Challenge Infection of Healthy Adult Volunteers With RSV A2
Phase: PHASE1
Conditions: Upper Respiratory Tract Infections
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Challenge Infection of Healthy Adult Volunteers with RSV A2 
NIH Protocol Number: 18-I-0039
Sponsored by: 
National Institute of Allergy and Infectious Diseases (NIAID)
IND Sponsor: 
Office of Clinical Research Policy and Regulatory Operations 
(OCRPRO), Division of Clinical Research (DCR), NIAID, 
National Institutes of Health (NIH)
Investigational New Drug (IND) #: 16440
RSV A2 Provided by: Laboratory of Infectious Diseases (LID), NIAID/NIH
Principal Investigator (PI): Lesia K. Dropulic, MD
Version Number: 9.0
Date: October 8, 2020

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Study Site:
NIH Clinical Center
10 Center Drive
Bethesda, MD 20892
Laboratory of Infectious Diseases RSV Lab
NIH Campus
Building 50
Bethesda, MD 20892
Immunological Monitoring Lab
Frederick National Laboratory for Cancer Research
Frederick, MD
Johns Hopkins RSV Lab
Center for Immunization Research
Bloomberg School of Public Health
624 N. Broadway
Baltimore, MD 21205

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Table of Contents
Table of Contents .............................................................................................................3
List of Tables ....................................................................................................................5
List of Abbreviations .........................................................................................................6
Protocol Summary ............................................................................................................9
Précis..............................................................................................................................11
1 Background Information and Scientific Rationale.......................................................12
1.1
Background Information ....................................................................................12
1.1.1
Description of the Study Agent/Intervention ...............................................14
1.1.2
Summary of Previous Pre-Clinical Studies .................................................15
1.1.3
Summary of Relevant Clinical Studies........................................................17
1.2
Rationale ...........................................................................................................21
2 Study Objectives.........................................................................................................22
2.1
Primary Objective ..............................................................................................22
2.2
Secondary Objectives........................................................................................22
2.3
Exploratory Objectives.......................................................................................23
3 Study Design ..............................................................................................................23
3.1
Description of the Study Design ........................................................................23
3.1.1
Definitions of RSV Illness............................................................................24
3.1.2
Study Phases..............................................................................................26
3.2
Rationale for Study Design................................................................................27
3.3
Study Endpoints ................................................................................................27
3.3.1
Primary Endpoint ........................................................................................27
3.3.2
Secondary Endpoints..................................................................................27
3.3.3
Exploratory Endpoints.................................................................................28
4 Study Population.........................................................................................................28
4.1
Rationale for Subject Selection .........................................................................28
4.2
Recruitment Plan ...............................................................................................28
4.3
Subject Inclusion Criteria...................................................................................29
4.4
Subject Exclusion Criteria..................................................................................29
4.5
Justification for Exclusion of Special Populations..............................................32
4.6
Recruitment of NIH Employees .........................................................................32
5 Study Agent/Interventions...........................................................................................33
5.1
Disposition and Dispensation ............................................................................33
5.1.1
Formulation, Packaging, and Labeling .......................................................33
5.2
Study Agent Storage and Stability.....................................................................34
5.3
Preparation, Administration, and Dosage of Study Agent .................................34
5.4
Assessment of Subject Compliance with Study Agent ......................................34
5.5
Concomitant Medication and Procedures..........................................................35
5.6
Prohibited Medications and Procedures............................................................35
6 Study Schedule and Clinical Procedures....................................................................36
6.1
Screening ..........................................................................................................36
6.2
Inpatient Study Phase (Day −1 until discharge) ................................................37
6.2.1
Treatment Plan for Persistent Symptoms Associated with RSV Illness......42
6.2.2
Escalation of Care Plan ..............................................................................42

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6.2.2.1
Increased Severity of Clinical Illness....................................................42
6.3
Follow-up Visit (Day 28) ....................................................................................43
6.4
Final Study Visit (Day 56) ..................................................................................44
6.5
Interim Safety Visit.............................................................................................44
6.6
Early Termination Visit.......................................................................................45
6.7
Pregnancy and Follow-up Visit ..........................................................................45
7 Laboratory Evaluations...............................................................................................46
7.1
Clinical Laboratory Evaluations (all performed in the NIH Clinical Center): ......46
7.2
Research Laboratory Evaluations: ....................................................................46
8 Potential Risks and Benefits.......................................................................................47
8.1
Potential Risks...................................................................................................47
8.2
Potential Benefits...............................................................................................48
9 Research Use of Stored Human Samples, Specimens or Data .................................48
10Remuneration Plan for Subjects.................................................................................49
11Assessment of Safety.................................................................................................50
11.1 Definitions for Sponsor Reporting......................................................................50
11.2 Documenting, Recording, and Reporting Adverse Events ................................53
11.3 Investigator Assessment of Adverse Events .....................................................54
11.3.1
Severity....................................................................................................54
11.3.2
Causality..................................................................................................55
11.4 Investigator Reporting Responsibilities to the Sponsor.....................................56
11.4.1
Adverse Events .......................................................................................56
11.4.2
Serious Adverse Events ..........................................................................56
11.4.3
Unanticipated Problems ..........................................................................57
11.4.4
Pregnancy ...............................................................................................57
11.5 Additional Investigator Reporting Procedures ...................................................57
11.5.1
Reporting Procedures to the IRB.............................................................57
11.5.2
Reporting to the NIAID Clinical Director ..................................................58
11.6 Sponsor’s Reporting Responsibilities ................................................................58
11.7 Halting Rules for the Protocol............................................................................58
11.7.1
Reporting a Study Halt ............................................................................58
11.7.2
Resumption of a Halted Study.................................................................59
11.8 Withdrawal Criteria for an Individual Subject.....................................................59
11.9 Safety Oversight ................................................................................................60
11.9.1
Safety Review and Communications Plan...............................................60
11.9.2
Sponsor Medical Monitor.........................................................................60
11.9.3
Safety Monitoring Committee ..................................................................60
12Clinical Monitoring Structure.......................................................................................60
12.1 Site Monitoring Plan ..........................................................................................60
13Statistical Considerations ...........................................................................................61
13.1 Sample Size Justification...................................................................................62
13.2 Analysis Plan .....................................................................................................64
14Ethics/Protection of Human Subjects .........................................................................65
14.1 Informed Consent Process ................................................................................65
14.1.1
Non–English-Speaking Participants ........................................................66
14.2 Subject Confidentiality .......................................................................................67

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15Data Handling and Record Keeping ...........................................................................67
15.1 Data Capture and Management ........................................................................67
15.2 Record Retention...............................................................................................67
Appendix A: Scientific References..................................................................................68
Appendix B: Schedule of Procedures/Evaluations .........................................................71
Appendix C: Blood Volumes for Specimen Collection....................................................73
Appendix D: Summary of Prior Clinical Studies (2000-present) .....................................74
Appendix E: Toxicity Table .............................................................................................76
Appendix F: Patient-Reported FLU-PRO Symptom Questionnaire ................................83
List of Tables
Table 1
The in vivo phenotype of RSV A2 is similar to biologically derived wild-type 
(wt) RSV in chimpanzees (From: Whitehead et al.)........................................................15
Table 2
The in vivo replication, pathogenicity, and immunogenicity of recombinant 
RSV A2 is similar to that of biologically derived wild-type (wt) RSV in chimpanzees 
(From: Whitehead et al.)..........……………………………………………………………….16
Table 3
Statistical analysis for determining neutralizing antibody eligibility titer cut-off 
based on shedding at any time after challenge ..............................................................22
Table 4
Participant remuneration................................................................................50
Table 5
Criteria for continuing the study after cohort 3 and the final criteria for 
interesting shedding rate at the end of the study based on 3 different null and alternative 
shedding rates ................................................................................................................63
Table 6
Probability of stopping early under the specified criteria in Table 5 for 
uninteresting rates of activity ..........................................................................................64

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List of Abbreviations
AE
Adverse Event
AGM
African Green Monkey
ALT
Alanine Transaminase
ANC
Absolute Neutrophil Count
AR
Adverse Reaction
AST
Aspartate Aminotransferase
BMI
Body Mass Index
CBC
Complete Blood Count
CDC
Centers for Disease Control and Prevention
CFR
Code of Federal Regulations
CHI
Center for Human Immunology, Autoimmunity, and Inflammation
CI
Confidence Interval
CIR
Center for Immunization Research
CLIA
Clinical Laboratory Improvement Amendments
CRF
Case Report Form
CRIS
Clinical Research Information System
CRIMSON
Clinical Research Information Management System of the NIAID
CSO
Clinical Safety Office
CTM
Clinical Trial Material
CXR
Chest X-Ray
DAIDS
Division of AIDS
DCR
Division of Clinical Research
DLM
Department of Laboratory Medicine
DNA
Deoxyribonucleic Acid
cDNA
Complementary Deoxyribonucleic Acid
EBV
Epstein-Barr Virus
EKG
Electrocardiogram
FACS
Fluorescence-Activated Cell Sorting
FDA
Food and Drug Administration
FEV1
Forced Expiratory Volume in 1 Second
FVC
Forced Vital Capacity
GCP
Good Clinical Practice
HEK
Human Embryonic Kidney
HIV
Human Immunodeficiency Virus

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HLA
Human Leukocyte Antigen
HRPP
Human Research Protection Program
ICH
International Conference on Harmonisation
Ig
Immunoglobulin
IND
Investigational New Drug
IRB
Institutional Review Board
IUD
Intrauterine Device
JHU
Johns Hopkins University
LID
Laboratory of Infectious Diseases
NHLBI
National Heart, Lung, and Blood Institute
NIAID
National Institute of Allergy and Infectious Diseases 
NIH
National Institutes of Health
NK
Natural Killer
NP
Nasopharyngeal
NREVSS
National Respiratory and Enteric Virus Surveillance System
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations
OHRP
Office for Human Research Protections
PCR
Polymerase Chain Reaction
PFT
Pulmonary Function Test
PFU
Plaque Forming Unit
PI
Principal Investigator
PRO
Patient-Reported Outcome
RD
Recommended Dose
RNA
Ribonucleic Acid
RSV
Respiratory Syncytial Virus
RT-PCR
Reverse Transcription–Polymerase Chain Reaction
SAE
Serious Adverse Event/Serious Adverse Experience
SAM
Synthetic Absorptive Matrix
SAR
Serious Adverse Reaction
SCSU
Special Clinical Studies Unit
SERF
Safety Expedited Report Form
SMC
Safety Monitoring Committee
SRCP
Safety Review and Communication Plan
SUSAR
Suspected Unexpected Serious Adverse Reaction
UP
Unanticipated Problem
UPnonAE
Unanticipated Problem that is not an Adverse Event

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URI
Upper Respiratory Tract Infection
wt
Wild-type

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Protocol Summary
Full Title:
Challenge Infection of Healthy Adult Volunteers with RSV A2 
Short Title:
RSV Challenge Study II
Clinical Phase:
1
IND Sponsor:
OCRPRO, DCR, NIAID
Conducted by:
LID, NIAID, NIH
PI:
Lesia K. Dropulic, MD
Sample Size:
N = 25
Accrual Ceiling:
Up to 35 subjects may need to be enrolled to compensate 
for dropouts.
Study Population:
Healthy volunteers 18 to 50 years of age with low pre-
challenge respiratory syncytial virus (RSV)–specific 
neutralizing antibody titers.
Accrual Period:
2 years.
Study Design:
This is a study evaluating safety, viral shedding, and RSV 
illness after human challenge with high dose wild-type, 
recombinant respiratory syncytial virus A2 (RSV A2) 
delivered intranasally using an atomizer. Healthy adult 
volunteers will receive one dose of RSV A2 intranasally 
followed by clinical, virologic, and immunologic evaluations 
performed during an inpatient stay at the NIH Clinical 
Center. Subjects will be followed prospectively for 56 days.
Study Duration:
Start Date: November 2017  End Date: November 2021
Individual subjects will participate for 56 days from the time 
of inpatient admission to completion of outpatient follow-up. 
The inpatient portion of the study for each subject will last 
approximately 9-14 days.
Study Agent:
Live, recombinant, wild-type RSV A2
Intervention Description: Intranasal inoculation of healthy volunteers with a single   
dose of recombinant RSV A2 using a nasal atomizer.

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Primary Objective:            Determine the safety and infectability of high dose, 
recombinant RSV A2 administered by nasal atomizer to 
healthy adult volunteers.
Secondary Objectives:
1. Characterize the onset, duration, and magnitude of viral 
shedding after RSV A2 challenge infection.
2. Determine the frequency of mild to moderate upper 
respiratory illness in healthy volunteers challenged with RSV 
A2.
3. Evaluate the duration and severity of clinical illness after 
RSV A2 challenge infection.
4. Determine the relationship between the magnitude and 
duration of viral shedding and the severity and duration of 
RSV illness.
5. Characterize RSV-specific serum and mucosal antibody 
responses after RSV A2 challenge infection.
Primary Endpoints:
Assess expected and unexpected adverse events (AEs) 
throughout the trial and detect RSV shedding in nasal wash 
by FilmArray multiplex polymerase chain reaction (PCR) 
assay and by reverse transcription (RT)–quantitative PCR 
and quantitative viral culture after challenge with RSV A2.
Secondary Endpoints:
1. Determine day of onset of shedding, number of days of 
shedding, peak nasal wash viral titer, and mean sum of daily 
viral titers after challenge.
2. Calculate the number of subjects with mild to moderate 
upper respiratory illness after challenge, based on study 
definition of RSV illness.
3. Determine the number of days RSV illness persists after 
challenge and degree of illness based on study definition.
4. Correlate the magnitude and duration of viral shedding 
with the severity and duration of RSV-related symptoms.
5. Determine the frequency, magnitude, and duration of 
serum and mucosal (nasal wash and/or nasosorption) RSV-
specific antibody responses after challenge.

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Précis 
Respiratory syncytial virus (RSV) is the leading cause of pediatric lower respiratory tract 
infection. RSV also causes lower respiratory tract disease in the elderly and life-
threatening disease in immunocompromised hosts. An RSV monoclonal antibody 
(palivizumab) is currently available for passive immunoprophylaxis in high-risk infants. 
Vaccines and antiviral agents are under development for the treatment and prevention 
of RSV, but none are licensed. The ability to challenge healthy volunteers with RSV 
could rapidly facilitate efficacy studies of future antivirals and vaccines. In addition, 
challenge studies would provide critical information on viral pathogenesis, including 
types of cells infected, mucosal and systemic immune response, and alterations in 
respiratory microbiota. Clinical trial material for human challenge studies has been 
prepared from live recombinant (complementary DNA-derived) RSV of subgroup A 
(RSV A2).
This study will be a phase 1 study in healthy adult male and non-pregnant female 
subjects 18 years to 50 years of age. The main purpose of the trial is to define the 
safety profile, determine the frequency of RSV shedding in nasal wash, estimate RSV 
illness rates, and study immune responses in subjects given 1 dose of 107 PFU of RSV 
A2 challenge virus using a nasal atomizer. If RSV A2 is found to be sufficiently 
infectious in adults, then it may be used as a challenge virus in future studies evaluating 
antivirals or the protective efficacy of RSV vaccines, or in studies of the 
immunopathogenesis of RSV infection.
Subjects will be admitted to the NIH Clinical Center and receive a single intranasal dose 
of 106.3 PFU or 107 PFU of RSV A2. Subjects will remain at the Clinical Center for 
approximately 9-14 days after challenge infection undergoing sequential clinical 
evaluations. Research specimens, nasal washes and blood, will be collected for various 
research assays. Subjects will be discharged when their daily nasal wash RSV result is 
negative for two days in a row, and they do not have any signs or symptoms suggestive 
of possible RSV-associated lower respiratory tract disease. Subjects will return for 
follow-up evaluation 28 and 56 days after viral challenge. 

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1 Background Information and Scientific Rationale
1.1
Background Information
RSV is an enveloped, single-stranded, negative-sense RNA virus that belongs to the 
Pneumovirus genus of the subfamily Pneumovirinae of the family Paramyxoviridae.1 
RSV has two subgroups, A and B. Several genotypes exist within each subgroup. RSV 
subgroups can shift in predominance in 1- or 2-year cycles, which presumably is an 
advantage for heterologous strains in populations with prior immunity; however, 
reinfections by the same subgroup may occur frequently. RSV is spread by contact of 
the nasal or conjunctival mucosa with virus-contaminated large droplets or fomites. The 
incubation period is about 4 to 5 days (range 2 to 8 days). The virus initially replicates in 
the nasopharynx and frequently spreads to the lower respiratory tract, especially in 
primary infection. Viral shedding from the respiratory tract usually occurs from 3 to 14 
days, but may last for weeks in infants.1  
RSV is the most important cause of severe viral respiratory tract disease in the pediatric 
population worldwide. Approximately 50%-70% of infants are infected with RSV during 
the first year of life, and nearly all children have been infected by age 2-3 years.1 In the 
U.S., the National Respiratory and Enteric Virus Surveillance System (NREVSS) of the 
Centers for Disease Control and Prevention (CDC) monitors the seasonal occurrence of 
RSV. From 2012 to 2014, RSV was associated with an estimated 57,527 
hospitalizations and 2.1 million outpatient visits among children < 5 years of age.2 
Worldwide, in children < 5 years of age, RSV was estimated to account for at least 
3.4 million yearly hospitalizations for acute respiratory tract disease and up to 200,000 
deaths each year.3 Reinfection by RSV occurs throughout life without need for antigenic 
change. Reinfection is particularly common during the first few years of life.4 
RSV is an important cause of morbidity and mortality in the elderly. Among adults > 65 
years of age, RSV accounts for approximately 10,000 deaths annually in the U.S.5 RSV 
also causes wintertime respiratory illnesses among older adults living in long-term care 
or attending daycare, with infection rates of 5%-10% in nursing home residents per 
year, complicated by pneumonia and death at rates of 10%-20% and 2%-5%, 
respectively.6 In temperate climates, seasonal outbreaks begin in the fall and continue 
through winter and spring (November to April). There is presently no known viral 
reservoir. 

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Disease manifestations vary greatly, ranging from mild upper respiratory tract illness to 
fever, otitis media, and mild to severe lower respiratory illness with bronchitis, 
bronchiolitis (inflammation of the small airways), or pneumonia. In children under 1 year 
of age, RSV is the most common cause of bronchiolitis. When infants and young 
children are exposed to RSV for the first time, 25%-40% will show signs of a lower 
respiratory tract illness and 0.5%-2% will require hospitalization.1 Infants at highest risk 
of a serious infection are those who are premature or have congenital heart or lung 
disease or abnormalities of the airways, or neuromuscular disease that compromises 
management of pulmonary secretions. Reinfection in healthy older children and adults 
usually involves only a common cold syndrome, although there can be lower respiratory 
tract involvement. RSV is an important cause of severe respiratory illness in the elderly 
and in high-risk adults who have chronic lung and/or heart disease or compromised 
immune systems. 
An RSV monoclonal antibody (palivizumab) was licensed in June 1998 by the Food and 
Drug Administration (FDA) for passive immunoprophylaxis in high-risk infants. Vaccines 
and antiviral agents are under development for the treatment and prevention of RSV, 
but none are licensed. There have been a number of obstacles to developing antiviral 
drugs and vaccines.1 One obstacle is that RSV grows only to moderate titer in cell 
culture and is notoriously prone to lose infectivity during handling, which complicates 
non-clinical and clinical studies. RSV is only semi-permissive in available experimental 
animals, so evaluation of the properties and efficacy of vaccines and antiviral drugs 
depends on clinical studies. In this regard, the availability of a challenge model in adults 
would be an important advance. The development of vaccines for pediatric use has 
been impeded by reduced immune responses in infancy due to immunologic immaturity 
and the immunosuppressive effects of maternal antibodies. The development of 
effective vaccines also is impeded because RSV replicates and causes disease in the 
superficial cells of the airway epithelium, where immune protection is less effective. In 
addition, a formalin-inactivated RSV vaccine in trials in the 1960s caused immune-
mediated disease enhancement in RSV-naïve recipients, and the same appeared to be 
true for subunit RSV vaccines in experimental animals. Therefore, inactivated and 
subunit vaccines have been considered to be contraindicated for pediatric use, where 
the vaccine need is greatest. However, live-attenuated and vectored vaccines have 
been shown to be safe for pediatric use and are under development, but identifying a 
suitably attenuated and satisfactorily immunogenic strain has been challenging. 

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A better understanding of viral pathogenicity and the host response to RSV infection in 
humans would aid efforts for improved RSV therapy and immunoprophylaxis. The 
correlates of immunity to RSV infection are not clearly defined. In adults, protection 
against reinfection is believed to be due to: (a) cytotoxic T lymphocytes, which likely 
contribute a short-term protection against reinfection during the same season; (b) local 
secretory immunoglobulin A (IgA) antibodies, which appear to play the most important 
role in short-term and long-term protection; and (c) serum antibodies, which confer 
durable protection that ameliorates severity but incompletely prevents reinfection. 1
Data from this and future RSV challenge studies will enhance our understanding of the 
immune response to RSV infection and facilitate efficacy trials for RSV antiviral and 
vaccine candidates, thereby expediting their potential applications to clinical use. 
1.1.1
Description of the Study Agent/Intervention
The RSV sequence on which the study agent, RSV A2, is based was derived from RSV 
strain A2, initially isolated from a child in Melbourne, Australia, in 1961.7 RSV strain A2 
was isolated using human embryonic kidney cells (HEKs). The study agent of this IND 
is a recombinant version of RSV A2 that was derived de novo from synthetic 
complementary DNA (cDNA).8 Therefore, it is free from any adventitious agents that 
could have been present at the time of virus isolation and has a short, well-defined 
passage history. The sequence of the clinical trial material (CTM), RSV A2, corresponds 
to the sequence of the original isolate, passaged seven times in HEK cells, except for 
13 nucleotide differences.9 Eleven of these nucleotide differences are silent on the 
amino acid level and considered to be phenotypically inconsequential. Specifically, 4 of 
the changes involve nucleotide differences which are considered incidental (C6221T; 
T6386C; T10514G; T13900C). Six nucleotide differences were introduced deliberately 
to generate marker restriction sites in noncoding regions of the genome (insertion of a C 
residue [C1099.1], A1138C, G1139C, A5611G, A5615T, A7559C). Another change 
involves a C4G substitution that was introduced deliberately near the beginning of the 
genome because it improves recovery from cDNA but appears to have no effect on 
virus replication or gene expression. In addition to these 11 non-coding changes, there 
are 2 nucleotide substitutions that result in amino acid changes in the F protein (E66K 
and P101Q). These changes modestly increase replication in cell culture (and appear to 
represent an adaptation to cell culture).

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1.1.2
Summary of Previous Pre-Clinical Studies 
Chimpanzees are the only nonhuman host that approaches humans with respect to 
susceptibility to RSV infection, replication, and disease. Virus replication of recombinant 
RSV A2 of identical genomic sequence to the CTM of this study was compared to 
replication of a biologically derived RSV A2 in RSV-naïve chimpanzees in two historic 
studies.10,11 These studies were approved by the NIAID Animal Care and Use 
committee, and performed more than 10 years before the 2011 recommendations of the 
Institute of Medicine on restricting the use of chimpanzees in biomedical research. In 
both studies, replication of recombinant RSV was similar to that of the biologically 
derived RSV A2, both in the upper and lower respiratory tract. RSV A2 caused 
moderate rhinorrhea and cough in chimpanzees, similar to biologically derived RSV A2 
(Table 1 10 and Table 2 9). RSV A2 induced high serum neutralizing titers to RSV (Table 
2 9). These results showed that RSV A2 had an in vivo phenotype similar to that of 
biologically derived RSV A2. 
Table 1
The in vivo phenotype of RSV A2 is similar to biologically derived 
wild-type (wt) RSV in chimpanzees (From: Whitehead et al.)
Mean virus titer (log10 PFU/ml)
Nasal wash sample
Tracheal lavage 
sample
Rhinorrhea 
scored
Virus used to 
infect animala
Chimpanzee 
no.b
Peak titer Daily titerc Peak titer Daily titerc Peak
Mean
Days 
with 
cough
1
4.9
3.7
3.4
3.2
3
1.1
4
2
5.1
3.1
4.6
4.5
4
2.0
4
3
4.4
3.9
4.0
2.5
3
1.4
5
Recombinant 
RSV
A2 (D46)
4
4.6
3.5
4.7
3.0
4
2.6
5
Mean ± SE
4.8 ± 0.16 3.6 ± 0.15
4.2 ± 0.30
3.1 ± 0.33
3.5
1.8
4.0
5b
4.6
3.6
5.1
3.7
3
1.0
2
A2 wt 
(biologically 
derived)
6b
5.3
3.6
5.9
4.0
3
2.1
0
Mean ± SE
5.0 ± 0.35 3.6 ± 0.30
5.5 ± 0.40
3.8 ± 0.46
3.0
1.6
1.0
a 
RSV naïve chimpanzees were inoculated by the intranasal and intratracheal routes with 104 PFU of 
the indicated virus in a 1-ml dose per site. Nasal wash samples were collected daily for 10 days, and 
tracheal lavage samples were collected on days 2, 5, 6, 8, and 10. A2 (D46), recombinant RSV 
derived from plasmid D46.
b 
Historic control animal from the work of Crowe et al., 1994, Vaccine 12:783-90.
c 
Mean daily titers were computed for each chimpanzee by adding together the virus titers from days 
with detectable levels of virus (≥0.7 PFU/ml) and dividing the sum by the number of days with 
detectable virus titer. 
d    The amount of rhinorrhea was estimated daily and assigned a score (0 to 4) that indicated extent and 
severity. Mean rhinorrhea scores represent the sum of scores during the 8 days of peak virus 
shedding divided by 8.

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Table 2
The in vivo replication, pathogenicity, and immunogenicity of 
recombinant RSV A2 is similar to that of biologically derived 
wild-type (wt) RSV in chimpanzees (From: Whitehead et al.)
Mean peak virus titer 
(log10 PFU/ml)
Rhinorrhea 
scorec 
(range, 0–4)
Mean serum 
neutralizing antibody 
titer (reciprocal log2)
 on day:
Virus used to 
infect 
chimpanzeesa
No. of 
animals
Nasopharyngeal 
swab specimen
Tracheal 
lavage fluid Peak Mean
0
28
Recombinant RSV
A2 (D46)
2
4.9 ± 0.15
5.4 ± 0.05
2.5
1.3
<3.3
10.5
wt RSV A2 
(biologically 
derived)
2b
5.0 ± 0.35
5.5 ± 0.40
3.0
1.4
<3.3
11.2
a 
RSV naïve chimpanzees were inoculated by the intranasal and intratracheal routes with 104 PFU of 
the indicated virus in a 1-ml dose per site. Nasal wash samples were collected daily for 10 days, and 
tracheal lavage samples were collected on days 2, 5, 6, 8, and 10. A2 (D46), recombinant RSV 
derived from plasmid D46.
b 
Historic control animal from the work of Crowe et al., 1994, Vaccine 12:783-90.
c 
The amount of rhinorrhea was estimated daily and assigned a score (0 to 4) that indicated extent and 
severity. rhinorrhea scores represent the sum of scores during the 8 days of peak virus shedding 
divided by 8. 
The RSV A2 CTM and an experimental RSV A2 virus derived from the same cDNA as 
the CTM were also characterized in nonhuman primates studies of RSV vaccine 
candidates in RSV-seronegative African Green Monkeys (AGMs). AGMs (n = 4) were 
dosed by intranasal and intratracheal inoculation at a dose of 2 × 106 PFU (equivalent to 
106.3 PFU). Serum was obtained on days 0, 21, and 28 post-immunization to determine 
the neutralizing antibody response. Nasopharyngeal (NP) swabs were collected daily on 
days 0 through 10 and day 12, and tracheal lavage samples were collected every other 
day from day 2 through day 12 from all animals included in the study. In this study and 
in several independent studies, RSV A2 replicated over 6 to 11 days in the upper 
respiratory tract, and up to 13 days in the lower respiratory tract of AGMs. Virus 
shedding from the upper respiratory tract ranged from 3.9 to 5.4 log10 PFU/ml, and 
shedding from the lower respiratory tract ranged from 3.0 to 5.5 log10 PFU/ml. 
Compared to the attenuated RSV vaccine strains, RSV A2 replicated to higher titers in 
nonhuman primates. RSV A2 was immunogenic, inducing serum neutralizing antibody 
titers of 6.7-10 log2 in naïve AGMs by day 28.

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1.1.3
Summary of Relevant Clinical Studies
Eleven studies of safe experimental infection with RSV in healthy human volunteers 
have been reported in the literature, dating back to 1961.12-18;20-12;23-25 Experimental 
infection of healthy adult volunteers (with low pre-challenge RSV titers) using RSV 
strains A and B in the dose range of 3.7-5.4 log10 PFU have resulted in 70%-92% viral 
shedding from the upper respiratory tract. When reported, nasal, sinus, and ear 
symptoms were noted in 38%-70% of people with evidence of viral shedding. The 
experimental RSV challenge model produces a phenotype of respiratory illness that is 
similar to that seen in seasonally occurring RSV infection of healthy working adults. In 
177 previously healthy working adults with RSV infection, the most frequently reported 
symptoms involved the nose, ear, throat, and sinus, and 85% of subjects also reported 
cough.19
AEs reported during RSV human challenge studies have included transient mild 
pulmonary function test (PFT) abnormalities, suppression of neutrophils and platelets, 
and liver enzyme elevations. Headache was the most commonly reported systemic 
complaint. In detail, the alanine aminotransferase (ALT) level was abnormal in 2 of 12 
subjects receiving a 104.7-PFU dose of RSV A2.13 The ALT level normalized by day 28. 
AEs also occurred in subjects receiving a 104-PFU dose of RSV A.20 The subjects in 
this challenge study had minor PFT abnormalities (5/53), and mild lab abnormalities 
with elevated ALT level (5/53), elevated aspartate aminotransferase (AST) level (8/53), 
reduced absolute neutrophil count (ANC) (3/53), and reduced platelets (1/53). Fever 
was not reported in any prior challenge subjects (several studies allowed for 
acetaminophen treatment). Overall, prior challenge studies with RSV have revealed that 
participants acquire an upper respiratory illness similar to that seen in the community. 
Importantly, no significant AEs related to challenge infection with RSV have been 
reported.
We previously conducted a phase 1 challenge study with RSVA2 in healthy volunteers 
18-50 years of age (NIH protocol number 15-I-0148; Clinical Trials Identifier 
NCT02484417). Cumulatively, we challenged 25 volunteers. Three volunteers in cohort 
1 and 10 in cohort 2 were challenged with the low dose of RSV A2 (105 PFU) using 
intranasal droplets. Five volunteers in cohort 3a and 7 in cohort 3b were challenged with 
a higher dose of RSV A2 (106.3 PFU) using intranasal droplets. Seven of 13 participants 
in low dose cohorts 1 and 2 had RSV shedding detected by RT-PCR or culture of nasal 
wash on any day, day 2 or beyond, after challenge infection with RSV A2, resulting in a 

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shedding frequency of 54%. We observed a total of 3 RSV-associated upper respiratory 
tract infection (URI) events (1 moderate illness and 2 mild illnesses), as determined by 
our RSV illness study definition. Hence, the frequency of mild to moderate respiratory 
RSV illness for the low dose group was 23%. Our third cohort of healthy volunteers that 
received the high dose of RSV A2 (106.3 PFU) was admitted to the Special Clinical 
Studies Unit (SCSU) in two separate groups: cohort 3a + cohort 3b. Three of 11 
evaluable participants in the entire cohort 3 had RSV shedding detected in nasal wash 
by culture and/or RT-PCR for a shedding frequency of 27%. Only 2 of the 3 participants 
had respiratory symptoms associated with RSV shedding and met the study definition of 
RSV illness, resulting in an illness frequency of 18%. The most common expected AEs 
related to RSV illness were nasal congestion/rhinorrhea (50%), headache (42%), sore 
throat (29%), cough (25%), and sinus congestion/pain (17%). The majority of symptoms 
were of mild-moderate severity (Grade 1 or 2), except for 1 episode of sinus congestion 
that was Grade 3. Unexpected AEs consisted of other symptoms that may occur with an 
URI, such as sneezing (32%), nausea (24%), itchy or irritated throat (16% each), watery 
eyes (20%), and decreased appetite (16%) that were of mild (Grade 1) severity. Five 
participants had episodes of epistaxis (16%) of Grade 2 or 3 severity that were possibly, 
probably or unlikely related to the nasal wash, nasal swab, or RSV infection. No serious 
AEs (SAEs) occurred during the entire study. Per the study design, since the RSV 
illness criteria were not met for the high dose cohort (>3 subjects/10-12 volunteers), 
additional subjects were not dosed with the high dose. We concluded that challenge 
infection with 105 PFU and 106.3 PFU RSV A2 appears to be safe in healthy volunteers. 
Disease manifestations were limited in healthy adults because of pre-existing immunity. 
The participants with RSV shedding in nasal wash had a significantly lower pre-dose 
mean neutralizing antibody titer compared to participants with no shedding detected 
(mean neutralizing antibody titer, 6.7 reciprocal log2 versus 8.3 reciprocal log2, 
P=0.0011). The same applied to RSV illness: Participants with RSV illness had a 
significantly lower pre-dose mean neutralizing antibody titer compared to participants 
without RSV illness [6.3 reciprocal log2 (log(2)) versus 8.0 log(2) , P=0.0041]. The pre-
dose mean serum F protein IgG was not significantly different in patients with or without 
RSV shedding or illness. 
The rationale for selecting 105 PFU as the starting dose for our first RSV challenge 
study (NIH protocol number 15-I-0148) was based on existing safety data from prior 
human and non-human primate challenge studies. Buchman and colleagues challenged 
32 healthy volunteers with a 106-PFU dose of RSV B without reporting safety issues; 

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however, their study population was not examined for pre-existing immunity to RSV.21 
The highest dose reported in the literature for RSV challenge in healthy adults is 106 
PFU. The next highest RSV challenge dose is 105.4 PFU of RSV A Memphis strain. The 
35 healthy adults receiving this dose had RSV neutralization titers in the lower tertile of 
the population and the study finished without safety complications. Also in pre-clinical 
studies, no substantial AEs were seen in non-human primates challenged with RSV A2 
at doses of 104 PFU (chimpanzees) or 106.3 PFU (AGMs) (unpublished data 
Buchholz).10,11 
Two prior human adult challenge infection studies specifically using RSV A2 were 
conducted using doses of 103 PFU or 104.7 PFU.13,14 In the study by Noah and 
colleagues, 3 of 9 subjects (33%) developed symptomatic upper respiratory illness and 
had evidence of RSV replication in the nasal lavage fluid after challenge with 103 PFU.14 
In the study by Lee and colleagues using a higher dose (104.7 PFU ), 12 of 13 subjects 
(92%) with low serum neutralizing antibody activity shed RSV A2 after challenge.13 Of 
these subjects, 92% displayed upper respiratory symptoms but none developed lower 
respiratory disease. This study provides evidence that development of RSV-induced 
respiratory illness after challenge infection can be done successfully and safely in 
healthy adults with low pre-inoculation serum anti-RSV antibody titers without 
complications of lower respiratory tract disease. Please refer to Appendix D for a 
summary table of completed RSV challenge studies. 
We also selected a dose of 106.3 PFU for our first RSV challenge study, which is higher 
than the previously reported 106 PFU dose used in human challenge.21 Safety was 
acceptable for the 106.3 PFU dose of RSV A2 CTM in non-human primate toxicity 
studies (unpublished data, Buchholz). Since RSV A2 CTM is a recombinant virus 
passaged through Vero cells, there is theoretical concern that human infectivity may be 
reduced.22 Therefore, we chose to evaluate safety of the 106.3 PFU dose along with the 
105 PFU, in case a high titer challenge virus is necessary to overcome a potential loss of 
infectivity in the setting of pre-existing RSV immunity in healthy volunteers. In addition, 
prior chimpanzee studies have revealed the RSV A2 CTM to be phenotypically similar in 
vivo to the biologically derived RSV A2 strain. For this reason, we do not anticipate 
increased pathogenicity at this higher dose. 
In this study, we propose to infect healthy volunteers who have low pre-challenge RSV-
specific neutralizing antibody titers with recombinant RSV A2 using a nasal atomizer 
(MAD Nasal Atomizer, Teleflex). This atomizer has been used safely and successfully 

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to challenge healthy volunteers with wild-type influenza virus at the NIH Clinical 
Center.28 In addition, FluMist is a quadrivalent influenza vaccine that is administered 
with a nasal atomizer that produces a fine mist primarily deposited in the nose and 
nasopharynx. The MAD Nasal Atomizer has been developed as a safe and painless 
way to deliver medications with rapid absorption across mucosal membranes. Delivery 
via the intranasal route is relatively painless and easy to perform with minimal training. 
For example, intranasal medication delivery is an effective method for delivering 
analgesia, anxiolysis, and anticonvulsants to pediatric patients.26 
The MAD Nasal Atomizer creates a fine mist of particles 30-100 microns in size, which 
are optimized for deposition across a broad area of mucosa. Administering part of the 
dose in each nostril doubles the targeted mucosal surface that the challenge virus can 
infect. A soft conical plug that is part of the atomizer forms a seal with the nostril, 
preventing expulsion of some of the inoculum via the nostril, which occurred with a few 
participants in the first challenge study when the virus was administered intranasally 
using droplets. In addition, the atomization should prevent the dose from dripping down 
into the oropharynx, which was reported by several participants during the first 
challenge study. No AEs related to the MAD Nasal Atomizer occurred during the 
influenza virus challenge studies performed at the NIH Clinical Center (NCT01646138; 
NCT02594189).
 As noted above, our first challenge study resulted in an overall shedding frequency of 
42%, and frequency was higher in the cohorts that received the lower dose, 105 PFU, of 
RSV A2 (54%) versus the cohort that received the higher dose, 106.3 PFU (27%). The 
overall frequency of RSV illness for all cohorts was only 21%-23% for the low dose 
recipients and 18% for the high dose participants. Our goal is to increase the number of 
participants who shed RSV in nasal wash (RSV shedding is defined as RSV detected in 
NP wash by PCR and/or viral culture on 2 or more days from day 2 post-challenge or 
later)  or meet the RSV illness study definition (Section 3.1.2) so that our challenge 
model can be utilized to test antivirals and possibly vaccines in future studies. Our 
hypothesis is that including an RSV neutralizing antibody titer cut-off in the eligibility 
criteria for our study participants and administering a higher dose of RSV A2 using a 
nasal atomizer to provide a larger surface for infection will result in a higher incidence of 
RSV shedding and a higher frequency of RSV illness after challenge.

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1.2
Rationale
This RSV challenge model addresses several unmet scientific and medical needs: 1) to 
provide a better understanding of the pathogenesis of and immunity to RSV infection in 
humans, 2) to expedite efficacy trials of new RSV therapeutics and possibly vaccines, 
and 3) to characterize the nasal microbiota in healthy adults before and after infection 
with RSV. 
The common goals within the RSV research community are to ultimately reduce 
morbidity and mortality from RSV infection in vulnerable hosts (infants, elderly, immune 
compromised, and people with chronic cardiopulmonary diseases). In vitro models for 
RSV are well established. However, development of targeted RSV therapies and testing 
these agents’ potential for efficacy and toxicity are better studied in vivo in humans. 
Human, healthy volunteer challenge models offer the ability to generate high quality 
data, minimize confounding variables, and study viral kinetics and human immune 
responses to RSV. Prior RSV challenge studies have been conducted without 
significant AEs. Because healthy volunteers have naturally acquired RSV immunity that 
puts them at low risk for untoward outcomes, we aim to use data gathered in this 
population to enhance the development of RSV antivirals and possibly vaccines that will 
eventually benefit those at greatest risk for severe RSV disease. Data collected from 
this and future challenge studies will help guide development and testing of RSV 
antivirals, vaccine candidates, and potentially novel treatment strategies.
This new phase 1 RSV challenge study seeks to optimize and improve the use of RSV 
A2 as a challenge virus in healthy adult volunteers by: 1) increasing the titer of the virus 
challenge dose by approximately five-fold; 2) pre-screening potential participants using 
a RSV neutralization titer cut-off (a more stringent criterion for establishing 
susceptibility); and 3) intranasal delivery of virus via a nasal atomizer (instead of by 
liquid droplets). 
The neutralizing antibody titer cut-off was established based on data from our first RSV 
challenge study (15-I-0148) that showed a statistical association of low neutralizing 
antibody titer with RSV shedding and RSV illness, as described above. To determine 
the optimal cut-off for neutralizing antibody eligibility titer, the proportion of estimated 
shedders for various antibody titer cut-offs was determined along with the estimated 
proportion of people who would meet that criteria during screening (Table 3). We initially 
chose a neutralizing titer cut-off of < 6.8 log (2)  and then, < 7.04 log (2) to balance the 

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estimated number of volunteers that will need to be screened to achieve an acceptable 
RSV shedding rate. However, our screening RSV neutralizing antibody titer data reveals 
that only approximately 16% of our screened volunteers have an eligible RSV titer.  An 
increase of the eligibility titer cut-off to < 8.00 log (2)  will increase the percentage of 
potentially eligible volunteers to approximately 40%, enabling us to enroll the study and 
answer the question of whether RSV A2 is a safe and suitable virus for challenge 
studies.
Table 3 Statistical analysis for determining RSV neutralizing antibody eligibility 
titer cut-off based on shedding at any time after challenge
2 Study Objectives
2.1
Primary Objective
Determine the safety and infectability of high dose, recombinant RSV A2 
administered by nasal atomizer to healthy adult volunteers.
2.2
Secondary Objectives
1. Characterize the onset, duration, and magnitude of viral shedding after RSV A2 
challenge infection.
2. Determine the frequency of mild to moderate upper respiratory illness in healthy 
volunteers challenged with RSV A2.
Neutralizing antibody titer, 
[log2]
Estimated proportion of 
shedders who meet criteria 
(CI)
Estimated proportion of 
screened volunteers who 
will meet criteria (CI)
≤ 92 (6.53)
1 (1, 1)
0.208 (0.046, 0.371)
≤ 118 (6.88)
0.875 (0.646, 1)
0.333 (0.145, 0.522)
≤ 132 (7.04)
0.788 (0.506, 1)
0.375 (0.181, 0.569)
≤ 151(7.24)
0.667 (0.4, 0.94)
0.5 (0.3, 0.70)

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3. Evaluate the duration and severity of clinical illness after RSV A2 challenge 
infection.
4. Determine the relationship between the magnitude and duration of viral shedding 
and the severity and duration of RSV illness.
5. Characterize RSV-specific serum and mucosal antibody responses after RSV A2 
challenge infection.
2.3
Exploratory Objectives
1. Characterize cell-mediated immune responses after RSV A2 challenge infection.
2. Determine cytokine and chemokine levels in serum and NP wash samples after 
RSV A2 challenge infection.
3. Characterize RNA gene expression patterns after RSV A2 challenge infection.
4. Characterize the phenotype of peripheral blood mononuclear cells after RSV A2 
challenge infection.
5. Characterize the nasal microbiome variations in healthy adults before and after 
RSV A2 challenge infection.
3 Study Design
3.1
Description of the Study Design
This is our second RSV challenge study with a goal of testing a higher dose of live 
recombinant RSV A2 and administering the dose using a nasal atomizer to healthy 18- 
to 50-year-old volunteers. The primary co-objectives are the safety of this approach and 
the frequency of shedding. The secondary objectives include description of the 
frequency and severity of illness and assessment of various immune responses after 
challenge. 
The first part of the study will be the challenge phase, during which the volunteers will 
be admitted to the NIH Clinical Center Hospital. The follow-up phase will consist of 
outpatient visits at Day 28 and Day 56 after challenge. 
We plan to administer 107 PFU of RSV A2 with a nasal atomizer to 21 healthy adult 
volunteers with low pre-challenge RSV neutralizing antibody titers. Since this is the first 
time we will use the nasal atomizer for administration of the 107 PFU dose, we will 
initially challenge 4 study participants with 106.3 PFU of RSV A2 (cohort 1), the highest 

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dose determined to be safe in the first RSV challenge study (15-I-0148). We will assess 
the participants for AEs and symptoms of RSV illness on a daily basis post-challenge. 
Nasal swabs, nasal washes, and blood samples will be obtained post-challenge for 
assessment of RSV shedding and immune responses, as noted in the schedule of 
procedures (Appendix B). The participants will remain as inpatients for a minimum of 9 
days and will be discharged after 2 negative RSV RT-PCR results using the FilmArray 
assay on consecutive days and if they do not have any symptoms suggestive of lower 
respiratory tract disease. 
After cohort 1 is discharged from the inpatient isolation unit, the safety data will be 
reviewed by the SMC.  If these participants do not experience any significant AEs 
related to the study agent or its administration, then our next 3 cohorts (cohorts 2-4) will 
be sequentially enrolled into the inpatient isolation unit.  Seven or fewer volunteers per 
group will be accrued for each inpatient admission until the maximum cohort size (n=7) 
for each cohort is achieved. Each participant in cohorts 2-4 will receive 107 PFU of RSV 
A2. After discharge of all participants in each cohort from the inpatient unit, the SMC will 
review the safety data for that particular cohort to determine if it is safe to proceed with 
the next cohort.  
Upon discharge from the inpatient unit, the participants will receive diary cards to enter 
symptoms or signs that arise in the follow-up period and will attend follow-up outpatient 
visits on Day 28 and Day 56 after challenge for assessment of safety and follow-up 
immune studies. 
3.1.1
Definitions of RSV Illness
RSV-associated upper respiratory illness for this study is defined as the presence of 
at least 1 major symptom or 2 minor symptoms that last 2 or more consecutive days. 
Additionally, there must be evidence of RSV shedding on 2 or more days as detected by 
culture and/or PCR of NP wash specimens collected on day 2 post-challenge or later.
Major symptoms were selected based on the frequent occurrence (>50%) of these 
upper respiratory symptoms during RSV infection among previously healthy working 
adults in a natural history study19 and include:

Nasal congestion or rhinorrhea

Sinus congestion or pain

Sore throat

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
Ear pain
Minor symptoms, based on the less frequent occurrence (<50%) of these symptoms in 
the natural history study, include:

Fever, with temperature ranging from 38°C (100.4°F) to 39.4°C (103°F)

Headache

Cough

Hoarseness
Mild upper respiratory illness: RSV-associated upper respiratory illness as defined 
above and upper respiratory signs or symptoms are no greater than Grade 1 severity. 
There is no evidence for clinically significant lower respiratory tract illness 
characteristics of which may include bronchospasm/wheezing, pneumonia, bronchiolitis, 
and respiratory distress that can manifest as increased work of breathing, increased 
respiratory rate, and oxyhemoglobin desaturation.
Moderate upper respiratory illness: RSV-associated upper respiratory illness as 
defined above and upper respiratory signs or symptoms are of Grade 2 or Grade 3 
severity. There is no evidence for clinically significant lower respiratory tract illness, as 
described above.
Severe respiratory illness: 

Any symptom of Grade 4 severity

Any evidence for clinically significant lower respiratory tract disease as noted 
above

Any acute changes in health that are deemed clinically significant by the 
examining physician. 
Sub-clinical illness: Presence of RSV shedding (detected in NP wash by RT- PCR or 
viral culture for 2 or more days on Day 2 post-challenge or later) and no clinical 
symptoms.
Symptom Flu-PRO questionnaire: We will use the FLU-PRO (the inFLUenza Patient-
Reported Outcome) instrument with additional questions (Appendix F) to evaluate the 
suitability of this comprehensive patient-reported outcome (PRO) measure to assess 
RSV illness symptoms in clinical research. The questionnaire will be administered to 

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study participants on Day -1 to establish a baseline and then on a daily basis to each 
patient while inpatient, beginning with Day 1. Participants will receive an email 
generated by the REDCaps project into their personal email accounts (the REDCaps 
project will be managed by CRIMSON personnel).  The email will contain a link to the 
questionnaire.  If a participant is not able to access the questionnaire via their email 
then a paper questionnaire will be provided to them and the results entered by the 
research team into the REDCaps database.  A recent study revealed that the 37-item 
FLU-PRO questionnaire is a valid measure of influenza symptoms in adults with a 
confirmed diagnosis of influenza.27 We will begin to assess the suitability of FLU-PRO to 
evaluate symptoms of RSV illness, given the overlap of symptoms between RSV and 
influenza. Symptoms recorded on the FLU-PRO questionnaire by the study participants 
will not be utilized to assess and record adverse events.  Adverse events will be 
assessed during the daily interim history and physical exam when the participants will 
be asked about the presence or absence of the expected adverse events and will be 
asked open-ended questions to elicit other symptoms.
3.1.2
Study Phases
Inpatient phase: 
A volunteer participant is considered enrolled if eligibility criteria continue to be met at 
Day 0.  Subjects will receive one intranasal inoculation of RSV A2 using a nasal 
atomizer on Day 0 and will be evaluated daily for microbiologic evidence of RSV 
infection and for clinical signs and symptoms. Daily safety assessments will also be 
performed. In addition, the RSV RT-PCR results (FilmArray) obtained from NP washes 
will be double-blinded from Day 0 through the duration of the inpatient stay to minimize 
bias during assessment of symptoms post-challenge.  
If a participant is withdrawn for any reason within the 3 business days prior to 
admission, then efforts will be made to maintain the intended cohort size by identifying 
another volunteer from a reserve pool of qualified participants. An optional “stand-by” 
participant may be enrolled in the inpatient cohort. The “stand-by” participant will be an 
eligible volunteer who is able to come on Day -1 for an abbreviated, outpatient, clinical 
evaluation, during which a nasal swab and wash will be performed under the LID 
screening protocol (#12-I-0121, Screening of Volunteers for Clinical Trials of 
Investigational and Licensed Vaccines, Antiviral Products, or Live Virus Challenge 
Studies) for which the participant has already been consented. If the nasal wash is 

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negative for respiratory pathogens as determined by the FilmArray assay and a 
scheduled volunteer withdraws from the cohort on Day -1 for any reason, after signing 
informed consent for the RSV study, the “stand-by” participant may be offered a position 
in the cohort on Day -1. 
Outpatient phase: 
All subjects will return for outpatient follow-up at 28 days and 56 days after viral 
challenge. 
3.2
Rationale for Study Design
In our first challenge study, we tested two different doses of RSV A2 in a dose-
escalation manner. Neither of these doses caused sufficient infectivity or illness in 
pre-immune healthy adults aged 18-50 years to establish a reliable challenge model. 
Therefore, we have decided to try a higher dose of RSV A2 and administer this dose 
using a nasal atomizer to attempt to increase the frequency of viral shedding and mild-
moderate illness amongst our healthy volunteers. We plan to challenge 25 healthy 
volunteers who have lower RSV neutralizing antibody titers, which correlated with viral 
shedding and RSV illness in our first challenge study. We hope that, with this sample, 
we will detect shedding in at least 55% of our study volunteers and have a higher 
probability of detecting SAEs in our study population (see Section 13.1). Our goal is to 
test that the higher dose administered using an atomizer is safe and to establish a 
sufficient frequency of RSV shedding in our study population to indicate that this 
approach may be useful to evaluate potential antivirals and vaccines in future studies.
3.3
Study Endpoints
3.3.1
Primary Endpoint
Assess expected and unexpected adverse events (AEs) throughout the trial and 
detect RSV shedding in nasal wash by FilmArray multiplex polymerase chain 
reaction (PCR) assay and by reverse transcription (RT)–quantitative PCR and 
quantitative viral culture after challenge with RSV A2
3.3.2
Secondary Endpoints
1. Determine day of onset of shedding, number of days of shedding, peak nasal 
wash viral titer, and mean sum of daily virus titers after challenge.

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2. Calculate the number of subjects with mild to moderate upper respiratory illness 
after challenge, based on study definition of RSV illness.
3. Determine the number of days RSV illness persists after challenge and degree of 
illness based on study definition.
4. Correlate the magnitude and duration of viral shedding with the severity and 
duration of RSV-related symptoms.
5. Determine the frequency, magnitude, and duration of serum and mucosal (nasal 
wash and/or nasosorption) RSV-specific antibody responses after challenge.
3.3.3
Exploratory Endpoints 
1. Perform transcriptome profiling of whole blood after RSV A2 challenge infection.
2. Perform fluorescence-activated cell sorting (FACS) analysis of peripheral blood 
mononuclear cells after RSV A2 challenge infection.
3. Perform next-generation sequencing using NP wash specimens before and after 
RSV A2 challenge to identify communities of microorganisms.
4. Perform functional RSV-specific T-cell assays to evaluate T-cell responses after 
challenge.
5. Measure cytokine and chemokine concentrations in serum and NP wash 
samples and correlate with RSV shedding, illness, and clearance of RSV.
4 Study Population
4.1
Rationale for Subject Selection
Because we will be infecting subjects with a virus that has the potential to cause 
substantial illness in people with underlying pulmonary or chronic debilitating conditions, 
we will enroll only healthy volunteers. We have selected the age group from 18 to 50 
years because during this stage, most people have mature and robust immune 
responses. All adults have experienced prior natural RSV infections and are, as a result, 
seropositive to RSV. 
4.2
Recruitment Plan 
Healthy subjects will be recruited from within a 350-mile radius of the NIH Clinical 
Center. Potential participants who live outside this radius may be eligible to participate, 
per the PI’s discretion, if they provide evidence of an ability to comply with follow up 
visits. For example, financial means are available, or family or friend support in the area.  
Subjects will respond to posted flyers, newspaper advertisements, Craigslist, Research 

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Match, Clinical Connection, the NIAID study web page, NIH employee list serve emails, 
and by telephone call or email to the clinical trial site. The clinical research staff will 
conduct an initial phone pre-screening, provide background information about the trial, 
and review basic inclusion and exclusion criteria before inviting potential subjects to 
undergo screening.
4.3
Subject Inclusion Criteria
1. Age 18-50 years inclusive.
2. General good health, without significant medical illness, physical exam findings, 
or significant laboratory abnormalities as determined by the investigator.
3. Willingness to stay confined to the inpatient unit for required study duration.
4. Willingness to have samples stored for future research.
5. Female subjects must be of non-childbearing potential (e.g., surgically sterilized 
[bilateral oophorectomy, bilateral tubal ligation, hysterectomy] or menopausal 
defined as 50 years old with amenorrhea for at least 1 year) or, if of child-bearing 
potential, they must agree to remain abstinent or if sexually active with a partner 
who can get them pregnant, they must have in place an effective method of 
contraception for at least 30 days prior to administration of the challenge virus 
and until 30 days after challenge virus administration:

intrauterine device (IUD) or equivalent

hormonal contraceptives (e.g., consistent, continuous use of contraceptive 
pill, patch, ring, implant, or injection)
o
if participant uses contraceptive pill, patch, or ring, they must also use a 
barrier method at the time of potentially reproductive sexual activity (e.g., 
[male/female condom, cap, or diaphragm] plus spermicide)

be in a monogamous relationship with a partner who has undergone a 
vasectomy at least 180 days prior to first dose of study agent
6. A plaque reduction RSV neutralization titer < 8.00 log (2).
4.4
Subject Exclusion Criteria 
The presence of any one of the following criteria is sufficient to exclude a prospective 
subject from enrolling in this study:
1. Subject who was previously challenged with RSV A2.

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2. Female subject who is pregnant or lactating OR planning to become pregnant 
from 30 days prior to inoculation through 30 days after inoculation.
3. Presence of self-reported or medically documented significant medical 
condition(s) including but not limited to:
a. Respiratory disease (e.g., chronic obstructive pulmonary disease, 
emphysema, rhinitis, sinusitis) in adulthood, and additionally:
i.
A history of asthma within the past 5 years, or a current 
diagnosis of asthma or reactive airway disease associated with 
exercise, seasonal hay fever or allergic rhinitis 
ii.
Presence of any febrile illness or symptoms suggestive of a 
respiratory infection within 2 weeks prior to inoculation.
b. Any significant abnormality of the nose or nasopharynx, including 
recurrent epistaxis within 90 days prior to viral inoculation or nasal or 
sinus surgery within 180 days prior to viral inoculation.
c. Chronic cardiovascular disease (e.g., congestive heart failure, 
cardiomyopathy, ischemic heart disease).
d. Chronic neurological or neurodevelopmental conditions (e.g., cerebral 
palsy, epilepsy, stroke, seizures).
e. Ongoing malignancy.
f.
Chronic medical condition requiring close medical follow-up or 
hospitalization during the past 5 years (e.g., diabetes mellitus, renal 
dysfunction, hemoglobinopathy, autoimmune disease).
g. An immunodeficiency.
4. Use of systemic corticosteroids exceeding 10 mg/day of prednisone equivalent 
and nasal steroid preparations or immunosuppressive drugs within 30 days 
before inoculation and within 60 days after. Low dose topical steroid preparations 
used for a discrete period of time are permitted.
5. Inhaled bronchodilator or inhaled steroid use within the last 360 days or use after 
upper respiratory tract infections.  
6. Behavioral or cognitive impairment or psychiatric disease that in the opinion of 
the investigator affects the ability of the subject to understand and cooperate with 
the study protocol.
7. Complete blood count (CBC), AST, ALT, creatinine values or other screening 
labs or tests (e.g. electrocardiogram [EKG], chest x-ray [CXR]) are outside of the 

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NIH Department of Laboratory Medicine normal reference range and deemed 
clinically significant by the PI. 
8. Positive FDA-approved HIV test obtained during screening procedures.
9. Positive serology for hepatitis C virus obtained during screening period.
10.Presence of hepatitis B surface antigen obtained during screening period. 
11.A smoker of tobacco products or a routine marijuana smoker currently or in the 
past year. 
12.Current alcohol abuse or addiction.
13.Current illicit drug abuse or addiction. 
14.Receipt of a licensed vaccine within 30 days  prior to RSV A2  inoculation and 
planned vaccination within 60 days after inoculation.
15.Receipt of blood or blood-derived products (including immunoglobulin) within 180 
days prior to viral inoculation. Receipt of packed red blood cells given for an 
emergent indication in an otherwise healthy person, and not required as ongoing 
treatment, is not exclusionary. 
16.Receipt of an investigational agent or vaccine within 90 days prior to scheduled 
RSV A2  inoculation and planned receipt within 60 days after inoculation.
17.A body mass index (BMI) ≤ 18.5 or ≥ 37.0.
18.A medical, occupational, or family problem that would preclude the participant 
from complying with all study requirements.
19.Shares household, works closely with, or has routine contact with a child 
(children) < 5 years of age or with immunocompromised individual(s), adult(s) 
with significant cardiopulmonary disease or asthma, institutionalized persons or 
persons with functional disability, or any other individual that, in the judgment of 
the PI, might be at increased risk for complications if exposed to RSV.
20.Deprived of freedom by an administrative or court order or in an emergency 
setting.
21.Any condition that in the opinion of the PI would jeopardize the safety or rights of 
a person participating in the trial or would render the person unable to comply 
with the protocol.
Co-enrollment Guidelines: For the first month of the study, co-enrollment in other trials 
is restricted. Co-enrollment in other trials examining investigational agents or devices 
within 90 days  prior to or 60 days following viral inoculation is restricted. After a subject 
completes the first month, they may co-enroll in trials that do not involve investigational 
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the immune system or upper and lower respiratory tract function. Study staff should be 
notified of subject’s desire to participate in another study as it may require approval by 
the PI.  
4.5
Justification for Exclusion of Special Populations
Exclusion of Women:
1. Pregnancy: Pregnant women are excluded from this study because the effects 
of RSV A2 on the developing human fetus are unknown with the potential for 
teratogenic or abortifacient effects. Although there is no evidence of an 
association between RSV infection and problems during pregnancy or harm to 
the fetus, for the sake of caution pregnant women will be excluded.
2. Breastfeeding: Because there is an unknown but potential risk for AEs in 
nursing infants secondary to challenge infection of the mother with RSV A2, 
breastfeeding women are excluded from this study.
Exclusion of Children: Because there are insufficient data regarding dosing or AEs 
available in adults to judge the potential risk in children, children are excluded from this 
study.
4.6
Recruitment of NIH Employees
NIH employees and members of their immediate families may participate in this 
protocol. We will follow the Guidelines for the Inclusion of Employees in NIH Research 
Studies and will give each employee a copy of the “NIH information sheet on Employee 
Research Participation.”

NIH staff may be a vulnerable class of participants.

Neither participation nor refusal to participate will have an effect, either beneficial 
or adverse, on the participant’s employment or work situation.

The NIH information sheet regarding NIH employee research participation will be 
distributed to all potential subjects who are NIH employees.

The employee subject’s privacy and confidentiality will be preserved in 
accordance with NIH Clinical Center and NIAID policies, which define the scope 
and limitations of the protections. 

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
For NIH employee subjects, consent will be obtained by an individual 
independent of the employee’s team. Those in a supervisory position to any 
employee and co-workers of the employee will not obtain consent. 

The importance of maintaining confidentiality when obtaining potentially sensitive 
and private information from co-workers or subordinates will be reviewed with the 
study staff at least annually and more often if warranted.
5 Study Agent/Interventions 
5.1
Disposition and Dispensation 
The RSV A2 challenge virus was manufactured by Meridian Life Sciences. RSV A2 is 
stored at Fisher BioServices (a NIAID-designated commercial repository). About 2 
weeks prior to admitting a cohort of healthy volunteers, the NIH Investigational Drug 
Management and Research Section pharmacist or PI will submit a Challenge Virus 
Request form to OCRPRO, who will request a shipment from Fisher BioServices. Upon 
receipt of the shipment by the NIH Pharmacy, the study agent will be stored in an 
access-controlled freezer at –80°C ± 15°C in the NIH Pharmacy. The RSV A2 will be 
diluted to dose with Lactated Ringer’s Solution for Injection (USP), obtained from the 
NIH Central Supply room and will be dispensed by the pharmacist on Day 0 as a one-
time dose (0.5 ml per nostril) using a 1-ml MAD Nasal Intranasal Atomizer (Teleflex, 
MAD300 atomizer). The study agent will be released to the study team after it is ordered 
in the Clinical Research Information System (CRIS) by the PI or by a designated study 
investigator and approved by a pharmacist. The challenge virus doses will be 
transported on wet ice to the inpatient unit and administered by the PI or by a 
designated study investigator. 
5.1.1
Formulation, Packaging, and Labeling 
Each 1-ml atomization device will be individually labeled with the subject ID number, 
expiration date, and time. The device carrier bag will be labeled with subject’s study ID 
number, the date the dose was dispensed, dosing instructions, recommended storage 
conditions, the expiration date and time, the name and address of the manufacturer, 
and the Investigational Use Statement (“Caution: New Drug – Limited by Federal [USA] 
Law to Investigational Use”).

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5.2
Study Agent Storage and Stability 
The challenge virus will be stored in an access-controlled –80°C freezer in the NIH 
Pharmacy at –80°C ± 15°C. The diluent (Lactated Ringer’s Solution for Injection, USP) 
will be stored at room temperature, as recommended by the supplier. Once thawed, 
RSV A2 must be diluted and used within 4 hours of thaw.
5.3
Preparation, Administration, and Dosage of Study Agent
Preparation: The dose of study agent will be prepared by the NIH research pharmacy 
as described in the Drug Fact Sheet developed by the research pharmacy. The 
concentration of stock RSV A2 is 107.3 PFU/ml. Lactated Ringer’s Solution for Injection, 
USP, will be used as  diluent.  Pharmacy personnel will prepare the dose of challenge 
virus for each study participant using aseptic technique under Biosafety Level 2 
conditions. The diluted challenge virus dose, 106.3 PFU for cohort 1 or 107 PFU for 
cohorts 2-4, will be drawn up in the atomization device 1-ml syringe.
Dosing and Administration: The research pharmacist will prepare and dispense one 
dose of either 106.3 PFU or 107 PFU for each enrolled participant in cohort 1 or cohorts 
2-4, respectively. In detail, the virus will be diluted to the appropriate concentration in 
Lactated Ringer’s Solution for Injection, USP.  Doses will be delivered using a 1-ml 
syringe atomization device. While supine, subjects will be dosed intranasally with 0.5 ml 
per nostril, administering a total RSV A2 dose of 106.3 PFU in one ml or 107 PFU in one 
ml. Doses will be administered by study staff trained to administer the study agent. 
Participants will be observed for 30 minutes after inoculation. 
Duration of Therapy: Single dose given on Day 0.
Use of Ancillary Medications/Over-the-Counter Products/Foods: Study staff will 
administer any concomitant medications, vitamins, or supplements as appropriate. 
Disposal of Used, Partially Used, and Unused Virus: Each vial of stock virus will be 
used to prepare doses only once and any unused agent will be discarded after use. 
Unused vials that have been thawed will also be discarded. Vials will be discarded by 
incineration following standard operating procedures of the NIH Pharmacy.
5.4
Assessment of Subject Compliance with Study Agent

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A one-time challenge virus dose will be directly administered to each subject 
intranasally using a nasal atomizer by the PI or other trained clinician on the study team. 
Doses of RSV A2 will be ordered in CRIS. The Clinical Research Information 
Management System of the NIAID (CRIMSON) will be the primary source for dose 
administration, where it will be documented in case report forms (CRFs). The dose 
administration will also be documented in CRIS.
5.5
Concomitant Medication and Procedures 
All concomitant prescription medications taken during study participation will be entered 
into CRIMSON. For this protocol, a prescription medication is defined as a medication 
that can be prescribed only by a properly authorized/licensed clinician. Medications to 
be documented in CRIMSON are concomitant prescription medications, over-the-
counter medications, and non-prescription medications taken at the time of AEs (all 
grades).
5.6
Prohibited Medications and Procedures 
Prohibited medications during the inpatient hospitalization include decongestants, nasal 
sprays (including nasal saline), antihistamines, expectorants, cough suppressants, anti-
inflammatory medications, throat lozenges, herbal remedies, or naturopathic remedies. 
Any participant requesting symptomatic relief for persistent symptoms associated with 
RSV illness that significantly interfere with usual activities will be evaluated as outlined 
in Section 6.2.1.
The interventions listed below have the potential to interfere with virus-induced immune 
responses and will be reviewed with the subjects during each outpatient visit. If a 
subject uses or receives any of these interventions during the study, then it will be noted 
in the subject's record. 
1. Use of any investigational drug or investigational vaccine other than the study 
article within 60 days after inoculation with RSV A2. 
2. Receipt of a licensed vaccine within 60 days after inoculation with RSV A2. 
3. Receipt of immunoglobulins and/or blood derived products, with the exception 
that packed red blood cells are not prohibited if/when given with emergent 
indications and not needed for ongoing therapy.
4. Administration of systemic corticosteroids exceeding 10 mg/day of prednisone 
equivalent within 60 days after inoculation with RSV A2.

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5. Nasal corticosteroids are prohibited after inoculation with RSV A2, but may be 
used after the Day 28 study visit.
6. Receipt of any immune suppressive or immune modulatory medications within 60 
days after inoculation with RSV A2.
6 Study Schedule and Clinical Procedures
The study schedule is provided in tabulated form in Appendix B. Total volumes of blood 
to be collected for research purposes are provided in Appendix C.
6.1
Screening 
Potential volunteers for the RSV challenge protocol will undergo screening after 
enrollment into the LID screening protocol #12-I-0121 (Screening of Volunteers for 
Clinical Trials of Investigational and Licensed Vaccines, Antiviral Products, or Live Virus 
Challenge Studies). 
To summarize, the following screening procedures will occur while the volunteer is 
enrolled in the screening protocol: 
Screen 1 Visit. (Day -180 to Day -14)

Review health history questionnaire

Vital signs (blood pressure, temperature, heart rate, respiratory rate, and pulse 
oximetry), height and weight

CBC with differential, acute care panel, mineral panel, and hepatic panel

Urine drug screen

Urine pregnancy test (females)

RSV-specific neutralizing antibody titer

HIV, hepatitis B, and hepatitis C testing

EKG

CXR
If any of the above tests were performed at NIH within 30 days of the Screen 1 Visit, 
and the investigators deem the results to be within limits of eligibility for the study, then 
duplicate screening labs do not need be repeated at the Screen 1 Visit. Volunteers 
deemed to be healthy and who meet eligibility criteria for the challenge study will 
continue to the Screen 2 Visit.

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Screen 2 Visit. This visit will occur between Days −56 to −2. The following procedures 
will be performed on this visit:

Complete medical history and physical examination 

Vital signs (blood pressure, temperature, heart rate, respiratory rate, and pulse 
oximetry)

Review and signing of reproductive counseling form.

PFT using standard equipment to measure forced vital capacity (FVC) and forced 
expiratory volume in 1 second (FEV1)

FLU-PRO symptom questionnaire (baseline)

NP wash for baseline cytokine assessment (see Section 7)

NP swab sampling may be performed for microbiome analysis (see Section 7)

Labs from the screen 1 visit may be repeated to follow-up on results outside of 
the laboratory reference range.
Screen 1 visit must occur prior to Screen 2 visit for all participants.
Volunteers will be contacted 2 weeks prior to Day −1 via email to provide instructions for 
arrival on Day −1. In addition, baseline health will be re-assessed by inquiring via email 
or phone whether there have been any changes in a volunteer’s health (e.g. new 
medical diagnoses/surgeries, unexpected visits to the doctor’s office, urgent care or ER) 
and if any new medications have been started.
 A volunteer who reports symptoms of an URI during the 2 weeks prior to inpatient 
enrollment may be offered an additional screening visit for NP wash and multiplex 
FilmArray PCR evaluation. If the volunteer has a positive result for a respiratory 
pathogen, then the volunteer may be offered a position in a future cohort, and the open 
spot in the upcoming cohort will be offered to another volunteer from a reserve pool of 
qualified participants. 
If the volunteer meets eligibility criteria for this study, then signed informed consent will 
be obtained at the screen 2 visit or afterwards, but prior to protocol-specific testing and 
procedures.
6.2
Inpatient Study Phase (Day −1 until discharge)
Subjects will be admitted to the Special Clinical Studies Unit in Clinical Center on study 
Day −1, where they will be familiarized with the study procedures and monitored for 

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signs or symptoms of acute illness that would preclude their further participation in the 
study. Visitors are not allowed in the Special Clinical Studies Unit where the study 
participants will remain hospitalized under isolation for the duration of the inpatient 
phase of the study.  Subjects manifesting any signs or symptoms of illness or who are 
now uncomfortable with or unwilling to adhere to study procedures will be discharged 
before administration of challenge virus. If a volunteer has a respiratory or febrile illness 
or a significant deviation of baseline health at the time of admission, as assessed by the 
PI, they will be discharged prior to virus administration. 
Admission to Clinical Center (Day −1)
1. Reconfirm or obtain signed informed consent, if not already obtained.
2. Record vital signs (blood pressure, temperature, heart rate, respiratory rate, and 
pulse oximetry), height, and weight.
3. Provide baseline FLU-PRO questionnaire to be filled out by subject.
4. Perform interim history and history-directed physical examination.
5. Obtain CBC with differential, acute care panel, and hepatic panel (these labs do 
not need to be repeated if they were obtained <56 days prior to Day −1)
6. Obtain urine drug screen.
7. Serum pregnancy test and pregnancy risk assessment (i.e., last menstrual period 
and any recent history of potentially reproductive sex that is too early to be 
detected) for subjects who are capable of becoming pregnant. A positive serum 
pregnancy test will exclude the subject from the trial. Suspicion of a possible very 
early pregnancy based upon recent unprotected, potentially reproductive sex 
may result in exclusion or deferral (i.e., until absence of pregnancy is assured) at 
the discretion of the PI. 
8. Review and signing of reproductive counseling form with female participants.
9. Obtain HIV rapid antibody screen.
10.Obtain hepatitis B and C serology testing, EKG, and CXR if obtained >120 days 
prior to Day -1; obtain PFT if performed > 56 days prior to Day −1.
11.Placement of synthetic absorptive matrices (SAM) in on naris for assessment of 
antibody and/or chemokine and cytokine levels in mucosal lining fluid.
12.Obtain NP wash for FilmArray assay, qualitative and quantitative viral culture, 
lab-based assay for RT-PCR, antibody assays, including secretory IgA, and 
cytokine/chemokine assays.
13.Obtain nasal swabs from each nares for microbiome analysis.
14.Obtain blood for HLA testing.

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15.Obtain blood for Epstein-Barr virus (EBV) serology (required for control for T-cell 
assays).
16.Obtain blood for quantitative immunoglobulins and research serum for total IgA 
and IgG.
17.Obtain blood for lymphocyte phenotyping panel T, B, and NK cell subsets.
18.Obtain research blood for RSV antibody and neutralization assays, T- and B-cell 
assays, transcriptome profiling, serum cytokine analyses, and for more extensive 
molecular immune cell phenotyping. 
Day of Inoculation (Day 0)
1. Perform interim history and directed physical examination, focusing on any acute 
complaints.
2. Obtain research blood for T- and B-cell assays, immune cell phenotyping, 
transcriptome profiling, and cytokine analysis. 
3. Record vital signs pre-inoculation and 30 ± 10 minutes post-inoculation.
4. RSV challenge virus inoculation: subjects in cohort 1 will receive 106.3 PFU of 
RSV A2 and subjects in cohorts 2-4 will be receive 107 PFU. Subjects will be 
supine and 0.50 ml of inoculum will be administered to each nostril. Subjects will 
be asked to remain supine for approximately 10 minutes.
5. Observe subjects for at least 30 minutes after inoculation with appropriate 
medical equipment available in case of a rare, unanticipated, anaphylactic 
reaction.
6. Assess AEs.  
Clinical Monitoring and Evaluation (Days 1 through 7)
See Appendix B for a tabular representation of study procedures. 
Study Days 1-6:
1. Perform focused history and history-directed physical examination focusing on 
any acute complaints and record findings (daily).
2. Record vital signs (daily). 
3. Assess AEs (daily).
4. Identify, provide, and record symptomatic clinical care as permitted per protocol.
5. Obtain NP wash for viral RNA detection, viral culture, and for cytokine assays. 
Qualitative RSV PCR by FilmArray will be performed on Days 1, 2, 3, and then 

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daily from Day 5 through day of discharge; quantitative RSV PCR. Viral culture 
will be performed daily starting on Day 2 through day of discharge. Nasal 
cytokine assays will be performed on Day 1 only. 
6. Obtain cotton-tipped nasal swab for microbiome analysis (Days 2 and 4).
7. Subjects fill in FLU-PRO symptom questionnaire once daily in the evening.
8. On Day 1 only, obtain CBC with differential and T, B, and NK cell lymphocyte 
immunophenotyping; and research blood for immune cell phenotyping, 
transcriptome profiling, and cytokine analysis. 
9. On Day 3 only, obtain CBC with differential, acute care panel, and hepatic panel. 
Study Day 7:
1. Perform focused history and history-directed physical examination focusing on 
any acute complaints and record findings.
2. Record vital signs. 
3. Assess AEs.
4. Subjects fill in FLU-PRO symptom questionnaire in the evening.
5. Obtain CBC with differential, acute care panel, and hepatic panel.
6. Obtain lymphocyte immunophenotyping, TBNK.
7. Obtain SAM strip for mucosal cytokines and/or antibody analysis
8. Obtain nasal swab for microbiome sequencing and culture.
9. Obtain NP wash for RSV fresh and quantitative culture, viral RNA detection by 
quantitative RT-PCR and FilmArray, nasal secretory IgA, and cytokine assays.
10.Obtain research blood for RSV antibody and neutralization assays.
11.Obtain research blood for B- and T-cell assays, immune cell phenotyping, 
transcriptome profiling and cytokine analysis. 
12.Evaluation for discharge criteria:

The participant must have 2 consecutive negative NP washes for RSV 
RNA, as detected by FilmArray assay (participant and study team will 
remain blinded to the RSV PCR results and an honest broker, designated 
by the PI, will notify the study team when a participant’s PCR results 
satisfy the discharge criteria)

The participant does not have any signs or symptoms of possible RSV-
associated lower respiratory tract illness
We expect that a healthy volunteer who remained well throughout Day 7 after challenge 
and who was not shedding RSV by Day 7 would not proceed to develop a symptomatic 

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respiratory illness thereafter or present risk for virus transmission after discharge. In 
healthy adults, the duration of viral shedding associated with onset of symptomatic 
infection in the majority of adults lasts less than 7 days, with an average shedding 
duration (detected using viral culture) of 3.9 days.19 For discharge, we rely on RT-PCR 
for documentation of clearance of viral shedding. Molecular detection of RSV by RT-
PCR is more sensitive than by culture, as seen in prior RSV challenge studies 
(Appendix D). The mean duration of RSV shedding by RT-PCR is 8-10 days. 
In the event that a participant who is no longer shedding RSV has persistent mild-to-
moderate upper respiratory illness by Day 7, then we will continue to provide clinical 
evaluation and recommendations for treatment, according to the standard of care and 
discharge the subject. In the event that a participant who is no longer shedding RSV 
has lower respiratory tract symptoms by Day 7, we would evaluate for indications to 
escalate care or continue standard of care and evaluate for discharge only when the 
subject is clinically stable.
Subjects remaining on inpatient care after study Day 7:
1. Perform focused history and history-directed physical examination focusing on 
any acute complaints and record findings.
2. Record vital signs. 
3. Assess AEs.
4. Subjects fill in FLU-PRO symptom questionnaire once daily in the evening up to 
and including Day 14.
5. Obtain SAM strip on Day 10.
6. Obtain nasal swabs for microbiome sequencing and culture on Day 10.
7.
Obtain NP wash for RSV culture and viral RNA detection and research blood for 
mucosal cytokine and antibody and lymphocyte assays, as per table in 
Appendix B.  For Day 10: Obtain research blood for B- and T-cell assays and 
quantitative and molecular immune cell phenotyping, serum for RSV-specific 
antibody testing and total serum IgG and IgA, serum for cytokine analysis.  Day 
11 onward, on a daily basis, nasal research procedures consist of nasal wash 
tested by FilmArray PCR for respiratory viruses, quantitative RT-PCR, 
quantitative viral culture, and fresh viral culture.
8. Evaluate volunteer for discharge criteria, as noted above, on a daily basis 

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6.2.1
Treatment Plan for Persistent Symptoms Associated with RSV Illness
Participants reporting persistent and significant symptoms associated with RSV illness 
may be offered symptomatic relief after clinical evaluation. If symptoms are refractory to 
single doses of over-the-counter acetaminophen (325-650 mg) and significantly, 
adversely impact daily activities (as assessed by the PI), such as the ability to sleep, 
then ibuprofen (200-600 mg), pseudoephedrine (30-60 mg), or throat lozenges may be 
provided, after clinical evaluation, for symptoms such as pain, persistent cough, nasal or 
sinus symptoms interfering with sleep, or fever when associated with significant 
discomfort.
Indications for offering symptomatic treatment to participants who have not yet met a 
study definition of RSV-associated upper respiratory illness and/or have not completed 
study evaluations (including research labs) through Day 7 will be determined by the PI 
applying Good Clinical Practice (GCP) principles.
6.2.2
Escalation of Care Plan 
Subjects who continue to shed virus at Day 7 will be strongly encouraged to remain in 
the Clinical Center until they have 2 consecutive negative NP washes for RSV. A 
prolonged stay in the Clinical Center required as a result of extended shedding will not 
be considered a SAE. Should a subject elect to leave the inpatient unit at any point prior 
to meeting per-protocol discharge criteria, appropriate precautions will be discussed in 
detail with the subject to minimize risks to the subject and any contacts (e.g. hand 
hygiene, avoidance of persons at high risk for complication from RSV, return to clinic for 
nasal wash follow-up to determine if RSV shedding continues). The Clinical Safety 
Office (CSO)/Sponsor Medical Monitor will be notified immediately if this situation 
occurs.  
6.2.2.1 Increased Severity of Clinical Illness
Any change in clinical status deemed important by the examining physician in 
consultation with the PI will initiate an escalation of care for that individual. This will be 
considered an SAE. Indicators for increased care may include the following:
1. Severe respiratory illness (Section 11.1).
2. Fever with rigors.

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3. Any clinically significant deterioration of a participant’s overall hemodynamic 
status.
4. Any other change noted in the physician’s exam or symptoms/signs deemed 
significant by the responsible medical and/or study staff in conjunction with the 
PI. This includes any untoward signs or symptoms that are inconsistent with the 
expected course of RSV in a generally healthy individual. 
Escalation of care will be individualized to address the specific needs of the subject and 
immediate steps will be taken. If deemed appropriate, standard-of-care treatments will 
be started immediately as deemed necessary by the PI. Appropriate laboratory, 
radiographic, and microbiological diagnostic testing will be performed to evaluate the 
participant and diagnose any complicating illness.
6.3
Follow-up Visit (Day 28)
A follow-up outpatient visit will be scheduled for Day 28 (-1 to +10 days). The following 
procedures will be conducted:
1. Obtain interim history and physical to assess for AEs.
2. Obtain and record vital signs.
3. Obtain diary card. Subjects will record symptoms starting the day after discharge 
and leading up to this visit. 
4. Obtain SAM strip for mucosal cytokines and/or antibody analysis.
5. Obtain nasal swab for microbiome sequencing and culture. 
6. Obtain NP wash for immunoglobulins, secretory IgA, and cytokine analysis. 
7. Obtain fasting, research blood for T-cell assays, immune cell phenotyping, 
transcriptome profiling, cytokine analysis, RSV antibody and neutralization 
assays. 
8. Obtain T, B, and NK cell flow cytometry test.
9. Obtain blood for CBC with differential, acute care panel, and hepatic panel. 
10.Assess for treatments that could potentially interfere with RSV-induced immunity 
(Section 5.6).
If a close contact of the subject becomes ill with a respiratory tract infection during 
Days 7-28, then the subject will be asked to request that the close contact inform the 
study team and enroll in the NIH protocol 11-I-0109, “Viral Infections in Healthy and 
Immunocompromised Hosts,” to determine if their illness is being caused by RSV, and 

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then to identify the virus. All efforts will be made to document transmission of virus that 
may have occurred after discharge.
6.4
Final Study Visit (Day 56)
A final study visit will be scheduled for Day 56 (-5 to +15 days). The following 
procedures will be conducted:
1. Obtain interim history and physical to assess for AEs.
2. Obtain and record vital signs. 
3. Obtain diary card.
4. Obtain SAM strip for mucosal cytokines and/or antibody analysis.
5. Obtain nasal swab for microbiome sequencing and culture. 
6. Obtain NP wash for immunoglobulins, secretory IgA, and cytokine analysis, RSV 
antibody and neutralization assays, immune cell phenotyping, transcriptome 
profiling . 
7. Obtain blood for CBC with differential, acute care panel, and hepatic panel.
8. Obtain T, B, and NK cell flow cytometry test.
9. Assess for treatments that could potentially interfere with RSV-induced immunity 
(Section 5.6).
6.5
Interim Safety Visit
Interim safety visit(s) will be scheduled for any participant who has a clinically significant 
AE for which follow-up is required prior to Day 28 or Day 56 or for a participant who 
withdraws from the inpatient phase of the study prior to meeting discharge criteria and is 
known to have a NP wash positive for RSV on the day of discharge. 
The following procedures may be performed:
1. Obtain interim history and physical to assess AEs.
2. If a prior respiratory pathogen panel result was abnormal, an NP wash may be 
obtained for respiratory pathogen panel, RSV culture, and quantitative viral RNA 
detection. Surplus NP wash will become a stored research specimen (Section 9).
3. If a prior laboratory result was abnormal, this abnormality may be re-evaluated 
with a repeat blood draw.
4. Any other event that the PI deems to require follow-up will be evaluated during 
the interim safety visits.

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6.6
Early Termination Visit 
A termination visit will be performed at the time of subject withdrawal. Interim safety 
visits or safety visits on Day 28 and/or Day 56 will be recommended to any subject who 
has received study virus and withdraws from the inpatient phase of the study 
prematurely or who has significant AEs that require follow-up. Study discontinuation will 
be determined on a case-by-case basis.
An individual may be withdrawn for any of the following reasons:
1. An individual subject’s decision.
2. A clinical AE, lab abnormality, or medical condition for which continued 
participation in the study would not be in the best interest of the subject.
3. Non-compliance with study procedures to the extent that it is harmful to the 
subject or integrity of study data.
4. Use of any investigational drug or investigational vaccine other than the study 
agent within 60 days after inoculation with RSV A2.
5. Receipt of a licensed vaccine within 60 days after inoculation with RSV A2.
6. Receipt of immunoglobulins and/or blood-derived products during the study 
period, with exception that packed red blood cells are not prohibited if/when 
given with emergent indications and not needed for ongoing therapy.
7. Chronic administration of immunosuppressant(s), defined as > 14 days (if 
corticosteroid, then dose is ≥ 10 mg/day of prednisone equivalent), or other 
immune modifying drug(s) taken during the study period. (Nasal corticosteroids 
are prohibited after inoculation with RSV A2, but may be used after the Day 28 
study visit.)
8. Any confirmed or suspected immunosuppressive condition or immunodeficient 
condition.
6.7
Pregnancy and Follow-up Visit 
If pregnancy is discovered before virus inoculation, then the subject will be released 
before participation and no further follow-up is needed. If pregnancy is discovered after 
virus inoculation, then the subject will be advised to notify obstetrician of study agent 
exposure, and we will continue to follow the participant for safety and pregnancy 
outcomes. Pregnancy events will also be reported to the sponsor medical monitor, the 
Safety Monitoring Committee (SMC), and/or the institutional review board (IRB).

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7 Laboratory Evaluations
7.1
Clinical Laboratory Evaluations (all performed in the NIH Clinical 
Center):
1.    CBC with differential.
2.    Acute care panel that includes sodium, potassium, chloride, bicarbonate, serum 
creatinine, and urea.
3.    Mineral panel that includes albumin, calcium, magnesium, and phosphorus.
4.    Hepatic panel that includes alkaline phosphatase, ALT, AST, and total and 
direct bilirubin.
5.    Urine or serum pregnancy test.
6.
T, B, and NK cell flow cytometry test.
7.    NP wash for qualitative RSV PCR. Before viral challenge and during the 
specified time points post-inoculation, NP wash specimens will be tested with 
the FilmArray® Respiratory Panel (BioFire Diagnostics, Inc. [Salt Lake City, UT, 
USA]). The assay will be performed by the NIH Clinical Center microbiology 
lab. This assay is a Clinical Laboratory Improvement Amendments (CLIA)–
certified laboratory diagnostic test and will be used to determine discharge 
eligibility. 
7.2
Research Laboratory Evaluations:
NP washes/swabs will be conducted as described in Section 6. Samples will be tested 
for the following: 
1. RSV detection by quantitative RSV RT-PCR and viral culture. Starting on Day 1, 
daily nasal wash specimens will be tested by FilmArray assay in the DLM.   
Starting on Day 1, nasal wash specimens will be tested for RSV by quantitative 
viral titer and culture (CIR at JHU; Baltimore, MD, USA). 
2. Cytokine and chemokine assays will be conducted at the LID (NIAID) and the 
Center for Human Immunology, Autoimmunity, and Inflammation (CHI, National 
Heart, Lung, and Blood Institute [NHLBI]).
3. RSV mucosal immunity will be done by measuring secretory IgA antibody levels. 
4. Nasal microbiome analysis (NIAID Microbiome Initiative).

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Blood samples will be analyzed as follows:
1. Serum RSV neutralization assays for RSV (CIR/JHU).
2. RSV-specific T-cell responses/T-cell repertoire to be conducted by Dr. Cyril Le 
Nouen, PhD (LID/NIAID). 
3. Molecular and quantitative immune cell phenotyping to include FACS analysis to 
be conducted by CHI.
4. Serum cytokine analysis to be conducted by CHI.
5. Transcriptome profiling using PAXgene (CHI/NHLBI).
The amount of blood drawn for research purposes will be within the limits allowed for 
adult research subjects by the NIH Clinical Center (Medical Administrative Policy 95-9, 
Guidelines for Limits of Blood Drawn for Research Purposes in the Clinical Center: 
http://cc-internal.cc.nih.gov/policies/PDF/M95-9.pdf).
8 Potential Risks and Benefits
8.1
Potential Risks
Study agent inoculation
Complications or severe illnesses associated with RSV A2 challenge inoculation are 
rare and include:

Pneumonia

Secondary bacterial infections

Hypoxemia/respiratory failure

Anaphylaxis
Also, as with any investigational product, there is a theoretical possibility of risks about 
which we have no present knowledge. Subjects will be informed of any such risks 
should further data become available.
We expect that subjects will develop RSV illness. These expected events are listed in 
Section 11.1.  Subjects will not be allowed to self-medicate for symptom relief; 
symptomatic treatment may be provided by study staff after clinical evaluation as 
needed for pain, persistent cough, nasal symptoms interfering with sleep, and/or fever 
when associated with significant discomfort. 

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Subjects may also be inconvenienced by being confined to the isolation unit for the 
duration of the hospital stay.
Acetaminophen
Acetaminophen is well tolerated and safe in recommended doses. Potential 
acetaminophen toxicity may occur at high doses exceeding the therapeutic limit.
NP washes/swabs
Potential risks associated with NP washes and/or swabs include pain or discomfort, a 
gag reflex, and very rarely, epistaxis.
Venipuncture
Risks occasionally associated with venipuncture include pain and bruising at the site of 
venipuncture, bleeding, lightheadedness, and, rarely, infection or syncope.
Pulmonary function tests
There are minimal risks associated with PFTs for healthy volunteers. It may cause mild 
shortness of breath and fatigue, but is otherwise noninvasive and safe. 
8.2
Potential Benefits
Subjects will not receive any direct benefit from participation in this study. 
Through a healthy volunteer’s participation in this study, the scientific and medical 
community may gain new knowledge and insights to guide development and testing of 
RSV antivirals, vaccine candidates, and potentially novel treatment strategies. This 
knowledge may advance efforts to bring benefit to populations of patients most 
vulnerable to severe RSV complications. 
9 Research Use of Stored Human Samples, Specimens or 
Data

Intended Use: Samples and data collected under this protocol may be used to 
study RSV infection and response in healthy adults. Human genetic testing will 

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include HLA typing. Also, human DNA may be isolated from stored research 
samples in the future for genetic testing. 

Storage: Access to stored samples will be limited using a locked freezer in a 
locked laboratory. Samples and data will be stored using codes assigned by the 
investigators. Data will be kept in password-protected computers. Only 
investigators will have access to the samples and data. 

Tracking: Samples will be stored at the National Laboratory for Cancer 
Research in Frederick, MD. Samples will be tracked using a database located on 
a password-protected computer, which will be maintained by the investigators 
and their designees. Only investigators and their designees will have access to 
this database.

Disposition at the Completion of the Protocol: 

In the future, other investigators (both at NIH and outside) may wish to use 
these samples and/or data for research purposes. If the planned research 
falls within the category of “human subjects research” on the part of the 
NIH researchers, NIH IRB review and approval will be obtained. This 
includes the NIH researchers sending out coded and linked samples or 
data and getting results that they can link back to their subjects.

Reporting the Loss or Destruction of Samples/Specimens/Data to the IRB:

Any loss or unanticipated destruction of samples or data (for example, due 
to freezer malfunction) that meets the definition of a reportable event will 
be reported to the NIH IRB according to NIH Human Research Protection 
Program (HRPP) Policy 801. 

Additionally, subjects may decide at any point not to have their samples 
stored. In this case, the PI will destroy all known remaining samples and 
report what was done to both the subject and to the IRB. This decision will 
not affect the subject’s participation in this protocol or any other protocols 
at NIH. 
10 Remuneration Plan for Subjects 
Participants will be compensated according to Table 4.

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Table 4 Participant remuneration
Study Day
Remuneration ($)
Inpatient Study Phase
−1
220
0
225
1
225
2
225
3
225
4
225
5
225
6
225
7
225
Initial Inpatient Subtotal
2020
Each Additional Inpatient Daya
325
Inpatient Completion Bonus
425
Outpatient Study Phase
Follow-up Visit
200
Final Study Visit
225
Maximum Follow-up Subtotal b
425
Total Compensation for Full Study Participationc
2870
a
Subjects will continue inpatient care if they continue to display RSV-related 
symptoms and are shedding virus after Day 7. Inpatient care will continue 
until discharge criteria are met (see Section 6.2).
b    If an interim visit is required, compensation for this visit will be $200
c
Full participation is defined as the initial inpatient phase (Days −1 to 7), 
1 follow-up visit (Day 28), and a final study visit (Day 56). 
11 Assessment of Safety
11.1 Definitions for Sponsor Reporting
The NIAID CSO is responsible for sponsor safety oversight of this study, and the 
definitions below comply with CSO requirements.
Adverse Event (AE): An AE is any untoward or unfavorable medical occurrence in a 
human subject, including any abnormal sign (e.g., abnormal physical exam or laboratory 
finding), symptom, or disease, temporally associated with the subject’s participation in 
the research, whether or not considered related to the research.
Expected Events: The following signs or symptoms are induced by or associated with 
RSV illness, and any combination of these (within the range from Grade 1 to Grade 3) is 
expected to occur with virus challenge. These symptoms will not be reported as 
unexpected AEs; they will be recorded as Expected Events of RSV infection in 
CRIMSON per protocol. Each individual with an expected AE consistent with RSV 
illness but of atypical duration or severity will be evaluated with good clinical judgment 

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for appropriate further management. In addition, the PI will monitor all subjects in 
aggregate, and any pattern of severity, or unexpectedly high frequency of a particular 
event, will be treated as a safety signal and potential safety concern and will prompt 
consultation with the sponsor.

Nasal congestion or rhinorrhea

Sore throat

Cough

Headache

Sinus congestion or pain

Fever, with temperature ranging from 38°C (100.4°F) to 39.4°C (103°F)

Ear pain
Adverse Reaction (AR): An AE that is caused by an investigational agent (drug or 
biologic).
Suspected Adverse Reaction (SAR): An AE for which there is a reasonable possibility 
that the investigational agent caused the AE. ‘Reasonable possibility’ means that there 
is evidence to suggest a causal relationship between the drug and the AE. A SAR 
implies a lesser degree of certainty about causality than AR, which implies a high 
degree of certainty.
Serious Adverse Event (SAE): An SAE is an AE that results in one or more of the 
following outcomes:

death

a life threatening event (places the subject at immediate risk of death from the 
event as it occurred)

an inpatient hospitalization or prolongation of an existing hospitalization

a persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions

a congenital anomaly/birth defect

a medically important event
Medical and scientific judgment should be exercised in deciding events that may not be 
immediately life threatening or result in death or hospitalization but may jeopardize the 
subject or may require intervention to prevent one of the other outcomes listed above.

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Unexpected Adverse Event: An AE is unexpected if it is not listed at the specificity or 
severity that has been observed. It is the responsibility of the IND Sponsor to make this 
determination.
Serious and Unexpected Suspected Adverse Reaction (SUSAR): A SUSAR is a 
Suspected Adverse Reaction that is both serious and unexpected.
Unanticipated Problem (UP): A UP is any event, incident, experience, or outcome that 
is
1. unexpected in terms of nature, severity, or frequency in relation to
a. the research risks that are described in the IRB-approved research 
protocol and informed consent document or other study documents; and
b. the characteristics of the subject population being studied; and
2. possibly, probably, or definitely related to participation in the research; and
3. places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or 
recognized. (Per the IND Sponsor, an AE with a serious outcome will be 
considered increased risk.)
Serious Unanticipated Problem (UP): A UP that meets the definition of a Serious 
Adverse Event or compromises the safety, welfare or rights of subjects or others. 
Unanticipated problem that is not an Adverse Event (UPnonAE): An unanticipated 
problem that does not fit the definition of an adverse event, but which may, in the 
opinion of the investigator, involve risk to the subject, affect others in the research 
study, or significantly impact the integrity of research data.  These events may involve a 
greater risk of social or economic harm to subjects or others rather than 
physical/psychological harm. Such events would be considered a non-serious UP. 
Examples of a UPnonAE include a breach of confidentiality, accidental destruction of 
study records, or unaccounted-for study drug.
Unanticipated Problem that is not an Adverse Event (UPnonAE): An UP that does 
not fit the definition of an AE, but which may, in the opinion of the investigator, involve 
risk to the subject, affect others in the research study, or significantly impact the integrity 
of research data. Such events would be considered non-serious UPs. For example, we 
will report occurrences of breaches of confidentiality, accidental destruction of study 
records, or unaccounted-for study drug.

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Protocol Deviation: Any change, divergence, or departure from the IRB-approved 
research protocol. 
Major Deviations: Deviations from the IRB-approved protocol that have, or may have 
the potential to, negatively impact, the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study.
Minor Deviations: Deviations that do not have the potential to negatively impact the 
rights, safety, or welfare of subjects or others, or the scientific integrity or validity of the 
study.
Non-compliance: Failure of investigator(s) to follow the applicable laws, regulations, or 
institutional policies governing the protection of human subjects in research, or the 
requirements or determinations of the IRB, whether intentional or not. 
Serious Non-compliance: Non-compliance, whether intentional or not, that results in 
harm or otherwise materially compromises the rights, welfare and/or safety of the 
subject. Non-compliance that materially affects the scientific integrity or validity of the 
research may be considered serious non-compliance, even if it does not result in direct 
harm to research subjects. 
Continuing Non-compliance: A pattern of recurring non-compliance that either has 
resulted, or, if continued, may result in harm to subjects or otherwise materially 
compromise the rights, welfare and/or safety of subjects, affect the scientific integrity of 
the study or validity of the results. The pattern may comprise repetition of the same non-
compliant action(s), or different noncompliant events. Such non-compliance may be 
unintentional (e.g. due to lack of understanding, knowledge, or commitment), or 
intentional (e.g. due to deliberate choice to ignore or compromise the requirements of 
any applicable regulation, organizational policy, or determination of the IRB).
11.2 Documenting, Recording, and Reporting Adverse Events
All AEs occurring from the time of viral challenge through the final study visit will be 
documented, recorded, and reported.
At each contact with the subject, information regarding AEs will be elicited by 
appropriate questioning and examinations and will be:

immediately documented in the subject’s medical record/source document,

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
recorded in CRIMSON, and

reported as outlined below (e.g., IND Sponsor, IRB, and FDA).
If a diagnosis is clinically evident (or subsequently determined), the diagnosis rather 
than the individual signs and symptoms or lab abnormalities will be recorded as the AE.
A laboratory abnormality will not be reported as an adverse event if ALL of the 
following criteria are met:
•
It is no more than “Grade 1” or “Mild” per the protocol specified toxicity table 
(or investigator assessment if not listed on the table); AND
•
It does NOT require an intervention (e.g., discontinuation of treatment, 
dose reduction/delay, additional assessments, or treatment); AND
•
It is assessed by the PI as NOT related to the study agent(s) or study procedure(s); 
AND
•
It is assessed by the PI as NOT clinically significant (e.g., the abnormal value does 
NOT suggest a disease or organ toxicity).
All abnormal laboratory findings will be reviewed on a routine bases by the PI to identify 
potential safety signals. An abnormal lab not included on the toxicity table should be 
assessed in a similar fashion to the criteria above.
All clinically relevant AEs that are not resolved by the end of the final study visit are 
followed until resolution or until stability is achieved (unless the subject is lost to follow-
up).
11.3 Investigator Assessment of Adverse Events
The investigator will assess all AEs with respect to seriousness (criteria listed above), 
severity (intensity or grade), and causality (relationship to study agent and relationship 
to research) according to the following guidelines.
11.3.1
Severity
The Investigator will grade the severity of each AE according to toxicity table provided in 
Appendix E, and supplemented by the “Division of AIDS Table for Grading the Severity 
of Adult and Pediatric Adverse Events” Version 2.1, July 2017, which can be found at: 
https://rsc.tech-res.com/docs/default-source/safety/division-of-aids-(daids)-table-for-
grading-the-severity-of-adult-and-pediatric-adverse-events-corrected-v-2-1.pdf 
Some Grade 1 lab parameters on the DAIDS Toxicity Table (fibrinogen, potassium 
[low], uric acid [males only, elevated]) fall within the NIH lab reference range for normal 

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values. These normal values will not be reported as Grade 1 AEs. The Grade 1 values 
for these tests will be reported as follows:

Fibrinogen: 100-176 mg/dL

Potassium (low): 3.0-3.3 mmol/L

Uric acid (males): 8.7-10.0 mg/dL

Magnesium (low): 0.60-0.65 mmol/L
11.3.2
Causality
Causality (likelihood that the event is caused by the study agent(s)) will be assessed 
considering the factors listed under the following categories:
Definitely Related

reasonable temporal relationship

follows a known response pattern

clear evidence to suggest a causal relationship

there is no alternative etiology
Probably Related

reasonable temporal relationship

follows a suspected response pattern (based on similar agents)

no evidence of a more likely alternative etiology
Possibly Related

reasonable temporal relationship

little evidence for a more likely alternative etiology
Unlikely Related

does not have a reasonable temporal relationship
OR

good evidence for a more likely alternative etiology
Not Related

does not have a temporal relationship

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OR

definitely due to an alternative etiology
Note: Other factors should also be considered for each causality category when 
appropriate. Causality assessment is based on available information at the time of the 
assessment of the AE. The investigator may revise the causality assessment as 
additional information becomes available.
11.4 Investigator Reporting Responsibilities to the Sponsor
11.4.1
Adverse Events
AE data will be submitted to the IND Sponsor when requested for periodic safety 
assessments, review of IND annual reports, review of IND safety reports, and 
preparation of final study reports.
11.4.2
Serious Adverse Events
All SAEs (regardless of relationship and whether or not they are also UPs) must be 
reported on the Safety Expedited Report Form (SERF) and sent to the CSO by fax or e-
mail attachment. Deaths and immediately life threatening SAEs must be reported to the 
CSO within 1 business day after the site becomes aware of the event. All other SAEs 
must be reported within 3 business days of site awareness.
CSO CONTACT INFORMATION:
Clinical Safety Office
5705 Industry Lane
Frederick, MD 21704
Phone: 301-846-5301
Fax: 301-846-6224
E-mail: rchspsafety@mail.nih.gov 
SAEs that have not resolved by the end of the final study visit are followed until final 
outcome is known. If it is not possible to obtain a final outcome for an SAE (e.g., the 
subject is lost to follow-up), the reason a final outcome could not be obtained will be 
recorded by the investigator on the AE CRF and the SERF.

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SAEs that occur after the study follow-up period that are reported to and are assessed 
by the investigator to be possibly, probably, or definitely related to study drug must be 
reported to the CSO.
11.4.3
Unanticipated Problems
UPs that are also AEs must be reported to the CSO and sent by fax or e-mail 
attachment no later than 7 calendar days of site awareness of the event. UPs that are 
not AEs are not reported to the CSO.
11.4.4
Pregnancy
All pregnancies will be reported on the Pregnancy Notification/Outcome Form to the 
CSO within 1 business day from site awareness.
Pregnancy outcome data (e.g., delivery outcome, spontaneous or elective termination of 
the pregnancy) will be reported to the CSO within 3 business days of the site’s 
awareness.
Although pregnancy itself is not an AE, events that meet SAE criteria during pregnancy, 
delivery, or in the neonate (e.g., congenital anomaly/birth defect) are reportable on the 
SERF.
In the event of pregnancy:

Keep the subject in the study and follow for safety through delivery, including 
maternal and fetal outcomes.

Report to safety oversight committee (SMC).

Advise research subject to notify the obstetrician of study participation and study 
agent exposure.
11.5 Additional Investigator Reporting Procedures
11.5.1
Reporting Procedures to the IRB
Reportable events will be tracked and submitted to the IRB as outlined in Policy 801.

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11.5.2
Reporting to the NIAID Clinical Director
The PI will report UPs, major protocol deviations, and deaths to the NIAID Clinical 
Director according to institutional timelines.
11.6 Sponsor’s Reporting Responsibilities
SUSARs as defined in 21 Code of Federal Regulations (CFR) 312.32 and determined 
by the IND Sponsor will be reported to FDA and all participating investigators as IND 
Safety Reports.
The IND Sponsor will also submit an IND Annual Report of the progress of the 
investigation to the FDA as defined in 21 CFR 312.33.
11.7 Halting Rules for the Protocol
Halting the study requires immediate discontinuation of study agent administration for all 
subjects and suspension of enrollment until a decision is made whether or not to 
continue enrollment and study agent administration.
The halting rules are:
One or more subjects experience the same or similar SAEs that are possibly, probably, 
or definitely related to the study agent or procedures;
OR
Any safety issue that the PI and/or the CSO determines should halt the study.
The PI and/or CSO will determine if the study should be halted. In addition, the FDA 
may halt the study at any time following review of any safety concerns.
11.7.1
Reporting a Study Halt
If a halting rule is met, a description of the AE(s) or safety issue must be reported by the 
PI, within 1 business day, to the CSO, the IRB, and the SMC by fax or email.

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11.7.2
Resumption of a Halted Study
The IND Sponsor, in collaboration with the PI, the lead associate investigators, and the 
SMC, will determine if it is safe to resume the study.
The PI will notify the IRB of the decision on resumption of the study.
11.8 Withdrawal Criteria for an Individual Subject
An individual subject will be withdrawn for any of the following:

An individual subject’s decision. (The investigator should attempt to determine 
the reason for the subject’s decision.)

Non-compliance with study procedures to the extent that it is potentially harmful 
to the subject or to the integrity of the study data.

A confirmed diagnosis of another respiratory infection (other than RSV) during 
the inpatient portion of the study.

The investigator determines that continued participation in the study would not be 
in the best interest of the subject.
If a participant is withdrawn before inoculation with the challenge virus, then no further 
testing or follow-up will be performed. Also, if a participant is admitted to the study unit 
and then withdrawn on day of admission (Day -1) prior to receiving study virus (for any 
reason), they may be replaced with an eligible and available “stand-by” participant on 
Day -1 (Section 3.1). If a participant withdraws after Day -1, then they will not be 
replaced. 
Subjects will be strongly discouraged from withdrawing from the study after inoculation. 
We would encourage any participants who are shedding RSV to remain as an inpatient 
until viral shedding resolves completely. We will offer inpatient care with isolation 
conditions and use of any over-the-counter cold remedies as needed, and no further 
testing will be performed. If the participant insists on withdrawing completely and leaving 
the hospital after inoculation, then no further research testing will be performed. 
However, the participant will be encouraged to contact the PI or study coordinator after 
withdrawing if concerns or issues arise that are thought to be related to participation in 
this study. They will be offered the opportunity to present for a follow-up visit as well.

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11.9  Safety Oversight
11.9.1
Safety Review and Communications Plan
A Safety Review and Communications Plan (SRCP) has been developed for the 
protocol. The SRCP is an internal communications document between the PI and the 
CSO, which delineates the safety oversight responsibilities of the PI, the CSO, and 
other stakeholders. The SRCP also includes the overall plan for conducting periodic 
safety surveillance assessments.
11.9.2
Sponsor Medical Monitor
A medical monitor, representing the IND Sponsor (OCRPRO), has been appointed for 
oversight of safety in this clinical study. The sponsor medical monitor will be responsible 
for performing safety assessments as outlined in an SRCP.
11.9.3
Safety Monitoring Committee
The NIAID Intramural SMC includes independent experts that do not have direct 
involvement in the conduct of the study and have no significant conflicts of interests as 
defined by NIAID policy. The Committee will review the study data to evaluate the 
safety, efficacy, study progress, and conduct of the study. The Committee may convene 
additional reviews as necessary. These reviews will occur after each cohort  has 
completed the inpatient phase and prior to enrolling the subsequent cohorts. All SAEs, 
all UPs, and all IND Safety Reports will be reported by the PI to the SMC at the same 
time they are submitted to the IRB or IND Sponsor. Please see  Table 5 and Table 6 in 
Section 13.1 for criteria pertaining to continuing the study after cohort 3 and for criteria 
for stopping the study it too low of a rate of infectivity is encountered.  The PI will notify 
the SMC when halting criteria are met.  The PI will submit the written SMC summary 
reports with recommendations to the IRB.
12 Clinical Monitoring Structure
12.1 Site Monitoring Plan
As per International Conference on Harmonisation (ICH) GCP 5.18 and 21 CFR 312.50, 
clinical protocols are required to be adequately monitored by the study sponsor. This 
study monitoring will be conducted according to the “NIAID Intramural Clinical 

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Monitoring Guidelines. Monitors under contract to NIAID/OCRPRO will visit the clinical 
research site to monitor aspects of the study in accordance with the appropriate 
regulations and the approved protocol. The objectives of a monitoring visit will be: 1) to 
verify the existence of signed informed consent documents and documentation of the 
informed consent process for each monitored subject; 2) to verify the prompt and 
accurate recording of all monitored data points in CRIMSON, and prompt reporting of all 
SAEs; 3) to compare abstracted information entered into CRIMSON with individual 
subjects’ records and source documents (subjects’ charts, laboratory analyses and test 
results, physicians’ progress notes, nurses’ notes, and any other relevant original 
subject information); and 4) to help ensure investigators are in compliance with the 
protocol. The monitors also will inspect the clinical site regulatory files to ensure that 
regulatory requirements (Office for Human Research Protections [OHRP], FDA) and 
applicable guidelines (ICH-GCP) are being followed. During the monitoring visits, the 
investigator (and/or designee) and other study personnel will be available to discuss the 
study progress and monitoring visit.
The investigator will make study documents (e.g., consent forms) CRIMSON data 
abstracts and pertinent hospital or clinical records (including CRIMSON) readily 
available for inspection by the IRB, the FDA, the site monitors, and the NIAID staff for 
confirmation of the study data. 
A specific protocol monitoring plan will be discussed with the PI and study staff prior to 
enrollment. The plan will outline the frequency of monitoring visits based on such factors 
as study enrollment, data collection status and regulatory obligations.
13 Statistical Considerations
This study is a phase 1 study to primarily assess the safety and infectability of a high 
dose, recombinant RSV A2 challenge virus administered by nasal atomizer to healthy 
adults and secondarily assess RSV illness following experimental infection. We will 
assess RSV shedding and illness as primary and secondary outcomes, respectively.  
The sample size calculations are based on a specified frequency of RSV shedding.  The 
analyses performed will be descriptive. 

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13.1 Sample Size Justification
Sample size calculations are based on ensuring a high probability of correctly 
concluding the shedding rate is above 55% for the 107 PFU dose of RSV A2 using a 
nasal atomizer. We also will have a high probability of observing an SAE if the true SAE 
rate is greater than 9%. The total sample size is 25 participants with the first cohort of 
size 4 (106.3 PFU dose) and 3 cohorts of size 7. After cohorts 2 and 3 are completed, the 
number of participants who had viral shedding will be evaluated and if the number is too 
low, the study will be stopped for inactivity (in Table 5 below, row 2 gives the criteria for 
continuing the study after each evaluation time and the number of participants with 
shedding at the end of the study that will be used to conclude activity is sufficiently 
high). The study will have a high probability of correctly concluding the 107 PFU RSV A2 
dose results in activity above 55% when the true rate is 77% (80% power). The 
probability of incorrectly concluding the RSV A2 dose results in activity above 55% 
when the true rate is 55% or less is smaller than 0.10. Table 6 shows the probability of 
stopping early under uninteresting rates of activity. Row 2 shows the probability of 
stopping after 7 patients is 0.153 and the probability of stopping after 14 patients is 
0.512 (assuming you didn’t stop after 7 patients). Overall there is a 0.665 probability of 
stopping early if the true activity rate is 0.55.

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Table 5 Criteria for continuing the study after cohort 3 and the final criteria for 
interesting shedding rate at the end of the study based on 3 different null and 
alternative shedding rates
Uninteresting 
shedding rate 
(null rate)
True rate that 
provides at 
least 80% 
power 
(alternative 
rate)
Number of 
participants 
with shedding 
needed to 
continue study 
after first 7 
participants 
(cohort 2)
Number of 
participants 
with shedding 
needed to 
continue study 
after 14 
participants 
(cohorts 2 plus 
3)
Number of 
participants 
with shedding 
needed to 
conclude high 
activity at the 
end of the 
study (n=21; 
cohorts 2-4)
.5
.73
3
7
14
*.55
.77
3
9
15
.6
.81
3
9
16
* The null and alternative rates that best meet the needs of this study are .55 and .77 
and are shown in the middle row.

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Table 6  Probability of stopping early under the specified criteria in Table 5 for 
uninteresting rates of activity
Uninteresting 
shedding rate 
(null rate)
Probability of 
stopping after 7 
participants
*Probability of 
stopping after 
14 participants
Overall 
probability of 
stopping early
.5
.227
.209
.436
.55
.153
.512
.665
.6
.096
.540
.636
*This probability is assuming the study doesn’t stop after 7 participants.
For the safety endpoint of SAE there is a high probability (.89) of observing at least 1 
SAE out of 21 participants if the true SAE rate is 10%, and a probability of .65 of 
observing at least 1 event if the true SAE rate is 5%.
13.2 Analysis Plan 
At the end of the study, we will conclude whether the dose is active based on the criteria 
above. Confidence intervals (CIs) around the shedding rate will also be calculated.
Key characteristics of the study analyses are described here. A more comprehensive 
and detailed (separate) analysis plan will be developed prior to the conclusion of the 
trial. 
Description of the Statistical Methods to Be Employed: 

Where applicable, safety events based on continuous outcomes such as 
laboratory values will be reported as actual test values, which for study group 
summaries may also be depicted in dot plots or other descriptive displays.

Differences in quantitative variables, such as shedding frequency in this 
challenge protocol compared to the first challenge protocol, will be assessed by 
using a Wilcoxon test or perhaps a t or F test (if normality of the responses is a 
credible assumption).

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
Correlations will be assessed by Spearman’s rank correlation, but Pearson’s 
correlation as well as linear regression may be employed for data that are 
sufficiently normal.

CIs will be calculated for all summary means (of raw values or of their logs, 
where appropriate) as well as proportions.

Methods to adjust p-values to account for multiple comparisons (other than 
standard repeated measures analysis of variables that change over time) are not 
planned for use. 
Level of Significance to be Used: A two-sided p-value of 0.05 will be used to denote 
conventional statistical significance. 
Subpopulation Analyses of Intervention Effect: Symptoms among those who 
become infected.
Handling of Missing and Spurious Data: Missing data will be assumed to be at 
random, unless there is evidence to the contrary (e.g., drop-outs differ by dose or other 
variable(s)). The potential effects of missing data will be handled by sensitivity analysis 
for critical conclusions. 
14 Ethics/Protection of Human Subjects
14.1 Informed Consent Process
Informed consent is a process where information is presented to enable persons to 
voluntarily decide whether or not to participate as a research subject. It is an ongoing 
conversation between the human research subject and the researchers which begins 
before consent is given and continues until the end of the subject's involvement in the 
research. Discussions about the research will provide essential information about the 
study and include: purpose, duration, experimental procedures, alternatives, risks and 
benefits. Subjects will be given the opportunity to ask questions and have them 
answered. 
The subjects will sign the informed consent document prior to undergoing any research 
procedures. The subjects may withdraw consent at any time throughout the course of 
the trial. A copy of the informed consent document will be given to the subjects for their 
records. The researcher will document the signing of the consent form in the subject’s 
medical record. The rights and welfare of the subjects will be protected by emphasizing 

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to them that the quality of their medical care will not be adversely affected if they decline 
to participate in this study. 
Informed consent will be obtained during the Screen 2 Visit (between days −30 and −2). 
Alternatively, informed consent may also be obtained on Day −1 prior to any 
protocol-specific procedures, if it was not obtained during screening.  
14.1.1
Non–English-Speaking Participants
If a non–English-speaking participant is unexpectedly eligible for enrollment, the 
participant will be provided with the Clinical Center Short Written Consent Form for 
Non–English-Speaking Research Participants in the participant’s native language and a 
verbal explanation of the purpose, procedures and risks of the study as described in 
Medical Administrative Series Policy M77-2, NIH HRPP Standard Operating Procedure 
12, and 45 CFR 46.117(b)(2). The IRB-approved English consent form will serve as 
basis for the verbal explanation of the study. The investigator will obtain an interpreter 
unless the investigator is fluent in the prospective participant’s language. Preferably, the 
interpreter will be someone who is independent of the participant (i.e., not a family 
member). Interpreters provided by the Clinical Center will be used whenever possible. 
The interpreter will interpret all oral communications (English to target language and 
conversely) between the investigator and a limited English-proficient participant, 
facilitate discussions, and clarify information as necessary.
The IRB-approved English consent form will be signed by the investigator obtaining 
consent and a witness to the oral presentation. The Clinical Center Short Written 
Consent Form will be signed by the participant and a witness who observed the 
presentation of information. The interpreter may sign the consent document as the 
witness and, in this case, will note “Interpreter” under the signature line. A copy of both 
signed forms will be provided to the participant to take home.
The investigator obtaining consent will document the consent process in the 
participant’s medical record (CRIMSON), including the name of the interpreter. Further, 
all instances of use of the Clinical Center Short Written Consent Form will be reported to 
the IRB at the time of annual review. If the Clinical Center Short Written Consent Form 
is used three times or more for the same language, this will be reported to the IRB 
immediately.

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14.2 Subject Confidentiality
All records will be kept confidential to the extent provided by federal, state and local law. 
The study monitors and other authorized representatives of the sponsor may inspect all 
documents and records required to be maintained by the investigator, including but not 
limited to medical records. Records will be kept locked and all computer entry and 
networking programs will be done with coded numbers only. Clinical information will not 
be released without written permission of the subject, except as necessary for 
monitoring by IRB, the FDA, NIAID, and OHRP.
15 Data Handling and Record Keeping
15.1 Data Capture and Management 
Study data will be maintained in CRIMSON and collected directly from subjects during 
study visits and telephone calls, or will be abstracted from subjects’ medical records. 
Source documents include all recordings of observations or notations of clinical 
activities and all reports and records necessary to confirm the data abstracted for this 
study. Data entry into CRIMSON will be performed by authorized individuals. The 
investigator is responsible for assuring that the data collected are complete, accurate, 
and recorded in a timely manner.
15.2 Record Retention
The investigator is responsible for retaining all essential documents listed in the ICH-
GCP guidelines. Study records will be maintained by the PI for a minimum of 3 years 
and in compliance with institutional, IRB, state, and federal medical records retention 
requirements, whichever is longest. All stored records will be kept confidential to the 
extent required by federal, state, and local law.
Should the investigator wish to assign the study records to another party and/or move 
them to another location, the investigator will provide written notification of such intent to 
OCRPRO/NIAID with the name of the person who will accept responsibility for the 
transferred records and/or their new location. Destruction or relocation of research 
records will not proceed without written permission from NIAID/OCRPRO.

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Appendix A: Scientific References
1.
Collins P, Karron R. Respiratory Syncytial virus and Metapneumovirus. In: 
Knipe DM, Howley PM, eds. Fields virology. 6th ed. Philadelphia, PA: Wolters 
Kluwer/Lippincott Williams & Wilkins Health; 2013:1086-1129.
2.
Haynes AK, Prill MM, Iwane MK, Gerber SI, Centers for Disease C, 
Prevention. Respiratory syncytial virus--United States, July 2012-June 2014. 
MMWR. Morbidity and mortality weekly report. 2014;63(48):1133-1136.
3.
Nair H, Nokes DJ, Gessner BD, et al. Global burden of acute lower 
respiratory infections due to respiratory syncytial virus in young children: a 
systematic review and meta-analysis. Lancet. 2010;375(9725):1545-1555.
4.
Glezen WP, Taber LH, Frank AL, Kasel JA. Risk of primary infection and 
reinfection with respiratory syncytial virus. American journal of diseases of 
children. 1986;140(6):543-546.
5.
Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory 
syncytial virus infection in elderly and high-risk adults. The New England journal 
of medicine. 2005;352(17):1749-1759.
6.
Falsey AR, McElhaney JE, Beran J, et al. Respiratory syncytial virus and 
other respiratory viral infections in older adults with moderate to severe influenza-
like illness. The Journal of infectious diseases. 2014;209(12):1873-1881.
7.
Lewis FA, Rae ML, Lehman NI, Ferris AA. A syncytial virus associated 
with epidemic disease of lower respiratory tract in infants and young children. 
The Medical Journal of Australia. 1961;2:932-933.
8.
Collins PL, Hill MG, Camargo E, Grosfeld H, Chanock RM, Murphy BR. 
Production of infectious human respiratory syncytial virus from cloned cDNA 
confirms an essential role for the transcription elongation factor from the 5' 
proximal open reading frame of the M2 mRNA in gene expression and provides a 
capability for vaccine development. Proceedings of the National Academy of 
Sciences of the United States of America. 1995;92(25):11563-11567.
9.
Connors M, Crowe JE, Jr., Firestone CY, Murphy BR, Collins PL. A cold-
passaged, attenuated strain of human respiratory syncytial virus contains 
mutations in the F and L genes. Virology. 1995;208(2):478-484.
10.
Whitehead SS, Bukreyev A, Teng MN, et al. Recombinant respiratory 
syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in 
chimpanzees. Journal of virology. 1999;73(4):3438-3442.
11.
Whitehead SS, Juhasz K, Firestone CY, Collins PL, Murphy BR. 
Recombinant respiratory syncytial virus (RSV) bearing a set of mutations from 

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cold-passaged RSV is attenuated in chimpanzees. Journal of virology. 
1998;72(5):4467-4471.
12.
Hall CB, Douglas RG, Jr., Schnabel KC, Geiman JM. Infectivity of 
respiratory syncytial virus by various routes of inoculation. Infection and 
immunity. 1981;33(3):779-783.
13.
Lee FE, Walsh EE, Falsey AR, Betts RF, Treanor JJ. Experimental 
infection of humans with A2 respiratory syncytial virus. Antiviral research. 
2004;63(3):191-196.
14.
Noah TL, Becker S. Chemokines in nasal secretions of normal adults 
experimentally infected with respiratory syncytial virus. Clinical immunology. 
2000;97(1):43-49.
15.
Kravetz HM, Knight V, Chanock RM, et al. Respiratory syncytial virus. III. 
Production of illness and clinical observations in adult volunteers. Jama. 
1961;176:657-663.
16.
Hall CB, McBride JT. Respiratory syncytial virus--from chimps with colds 
to conundrums and cures. The New England journal of medicine. 
1991;325(1):57-58.
17.
McKay E, Higgins P, Tyrrell D, Pringle C. Immunogenicity and 
pathogenicity of temperature-sensitive modified respiratory syncytial virus in adult 
volunteers. Journal of medical virology. 1988;25(4):411-421.
18.
Hall CB, Walsh EE, Hruska JF, Betts RF, Hall WJ. Ribavirin treatment of 
experimental respiratory syncytial viral infection. A controlled double-blind study 
in young adults. Jama. 1983;249(19):2666-2670.
19.
Hall CB, Long CE, Schnabel KC. Respiratory syncytial virus infections in 
previously healthy working adults. Clinical infectious diseases : an official 
publication of the Infectious Diseases Society of America. 2001;33(6):792-796.
20.
DeVincenzo JP, Whitley RJ, Mackman RL, et al. Oral GS-5806 activity in 
a respiratory syncytial virus challenge study. The New England journal of 
medicine. 2014;371(8):711-722.
21.
Buchman CA, Doyle WJ, Pilcher O, Gentile DA, Skoner DP. Nasal and 
otologic effects of experimental respiratory syncytial virus infection in adults. 
American journal of otolaryngology. 2002;23(2):70-75.
22.
Simoes EA, DeVincenzo JP, Boeckh M, et al. Challenges and 
opportunities in developing respiratory syncytial virus therapeutics. The Journal 
of infectious diseases. 2015;211 Suppl 1:S1-S20.

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23.
Habibi MS, Jozwik A, Makris S, et al. Impaired Antibody-mediated 
Protection and Defective IgA B Cell Memory in Experimental Infection of Adults 
with Respiratory Syncytial Virus. American journal of respiratory and critical care 
medicine. 2015.
24.
DeVincenzo J, Lambkin-Williams R, Wilkinson T, et al. A randomized, 
double-blind, placebo-controlled study of an RNAi-based therapy directed against 
respiratory syncytial virus. Proceedings of the National Academy of Sciences of 
the United States of America. 2010;107(19):8800-8805.
25.
DeVincenzo JP, Wilkinson T, Vaishnaw A, et al. Viral load drives disease 
in humans experimentally infected with respiratory syncytial virus. American 
journal of respiratory and critical care medicine. 2010;182(10):1305-1314.
26.      R Wolfe, D Braude. “Intranasal Medication Delivery for Children: A Brief 
Review and Update.” Pediatrics. 2010
27.      Powers JH et al. Development of the Flu-PRO: a patient-reported 
outcome (PRO) instrument to evaluate symptoms of influenza.  BMC Infect Dis.  
2016;16: 1-11.
28.      Memoli MJ, Czajkowski L, Reed S, et al.  Validation of the wild-type                      
influenza     A human challenge model H1N1pdMIST: an A(H1N1)pdm09 dose-
finding investigational new drug study.  Clin Infect Dis.  2015;60:  693-702.
    

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Appendix B: Schedule of Procedures/Evaluations
Inpatient
Outpatient
Study Day
Clinical Evaluations
−
1
0
1
2
3
4
5
6
7 a
8-9
10
11+
28 (-1 to +10)
56 (-5 to +15)
Informed consent, if not signed during screen 2 visit 
or after
X
Height and weight
X
Vital signs
X
X
X
X
X
X
X
X
X
X
X
X
X
X
FLU-PRO symptom questionnairef
X
X
X
X
X
X
X
X
X
X
X
Interim medical history and physical exam
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Review/sign reproductive counseling form
X
Diary card
X
X
Assessment of AEs
X
X
X
X
X
X
X
X
X
X
X
X
X
CBC with differential
X
X
X
X
X
X
Acute care panel
Xb
X
X
X
X
Hepatic panel
Xb
X
X
X
X
Urine qualitative drug screen
X
Pregnancy testing, serum
Xc
HIV 1/2 rapid antibody test
X
FilmArray PCRd , respiratory virus panel
X
X
X
X
X
X
X
X
X
X
HLA typing
X
EBV IgG
X
Quantitative immunoglobulins
X
Lymphocyte phenotyping, TBNK
X
X
X
X
X
Hepatitis B and C serology
Xg
EKG
Xg
CXR
Xg
PFT
Xb
Evaluate discharge criteriae
X
X
X
X
Research Evaluations
RSV A2 inoculation (0.5 ml per nare)
X
Nasosorption using SAM strips (LID RSV lab)
X
X
X
X
X
Nasal swab – microbiome (sequencing) (LID RSV 
lab)
X
X
X
X
X
X
X
Nasal swab – microbiome (culture) (LID RSV lab)
X
X
X
X
X
X
NP wash – research lab–based quantitative RT-
PCR, quantitative viral culture (JHU RSV lab)
X
X
X
X
X
X
X
X
X
X
NP wash – fresh viral culture (LID RSV lab)
X
X
X
X
X
X
X
X
X
X
NP wash – cytokines/chemokines (LID RSV lab)
X
X
X
X
X
X

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NP wash – immunoglobulins (e.g.secretory IgA) (LID 
RSV lab)
X
X
X
X
X
Serum for total IgA or IgG (LID RSV lab)
X
X
X
Serum for neutralizing antibody titer (PRNT), anti-
RSV IgG, anti-F and/or anti-G (JHU RSV lab)
X
X
X
X
Cellular immune response T- and B-cell assays 
(Frederick IML PBMCs)
X
X
X
X
X
Quantitative & molecular immune cell phenotyping 
(Frederick IML)
X
X
X
X
X
X
Serum cytokine analysis (Frederick IML) 
X
X
X
X
X
Transcriptome PAXgene (LID RSV lab)
X
X
X
X
X
X
a
First possible discharge day.  If the subject is still shedding virus at this time, then inpatient hospitalization will continue until they have 2 consecutive 
negative NP washes for RSV by FilmArray assay (Respiratory Virus Panel).
b
Repeat labs and PFT on Day -1 if performed during screening period at >56 days prior to Day -1.
c
Subjects of child-bearing potential must have a negative result prior to proceeding with virus inoculation.
d
Multiplex PCR for respiratory viruses and bacteria (FilmArray, BioFire Diagnostics, Inc. [Salt Lake City, UT, USA]).
e
Discharge criteria fulfilled if subject has 2 consecutive negative NP washes for RSV RNA (Film Array) and no evidence for RSV-associated lower 
respiratory tract disease.
f      Administered using RED CAP; if RED CAP not working for participant, paper questionnaires will be administered once daily      
g     Perform on Day -1 if performed during screening at >120 days prior to Day -1
      
AE = adverse event; CBC = complete blood count; CHI = Center for Human Immunology, Autoimmunity, and Inflammation; CXR = chest x-ray; EBV = Epstein-
Barr virus; EKG = electrocardiogram; HLA = human leukocyte antigen; Ig = immunoglobulin; LID = Laboratory of Infectious Disease; JHU = Johns Hopkins 
University; IML = Immunologic Monitoring Lab; NP = nasopharyngeal; PCR = polymerase chain reaction; PFT = pulmonary function tests; PRNT = plaque 
reduction neutralizing titer; RSV = respiratory syncytial virus; SAM = synthetic absorptive matrix; X = to be performed.

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Appendix C: Blood Volumes for Specimen Collection
Blood volume collected (ml)
Evaluation
Day −1
0
1
2
3
4
5
6
7
10
28 
(-1 to +10)
56
(-5 to 
+15)
CBC with differential
2
2
2
2
2
2
Acute care/hepatic panel 
4
4
4
4
4
Pregnancy testing, females
4
HIV 1/2 rapid antibody test
4
HLA typing
10
EBV IgG
4
Quantitative immunoglobulins
4
Serum for total IgA/IgG; 
Neutralizing antibody titer: anti-RSV 
IgG, anti-F and/or anti-G
4
4
4
4
T, B, NK cell flow cytometry test
3
3
3
3
3
Cellular immune response assays 
30
30
30
30
30
Quantitative and molecular immune 
cell phenotyping 
30
30
35
30
30
35
Cytokine analysis
8
8
8
8
8
Transcriptome PAXgene
2.5
2.5
2.5
2.5
2.5
2.5
Daily volume
109.5
70.5
50.5
0
6
0
0
0
79.5
30
83.5
50.5
Cumulative volume
109.5
180
231
231
237
237
237
237
320
350
433.5
485

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Appendix D: Summary of Prior Clinical Studies (2000-present)
Study 
(reference)
RSV 
subtype
(strain)
Dose a
Pre-
challenge 
serum 
neutralizing 
titer b
N c
% Shedding 
d
% 
Symptomatic 
of infected 
subjects e
Mean
peak viral 
titer 
(log10 per ml)
Mean 
duration of 
shedding 
(days) 
Lee et al, 200413
A (A2)
4.7 
(TCID50)
<9.36 
13
92 (cx)
100 f
2.2 (cx)
4.4 (cx)
4.7 (TCID50)
NA
14
50 (cx)
38 g
NA
NA
3.7 (TCID50)
NA
14
21 (cx)
40 g
NA
NA
Noah and 
Becker, 200014
A (A2)
3.0 (PFU)
NA
9
33 (cx)
100 f
NA
NA
DeVincenzo et 
al, 201420,k
A
(Memphis-37)
4.0 (PFU)
NA
53
67.9 (PCR)
NA
6.0 - 6.4 
(PCR) h 
8 (PCR) i
DeVincenzo et 
al, 201024,k
A 
(Memphis-37)
4.7 (PFU)
NA
42
71.4 (cx)
85.7 (PCR)
NA
3.2 (cx) i
4.5 (PCR) i
7 (cx) i
9 (PCR) i
DeVincenzo et 
al, 201025
A 
(Memphis-37)
3.0 to 5.4 
(PFU)
NA 
35
60 (cx)
77 (PCR)
NA
4.5 (cx) i
6.5 (PCR) i
6.5 (cx) i
10 (PCR) i
Habibi et al, 
201523
A
(Memphis-37)
4.0 (PFU)
NA
61
56 (PCR)
68 f
NA
NA
Buchman et al, 
200221
B 
6.0 (PFU)
NA
32
47 (cx)
NA
NA
NA
a RSV dose administered (log10); PFU, plaque-forming units; TCID50, 50% cell culture infectious dose.
b microneutralization titer, reciprocal log2; NA, data not available.
c N, number enrolled.
d % of subjects shedding RSV challenge virus; cx, determined by quantitative viral culture; PCR, determined by quantitative reverse transcription polymerase chain 
reaction.

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e % of RSV challenge virus infected subjects exhibiting symptoms of upper respiratory tract infection. Lower respiratory tract symptoms were not detected in any 
study.
f infected defined as: RSV shedding detected. 
g infected defined as: 4-fold or greater rise in RSV serum antibody titer and/or RSV shedding detected.
h Separate models were used to compare with 3 different antiviral treatment cohorts, resulting in slight differences in adjusted means in each model.
i approximate values, obtained from graphical data.
k Data from RSV challenge virus recipients treated with investigational antiviral drug were excluded.

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Appendix E: Toxicity Table
The functional table below should be used to grade the severity of an AE that is not specifically identified in the grading 
table. In addition, all deaths related to an AE are to be classified as Grade 5.
Parameter
Definition
Grade 1
(Mild)
Grade 2
(Moderate)
Grade 3
(Severe)
Grade 4
(Life-
threatening)
Fever
Sustained rise in body 
temperature
37.7°C-38.6°C
38.7°C-39.3°C
39.4°C-40.5°C
>40.5°C
Nasal 
congestion or 
rhinorrhea
Congestion: stuffy or blocked 
nasal passages. May be observed 
on exam and/or cause changes in 
phonation
Rhinorrhea: nasal drainage not 
associated with NP washes. May 
be observed on exam and/or 
cause nose blowing >2x/hr
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities and 
significant 
interference with 
sleep 
N/A
Ear pain
Pain in the ear, may be 
associated with abnormal 
tympanic membrane landmarks
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities and 
requiring 
prescription 
strength 
medications 
N/A
Sinus 
congestion or 
pain
A sensation of fullness, pressure 
or pain on the face overlying a 
sinus cavity
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities and 
significant 
interference with 
sleep 
N/A
Sore throat
Pain in the throat, and/or difficulty 
swallowing. May also be 
accompanied by pharyngeal 
exudate, and/or enlarged tender 
lymph nodes.
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities and 
requiring 
prescription 
strength 
medications 
Hospitalization 

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Parameter
Definition
Grade 1
(Mild)
Grade 2
(Moderate)
Grade 3
(Severe)
Grade 4
(Life-
threatening)
and/or IV 
hydration 
Epistaxis
Bleeding from any part of the 
nose or nasopharynx
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities and 
requiring 
intervention such 
as nasal packing 
Hospitalization or 
operative 
intervention 
indicated
Headache
Pain located in any part of the 
head that can arise from different 
causes lasting >1 hour, as 
reported by subject
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities and 
requiring 
prescription 
strength 
medications
Symptoms 
causing inability 
to perform basic 
self-care 
functions OR 
Hospitalization 
indicated (other 
than emergency 
room visit) OR 
Headache with 
significant 
impairment of 
alertness or other 
neurologic 
function 
Cough
Episodes of forcefully expelling air 
from the lungs, often to clear 
irritants or secretions
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities and 
requiring 
prescription 
strength 
medications
Hospitalization 
Hoarseness
Sustained rough or harsh 
changes in usual voice, often with 
throat irritation, noted by subject 
or health professional
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities 
Hospitalization or 
operative 
intervention 
indicated to 
maintain patent 
airway

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Parameter
Definition
Grade 1
(Mild)
Grade 2
(Moderate)
Grade 3
(Severe)
Grade 4
(Life-
threatening)
Malaise
Generalized feeling of discomfort, 
illness, or lack of well-being.
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities 
Incapacitating 
malaise causing 
inability to 
perform basic 
self-care 
functions 
Chills
Feeling a sense of coldness 
accompanied by shivering
no or minimal 
interference with 
usual daily 
activities 
greater than 
minimal 
interference with 
usual daily 
activities 
inability to perform 
usual daily 
activities 
N/A
Usual daily activities: Everyday tasks, performance of which is required for personal self-care and independent living, 
such as eating, bathing, dressing, toileting, and transferring. 
Grading of AEs related to Vital Signs and Laboratory Values: Adapted from FDA Guidance for Industry Toxicity 
Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials, 2007. Changes 
to the FDA tables are annotated as footnotes.

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1 Subject should be at rest for all vital sign measurements. 
2 Use clinical judgment when characterizing bradycardia among some healthy subject populations, for example, conditioned athletes. 
3 Severity grading definition for hypotension includes added clarifications such that an asymptomatic low blood pressure reading is not an adverse 
event. 
4 Removed ER visit as necessary criteria for Grade 4 AE

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1 Severity grading for hemoglobin decrease on the basis of the magnitude of decrease from baseline is not applicable at 

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the Grade 1 level; only absolute hemoglobin will be used to define Grade 1 decrease. Increases in hemoglobin are AEs 
only for values above the upper limit of normal and are graded by the systemic illness clinical criteria. 
# 1 X ULN was removed from the definition for PT increase. 
* Any laboratory value shown as a “graded” value in the table that is within the institutional normal range will not be 
severity graded or recorded as an adverse event. 
**”ULN” is the upper limit of the normal range.

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Appendix F: Patient-Reported FLU-PRO Symptom 
Questionnaire
Participant ID : _________
Participant Initials :__________Date : ____/____/___
RSV Illness Symptom Questionnaire
What time is it? ______  AM / PM  (please circle)
Please rate the extent to which you had each symptom during the past 24 hours. 
Not at all
A little bit
Somewhat
Quite a bit
Very much
Runny or dripping nose
☐
☐
☐
☐
☐
Congested or stuffy nose
☐
☐
☐
☐
☐
Sinus pressure
☐
☐
☐
☐
☐
Scratchy or itchy throat
☐
☐
☐
☐
☐
Sore or painful throat
☐
☐
☐
☐
☐
Difficulty swallowing
☐
☐
☐
☐
☐
Teary or watery eyes
☐
☐
☐
☐
☐
Sore or painful eyes
☐
☐
☐
☐
☐
Eyes sensitive to light
☐
☐
☐
☐
☐
Trouble breathing
☐
☐
☐
☐
☐
Chest congestion
☐
☐
☐
☐
☐
People experience the RSV illness in different ways. We would like to know about the symptoms 
you have been experiencing during the past 24 hours. For each symptom, please mark one box 
 under the response that best matches your experience. Mark the “Not at all” box, if you did not 
have that symptom in the past 24 hours.

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Please rate the extent to which you had each symptom during the past 24 hours. 
Not at all
A little bit
Somewhat
Quite a bit
Very much
Chest tightness
☐
☐
☐
☐
☐
Dry or hacking cough
☐
☐
☐
☐
☐
Wet or loose cough
☐
☐
☐
☐
☐
Felt nauseous (feeling like you 
wanted to throw-up)
☐
☐
☐
☐
☐
Stomach ache
☐
☐
☐
☐
☐
Felt dizzy 
☐
☐
☐
☐
☐
Head congestion
☐
☐
☐
☐
☐
Headache
☐
☐
☐
☐
☐
Lack of appetite
☐
☐
☐
☐
☐
Sleeping more than usual
☐
☐
☐
☐
☐
Body aches or pains
☐
☐
☐
☐
☐
Weak or tired
☐
☐
☐
☐
☐
Chills or shivering
☐
☐
☐
☐
☐
Felt cold
☐
☐
☐
☐
☐
Felt hot
☐
☐
☐
☐
☐
Sweating
☐
☐
☐
☐
☐
In the past 24 hours, how often have you had any of the following symptoms?
Never
Rarely
Sometimes
Often
Always

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Please rate the extent to which you had each symptom during the past 24 hours. 
Not at all
A little bit
Somewhat
Quite a bit
Very much
Sneezing
☐
☐
☐
☐
☐
Coughing
☐
☐
☐
☐
☐
Coughed up mucus or phlegm
☐
☐
☐
☐
☐
0 times
1 time
2 times
3 times
4 or more 
times
How many times did you vomit? 
☐
☐
☐
☐
☐
How many times did you have 
diarrhea?
☐
☐
☐
☐
☐
Items to be asked in the daily diary through to Day 14 along with the FLU-PRO items.
1.  Did you take any medication for your RSV illness symptoms today? (Please select one 
response only)
0  No
1  Yes
2.  Do you have asthma, COPD (chronic obstructive pulmonary disease) or both?
0  No
1  Yes
3.
[Only asked if answer to the question above is “yes”].  Did you use any rescue medication 
today for your asthma or COPD? (Please select one response only)
0  No
1  Yes
4.  Overall, how severe were your RSV illness symptoms today? (Please select one response 
only)

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0  No flu symptoms today
1  Mild
2  Moderate
3  Severe
4  Very severe
5.  Overall, how were your RSV illness symptoms today compared to yesterday? (Please select 
one response only)
1  Much better
2  Somewhat better
3  A little better
4  About the same
5  A little worse
6  Somewhat worse
7  Much worse
6.  How much did your RSV illness symptoms interfere with your usual activities today? (Please 
select one response only)
1  Not at all
2  A little bit
3  Somewhat
4  Quite a bit
5  Very much
7.  Have you returned to your usual activities today?
0  No
1  Yes
8.  In general, how would you rate your physical health today? (Please select one response only)
1  Poor
2  Fair
3  Good

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4  Very Good
5  Excellent
9.  Have you returned to your usual health today?
0  No
1  Yes