  
2. 
Blood for Safety Lab Evaluations (Follow-up): white blood cells (WBCs), absolute lymphocyte count, hemoglobin, platelets, and alanine transaminase (ALT), and 
creatinine (Cr).  
3. 
HLA testing may be performed on blood already drawn up to this timepoint with no increase in blood volume drawn 
4. 
Sera tested for antibodies 
5. 
Sera tested for cytokines 
6. 
Blood draw and testing will be done in at least a subset of participants 
7. 
A/Bethesda/MM2/H1N1-specific plasmablast responses (ASC) H1 and N1  
8. 
Immunophenotyping of innate cells 
9. 
Immunophenotyping of T and B cell subsets (including cTfh)  
10. A/Bethesda/MM2/H1N1-specific responses in T and B cell subsets (including cTfh) 
11. Drawn prechallenge 
 

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High-Risk Medical Conditions for Severe Influenza 
People with any of the following medical conditions are at increased risk for serious 
complications from influenza virus infection [56]. 
• 
Asthma 
• 
Neurologic and neurodevelopment conditions 
• 
Blood disorders (such as sickle cell disease) 
• 
Chronic lung disease (such as chronic obstructive pulmonary disease [COPD] and cystic 
fibrosis) 
• 
Endocrine disorders (such as diabetes mellitus) 
• 
Heart disease (such as congenital heart disease, congestive heart failure and coronary 
artery disease) 
• 
Kidney disorders 
• 
Liver disorders 
• 
Metabolic disorders (such as inherited metabolic disorders and mitochondrial disorders) 
• 
Obesity with a body mass index [BMI] of 40 or higher 
• 
Weakened immune system due to disease (such as people with HIV or AIDS, or some 
cancers such as leukemia) or medications (such as those receiving chemotherapy or 
radiation treatment for cancer, or persons with chronic conditions requiring chronic 
corticosteroids or other drugs that suppress the immune system) 
• 
Pregnant women 
 
 

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Flu-PRO Survey Instrument and Validation Diary 
 

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Version 3.0 
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Version 3.0 
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04 November 2020  
 
 
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DMID Protocol 18-0010 
Version 3.0 
H1N1v Virus Challenge Study in Healthy Subjects 
04 November 2020  
 
 
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