Hallmarks of Protective Immunity in Sequential Rhinovirus Infections in Humans
Phase: PHASE1
Conditions: Healthy Volunteers
Sponsor: University of Virginia

DMID Protocol 16-0055 
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Hallmarks of Protective Immunity in Sequential Rhinovirus 
Infections in Humans 
 
DMID Protocol Number:  16-0055 
 
DMID Funding Mechanism:  1U01AI125056-01 
PAR-13-151 
IND Sponsor:  Ronald B. Turner, MD 
Principal Investigators:  Judith A. Woodfolk, MBChB, PhD; Ronald B. Turner, MD 
DMID Clinical Protocol Manager:  Tena Knudsen, RN 
DMID Medical Monitor:  Venus Shahamatdar, MD 
 
 
Version Number:   2.0 
13 October 2017 
UVA IRB-HSR #18936, Version 2.0 
 

DMID Protocol 16-0055 
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STATEMENT OF COMPLIANCE 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by 
the following:  
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies: 
45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 54, 21 CFR Part 56, 21 CFR Part 11 and 
21 CFR Part 312 
• 
Conformity with the International Conference on Harmonization (ICH) E6(R2), and per 
FDA Guidance for Industry E6: Good Clinical Practice (GCP), Consolidated Summary   
• 
NIH Clinical Terms of Award 
Refer to:  http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.htm#46. 
http://www.fda.gov/cder/guidance/959fnl.pdf 
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-01-061.html 
 
Compliance with these standards provides public assurance that the rights, safety and well-
being of study subjects are protected, consistent with the principles that have their origin in 
the Declaration of Helsinki. 
All key personnel (all individuals responsible for the design and conduct of this study) will 
complete and maintain Human Subjects Protection Training. 
 

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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this trial will be conducted according to all 
stipulations of the protocol, including all statements regarding confidentiality, and according 
to local legal and regulatory requirements and applicable US federal regulations and ICH 
guidelines. 
 
Site Investigators: 
 
Signed: 
 
Date: 
 
 
Name 
Title 
 
 
 
 
Signed: 
 
Date: 
 
 
Name 
Title 
 
 
 

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TABLE OF CONTENTS 
Page 
Statement of Compliance 
ii 
Signature Page 
iii 
Table of Contents 
iv 
List of Abbreviations 
viii 
Protocol Summary 
x 
1 
Key Roles 
1 
2 
Background Information and Scientific Rationale 
3 
 
Background Information .................................................................................... 3 
 
Rationale ........................................................................................................... 7 
 
Potential Risks and Benefits .............................................................................. 8 
 
Potential Risks ....................................................................................... 8 
 
Known Potential Benefits ....................................................................... 8 
3 
Objectives 
9 
 
Study Objectives ............................................................................................... 9 
 
Primary Objectives ................................................................................ 9 
 
Exploratory Objectives ........................................................................... 9 
 
Study Outcome Measures ................................................................................. 9 
 
Primary Outcome Measures .................................................................. 9 
 
Exploratory Outcome Measures .......................................................... 10 
4 
Study Design 
11 
 
Description of the Study .................................................................................. 11 
 
Volunteers ....................................................................................................... 11 
5 
Study Enrollment and Withdrawal 
13 
 
Selection of the Study Population .................................................................... 13 
 
Subject Inclusion Criteria ................................................................................ 13 
 
Inclusion at Re-challenge .................................................................... 14 
 
Subject Exclusion Criteria ............................................................................... 14 
 
Exclusion at enrollment ....................................................................... 14 
 
Exclusion at Challenge ........................................................................ 15 
 
Exclusion at Re-challenge ................................................................... 15 
 
Treatment Assignment Procedures ................................................................. 15 
 
Randomization Procedures ................................................................. 15 
 
Masking Procedures ............................................................................ 15 
 
Reasons for Withdrawal....................................................................... 16 
 
Handling of Withdrawals ...................................................................... 16 
 
Termination of Study ........................................................................... 17 
6 
Investigational Product 
18 
 
Investigational Product Description ................................................................. 18 
 
Acquisition ........................................................................................... 18 

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Formulation, Packaging, and Labeling ................................................. 18 
 
Product Storage and Stability .............................................................. 18 
 
Dosage, Preparation and Administration of Investigational Product ................ 18 
 
Challenge and Re-challenge with RV .................................................. 18 
 
Modification of Investigational Product for a Participant ....................... 18 
 
Accountability Procedures for the Investigational Product(s) ........................... 19 
 
Assessment of Subject Compliance with Investigational Product .................... 19 
 
Concomitant Medications/Treatments ............................................................. 19 
7 
Study Schedule 
20 
 
Study Schedule ............................................................................................... 20 
 
Screening ........................................................................................................ 20 
 
Screening and Enrollment Visit (Study day -14 to -7, study visit 1) ...... 20 
 
First Virus Challenge ....................................................................................... 20 
 
Study day 0 (study visit 2) .................................................................... 20 
 
Study days 1-5 (study visits 3-7) .......................................................... 20 
 
Study day 7 (study visit 8) .................................................................... 21 
 
Study day 14 (study visit 9) .................................................................. 21 
 
Study day 28 ± 2 (study visit 10) .......................................................... 21 
 
Study day 63 ± 2, Week 9 (study visit 11) ............................................ 21 
 
15 Weeks after Virus Challenge (study visit 12) ................................... 21 
 
Second Virus Challenge .................................................................................. 21 
 
Study day RC0 (study visit RC 1) ........................................................ 21 
 
Study days RC1-RC5 (study visits RC 2-6).......................................... 22 
 
Study day RC7 (study visit RC 7) ........................................................ 22 
 
Study day RC14 (study visit RC 8) ...................................................... 22 
 
Study day RC28 ± 2 (study visit RC 9) ................................................. 22 
 
Study day RC63 ± 2, RC Week 9 (study visit RC 10) ........................... 22 
 
15 Weeks after Second Virus Challenge (study visit RC 11) ................ 22 
 
Follow-up ........................................................................................................ 22 
 
Final Study Visit .............................................................................................. 22 
 
Early Termination Visit .................................................................................... 23 
 
Unscheduled Visit ........................................................................................... 23 
8 
Study Procedures/Evaluations 
24 
 
Clinical Evaluations ......................................................................................... 24 
 
Medical history .................................................................................... 24 
 
Vital Signs ........................................................................................... 24 
 
Concomitant medications .................................................................... 24 
 
Study Procedures ........................................................................................... 24 
 
Nasal Lavage ...................................................................................... 24 
 
Symptom Scoring ................................................................................ 24 
 
Assessment of infection and illness ..................................................... 25 
 
Laboratory Evaluations ................................................................................... 25 

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Virology ............................................................................................... 25 
 
Serology .............................................................................................. 26 
 
Other Laboratory Evaluations .............................................................. 26 
 
Special Assays or Procedures ............................................................. 26 
 
Specimen Preparation, Handling, and Shipping ................................... 27 
 
Specimen Shipment ............................................................................ 27 
9 
Assessment of Safety 
28 
 
Methods and Timing for Assessing, Recording, and Analyzing Adverse Events 
and Serious Adverse Events ........................................................................... 28 
 
Adverse Event Definition ..................................................................... 28 
 
Serious Adverse Event Definition ........................................................ 28 
 
Assessment and Reporting of Adverse Events: ................................... 29 
 
Assessment and Reporting of Serious Adverse Events ....................... 30 
 
Reporting Plan for Serious Adverse Events, Unanticipated Problems, 
Protocol Deviations and Data Breaches .............................................. 31 
 
Reporting of Pregnancy ....................................................................... 33 
 
Methods and Timing for Assessment of the Performance of the Human 
Rhinovirus Challenge Model ........................................................................... 33 
 
Historical performance of the challenge model .................................... 33 
 
Evaluation of viral infection .................................................................. 34 
 
Evaluation of symptoms....................................................................... 34 
 
Halting Rules ....................................................................................... 34 
 
Safety Oversight (ISM plus DSMB) ................................................................. 35 
 
Independent Safety Monitor (ISM) ....................................................... 35 
 
Data and Safety Monitoring Board (DSMB) ......................................... 35 
 
Safety Monitoring................................................................................. 36 
10 
Clinical Monitoring 
37 
 
Site Monitoring Plan ........................................................................................ 37 
11 
Statistical Considerations 
38 
 
Study Hypotheses ........................................................................................... 38 
 
Study Outcome Measures ............................................................................... 38 
 Primary Objectives .............................................................................. 38 
 Exploratory Objectives ......................................................................... 38 
 Primary Outcome Measure .................................................................. 38 
 
Sample Size Considerations ........................................................................... 39 
 Sample Size Calculation Summary ...................................................... 39 
 Power Calculation Details .................................................................... 39 
 
Planned Interim Analyses ............................................................................... 40 
 Safety Review ..................................................................................... 40 
 Immunogenicity or Efficacy Review ..................................................... 40 
 
Analysis Plan .................................................................................................. 40 
 General Statistical Plan ....................................................................... 40 

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 Primary Outcome Analyses (Correlation between the number of pre-
existing RV-16 specific T-cells (15 wks.) and infection following re-
challenge with RV-39 as assessed by virus shedding in nasal lavage) 41 
 Potential analyses for exploratory endpoints........................................ 42 
 Mass Cytometry Data Analysis ............................................................ 43 
 Gene Expression Data Analysis .......................................................... 44 
12 
Source Documents and Access to Source Data/Documents 
46 
13 
Quality Control and Quality Assurance 
47 
14 
Ethics/Protection of Human Subjects 
48 
 
Ethical Standard .............................................................................................. 48 
 
Good Clinical Practice ..................................................................................... 48 
 
Institutional Review Board ............................................................................... 48 
 
Informed Consent Process .............................................................................. 49 
 
Exclusion of Women, Minorities, and Children (Special Populations) .............. 50 
 Participation of Women ....................................................................... 50 
 Participation of Minorities ..................................................................... 50 
 Participation of Children....................................................................... 50 
 
Subject Confidentiality ..................................................................................... 50 
 
Study Discontinuation ..................................................................................... 51 
 
Future Use of Stored Specimens .................................................................... 51 
15 
Data Handling and Record Keeping 
53 
 
Data Management Responsibilities ................................................................. 53 
 
Data Capture Methods .................................................................................... 53 
 
Types of Data.................................................................................................. 53 
 
Timing/Reports ................................................................................................ 54 
 
Study Records Retention ................................................................................ 54 
 
Protocol Deviations ......................................................................................... 54 
16 
Publication Policy 
55 
17 
Literature References 
57 
Appendix A: Schedule of Events 
61 
 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
AIDS 
Acquired Immunodeficiency Syndrome 
CBC 
Complete Blood Count 
CD 
Cluster of Differentiation 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CROMS 
Clinical Research Operations and Management Support 
DCC 
Data Coordinating Center 
DCF 
Data Collection Form 
DHHS 
Department of Health and Human Services 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, 
NIH, DHHS 
DSMB 
Data and Safety Monitoring Board 
eCRF 
Electronic Case Report Form 
FDA 
Food and Drug Administration 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
GEE 
Generalized Estimating Equation 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
HSR 
Health Sciences Research 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IEC 
Independent or Institutional Ethics Committee 
IgG 
Immunoglobulin G 
IND 
Investigational New Drug Application 
IP 
Investigational Product 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
JAMA 
Journal of the American Medical Association 
MedDRA 
Medical Dictionary for Regulatory Activities 
MHCII 
Major Histocompatibility Complex Class II 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
NCI 
National Cancer Institute, NIH, DHHS 
NDA 
New Drug Application 
NEJM 
New England Journal of Medicine 

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NIAID 
National Institute of Allergy and Infectious Diseases, NIH, 
DHHS 
NIH 
National Institutes of Health 
NKT 
Natural Killer T Cell 
NSAID 
Non-Steroidal Anti-inflammatory Drug 
OCRA 
Office of Clinical Research Affairs, DMID, NIAID, NIH, DHHS 
OHRP 
Office for Human Research Protections 
OHSR 
Office for Human Subjects Research 
ORA 
Office of Regulatory Affairs, DMID, NIAID, NIH, DHHS 
PCR 
Polymerase Chain Reaction 
PHI 
Protected Health Information 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
qPCR 
Quantitative Polymerase Chain Reaction 
RSV 
Respiratory Syncytial Virus 
RV 
Rhinovirus 
SADR 
Serious Adverse Drug Reaction 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
Tfh 
T Follicular Helper 
Teff 
T Effector 
Th 
T Helper 
US 
United States 
UVA 
University of Virginia 
WHO 
World Health Organization 
 
 
 

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PROTOCOL SUMMARY 
 
Title: 
Hallmarks of Protective Immunity in Sequential Rhinovirus 
Infections in Humans 
Phase: 
II 
Population: 
Healthy young adult volunteers both male and female from 
the University of Virginia (UVA) community. Approximately 
120 healthy, young adult volunteers with an antibody titer 
≤1:2 to both rhinovirus (RV) type 39 and RV type 16 will be 
recruited for participation in the study. Approximately 80 
subjects who meet all inclusion and exclusion criteria will be 
challenged with RV. 
Number of Sites: 
Single-site 
Study Duration: 
3 years (for clinical studies, total study duration is 5 years) 
Subject Participation 
Duration: 
Each subject will participate in two separate challenge 
cohorts over a period of 32 weeks. 
Description of Agent or 
Intervention: 
 Human RV types 39 and 16 by intranasal administration 
Objectives: 
 
The primary objective of this study is to assess the relationship between RV-specific 
T-cell immunity and the human host response to primary RV challenge and 
subsequent secondary challenge with either homologous or heterologous RV 
serotypes.  The overall hypothesis that will be addressed by the mechanistic studies 
in this proposal is that T helper (Th) and T follicular helper (Tfh) cells directed against 
conserved RV epitopes expand upon RV exposure and some of these cells persist as 
stable cross-reactive memory populations capable of displaying lineage-specific 
protective functions upon re-infection with related or unrelated strains of RV. The 
human specimens collected in this study will be analyzed with a variety of state-of-
the-art techniques to provide an in depth description of T-cell responses to RV 
infection, and the correlation of these responses with viral infection, antibody 
responses, and illness.  Beyond this objective, by using a systems biology approach, 
we aim to gain new insight into the role of diverse cell types involved in adaptive 
immunity to RV.  
 

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Estimated Time to 
Complete Enrollment: 
3 years 
 
Schematic of Study Design: 
 
 
-2wk.
T cells
Phenotype
Gene expression
Function
Antibodies
Specific Ab./neut. Ab.
Other
Nasal cytokines
RV cultures
Viral PCR
Symptoms
Challenge #1  (“primary”)
(RV-16)
0d.      1wk.      2wk.       4wk.                   9wk.             15wk.   16wk.    17wk.    19wk.  24wk  30wk.
Challenge #2 (“secondary”)
(RV-16 or RV-39)
√
√
√
√
√
√
√
√
√
√
√
√
 
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√√√√√√
√√√√√√
√√√√√√
√√√√√√
√
√
√√√√√√
√√√√√√
Challenge #1
RV-16
(N=80)
Screen
750
allergic
RV-16/39 seropositive
other exclusions (eg. HLA type)
Exclusions before
2nd challenge
Challenge #2
Targeted
enrollment
RV-16
(N=35)
RV-39
(N=35)
Primary effector phase
Primary memory phase
Secondary effector phase
Secondary memory phase
A
B
Model of experimental RV challenge (A) and targeted enrollment plan (B). T cells and other immune parameters will
be measured at designated time points during a primary response to RV-16 (Challenge #1) and secondary responses
to RV-16 or RV-39 (Challenge #2).

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1 
KEY ROLES 
Individuals: 
 
 
Principal Investigators:  
Judith A .Woodfolk, MBChB, PhD 
University of Virginia 
P.O. Box 801355 
Charlottesville, VA 22908 
Phone Number:  434-924-1293  
Fax Number 
Email:  jaw4m@eservices.virginia.edu 
 
Ronald B. Turner, MD 
University of Virginia 
P.O. Box 800386 
Charlottesville, VA 22908 
Phone Number:  434-243-9864 
Fax number:  434-982-4246 
Email:  rbt2n@virginia.edu 
 
DMID 
 
DMID Scientific Lead: 
Teresa M. Hauguel, Ph.D. 
Program Officer 
Respiratory Diseases Branch 
Division of Microbiology and Infectious Diseases 
NIAID/NIH/DHHS 
5601 Fishers Lane, Room 8E19 
Bethesda, MD 20892 
Phone:  240-627-3272 
Email:  hauguelt@niaid.nih.gov 
 
DMID Medical Officer:   
Trinka Coster, M.S., M.D. (C) 
Medical Officer, Respiratory Diseases Branch 
Division of Microbiology and Infectious Diseases 
NIAID/NIH/DHHS 
Room 8E25, 5601 Fishers Lane 
Bethesda, MD  20892 
Tel:  301 - 761-7736 
Fax: 240 - 627-3139 
Email:  trinka.coster@nih.gov 
 

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DMID Medical Monitor:  
Venus Shahamatdar, M.D. [C] 
Medical Monitor 
Office of Clinical Research Affairs 
DMID, NIAID, NIH 
Phone:  240-627-3369 
Email:  venus.shahamatdar@nih.gov 
 
DMID Clinical Project Manager: 
Tena Knudsen, B.S.N. 
Respiratory Diseases Branch 
DMID, NIAID, NIH 
5601 Fishers Lane, Room 8E36, MSC 9825 
Rockville, MD 20852-9825 
Phone:  240-627-3290 
Email:  knudsent@niaid.nih.gov  
 
DMID Pharmacovigilance Group: 
Clinical Research Operations and Management Support 
(CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 
(outside US) 
SAE FAX Phone Number: 1-800-275-7619 (US) or 1-301-897-
1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
 
Institutions: 
 
 
 
 
 
 
 
University of Virginia 
P.O. Box 800386 
Charlottesville, VA 22908 
Contact Person/Local Investigator:  Ronald B. Turner, MD 
Phone Number:  434-243-9864 
Fax number:  434-982-4246 
Email:  rbt2n@virginia.edu 
Institutional Review Board 
 
UVA Institutional Review Board for Health Sciences Research  
P.O. Box 800483  
Charlottesville, VA 22908 
Phone Number:  434-924-2620 
School of Medicine 
Clinical Trials Office 
 
Cobb Hall, Room 1033 
135 Hospital Drive 
Charlottesville, VA 22908-0786 
UVA Messenger Mail:  Box 800786 
Phone:  434-924-8530 

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2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
 
Background Information 
 
RV infections are one of the most common infections of man.  These infections are typically 
associated with upper respiratory symptoms but also cause lower respiratory disease in infants 
and children and immunocompromised hosts [1].  Approximately 70% of all asthma 
exacerbations in school-aged children are associated with RV infection and RV infections in 
young infants may influence the future development of asthma in susceptible hosts [2-4]. RV 
infections are also associated with exacerbations of chronic obstructive pulmonary disease [5].  
Although there has been considerable effort to develop effective antivirals for treatment of RV 
infections, there are no treatments currently available.   
 
Increased serum concentrations of neutralizing type-specific antibody are associated with 
protection from infection and illness [6-9].  
Although some neutralizing antibodies that 
cross react with different serotypes have 
been described [10], efforts to produce 
antibody-based vaccines have largely been 
abandoned due to the large number of 
different serotypes and the limited protection 
provided by the early vaccines [11, 12].    
 
Activation of RV-specific cluster of 
differentiation (CD) 4+ T cells is a critical 
antecedent to immunoglobulin G (IgG) 
responses; however, the nature of the T cells 
involved remains enigmatic owing to the lack 
of robust mouse models of RV infection, and 
challenges in identifying and tracking rare 
RV-specific T cells in humans.  It is now 
recognized that naïve CD4+ T cells develop 
into a variety of specialized T-cell subsets 
under the control of lineage-specifying 
transcription factors.  These subsets include 
“conventional” T effector (Teff) types (e.g., 
Th1, Th2 and Th17), as well as Tfh cells.  
Recent work in animal models indicates that 
Tfh cells, which provide help to B cells in 
germinal centers for antibody responses, 
persist as memory T cells following acute viral infection [13].   
 
1.  Identification of Conserved T-Cell Epitopes of RV-16:  This study protocol involves 
sequential RV challenges using the group A viruses, RV-16 and RV-39.  Through a 

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collaboration with Dr. William Kwok, PhD (Benaroya Research Institute), we have 
comprehensively mapped CD4+ T-cell epitopes of the RV-16 capsid proteins VP1 and VP2 in 
human leukocyte antigen (HLA)-diverse humans by in vitro (tetramer-guided epitope mapping) 
and in silico (computer prediction) methods [14-18]. The capsid proteins VP1 and VP2, encase 
the RNA genome and contain motifs known to bind neutralizing antibodies, as well as surface 
receptors for cell entry [19-22].  Thus, they provide candidate T-cell antigens. This work 
generated 10 unique peptide epitopes of RV-16 that are conserved across RV groups A, B and 
C (Fig. 1).  These epitopes bind to HLA class II molecules present in 85% of the general 
population (listed in Fig. 1A).  By a multi-step process that involved tetramer guided epitope 
mapping followed by direct ex vivo staining with tetramers [23-25], it was confirmed that 
circulating memory RV-16-specific CD4+ T cells present at the highest frequencies in healthy 
uninfected subjects preferentially target these epitopes (Figs. 1B & C).  Moreover, using a 
variety of computer prediction algorithms, we have shown that those epitopes identified by 
tetramer guided epitope mapping are highly conserved across RV-A species, and to a lesser 
extent across RV-B and C species (Table I).  Details of these findings are in a manuscript 
published in the Journal of Immunology [26]. 
 
 
 
2.  Patent:  Based on their cross-reactive potential, the UVA, through its affiliated Licensing and 
Ventures Group, has patented the use of these peptide epitopes as vaccines against RV 
infection.  This patent is declared in the consent form.  There is no income from this patent and 
it therefore does not constitute a financial conflict of interest under Public Health Service 
guidance. (http://grants.nih.gov/grants/policy/coi/coi_faqs.htm#3181) 
 

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3. Tracking RV-specific T Cells During Experimental 
Infection:  The current study will use major 
histocompatibility complex class II (MHCII) tetramers 
that display conserved RV-16 peptide epitopes to 
identify and track circulating RV-specific CD4+ T cells in 
subjects who receive sequential RV challenges with RV-
16 and RV-39 (Fig. 1 and Table I).  This will allow us to 
interrogate adaptive immunity to heterotypic RV strains.  
Preliminary work performed under a separate pilot study 
established the feasibility of this approach.  In that study, 
tetramer-guided epitope mapping [16] was used to 
develop two DRB1*0401 tetramers that displayed 
conserved RV-39 epitopes (one each for capsid protein 
VP1 and VP2).  The pilot study included 16 healthy 
DRB1*0401+ subjects (ages 18-60 years old) who participated in a RV-39 experimental challenge 
study. All subjects had no history of allergies and were seronegative for RV-39 (serum neutralizing 
antibody titer ≤1:4) (see enrollment, Fig. 2).  Challenge with RV-39 was administered intranasally 
(2 doses per nostril of a 0.25 mL solution containing 100 TCID50/mL).  RV-39 antibody titer was 
re-tested immediately before challenge (day 0) and during convalescence (day 21).  Those 
subjects experiencing ≥4-fold increase in RV-39 serum neutralizing antibody titer, or at least one 
RV-39 positive nasal culture, were considered infected.  Peripheral blood mononuclear cell 
(PBMCs) were isolated from DRB1*0401+ subjects before RV challenge (days -28 and 0), and 
during acute (day 5) and convalescent (day 21) phases (model, Fig. 2).  Samples were frozen 
and then thawed for analysis upon study completion.  Tetramer staining and flow cytometry 
analysis was performed using established methods.  A control tetramer containing an irrelevant 
peptide (GAD555-567) provided a negative control [23-25, 27, 28]. 
 
Of the 16 
DRB1*0401+ 
subjects 
enrolled, 12 
were infected 
and 4 were 
uninfected.  In 
post hoc tests, 3 
subjects had 
evidence of 
recent RV 
exposure based 
on positive 
serum 
neutralizing 
antibody titers or positive polymerase chain reaction (PCR) for virus on day 0.  In addition, two 
subjects who met criteria for infection based on positive nasal culture failed to seroconvert by 
day 21.  Thus, we could assess modulation of RV-specific CD4+ T cells in the context of 
different serology and exposure profiles.  Circulating CD4+ T cells specific for conserved regions 
of RV-39 capsid proteins were identified in all subjects immediately before inoculation.  Among 
Enrollment of DRB1 *0401+ subjects
RV-39 challenge model
Fig. 2. Experimental RV-39 challenge study. Enrollment of
DRB1*0401+ subjects and RV-39 challenge study design.
Arrows in the model denote PBMC sampling time points.
Fig. 3. Tracking RV-39-specific CD4+ T cells during experimental challenge. (A) Change in
the frequency of tetramer+ CD4+ T cells in infected (n=12) and uninfected (n=4) subjects.
*p<0.05,
**p<0.01.
(B)
Representative
data
from
an
infected
subject
with
RV-39
seroconversion. Control: GAD555 tetramer. (C) Relationship between the frequency of RV-
specific CD4+ T cells at day 0 and time to positive nasal RV culture post-inoculation. *p=0.03.

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infected subjects, expansion of circulating RV-specific CD4+ T cells was observed within 5 days 
of RV inoculation (Fig. 3A).  Moreover, T-cell expansion during the acute phase was present in 
infected individuals who had evidence of recent RV exposure, including one with a high T-cell 
frequency pre-inoculation (in red, Fig. 3A).  T-cell frequencies either contracted, or else 
continued to expand, after resolution of infection (Figs. 3A & B).  Subjects who failed to 
seroconvert post-inoculation, showed no expansion of circulating RV-specific CD4+ T cells 
during the acute phase, regardless of infection status (in blue, Fig. 3A).  Notably, higher 
frequencies of RV-specific T cells pre-inoculation were associated with lower infection rates as 
judged by time to positive RV culture (Fig. 3C) and delayed time to peak symptoms.  These 
observations, coupled with the link between high levels of RV shedding and more severe 
symptoms [29], imply a protective role for pre-existing T cells directed against conserved 
epitopes.  A manuscript describing these findings has been submitted for publication (Muehling 
LM, Turner RB, Brown KB, Wright PW, Lehtinen M, Kwok WW, Woodfolk JA.  Single-cell 
tracking of rhinovirus-specific CD4+ T cells during experimental infection).  MHCII tetramers that 
display conserved epitopes of RV-16 (shown in Fig. 1 & Table I) have recently been used by 
the Woodfolk group to identify, enumerate, and track RV-16-specific CD4+ T cells in healthy and 
asthmatic subjects following experimental challenge with RV-16 (work in progress).  Together, 
these findings establish our ability to track circulating RV-specific T cells during experimental 
infection. 
 
4.  Tracking the Molecular Signature of RV-Specific T Cells:  Using both RV-39 and RV-16 
challenge models, we can assess the molecular signature of circulating RV-specific T cells as 
follows:    
1.  RV-specific T cells that exist prior to infection include central memory Tfh cells. 
2.  During acute infection, RV-specific T cells expand in numbers and acquire an activated 
effector memory phenotype consistent with trafficking to inflamed sites. 
3.  Following infection, RV-specific T cells revert to a central memory phenotype. 
4.  RV-specific T cells express CCR5, a molecule implicated in T-cell homing to sites of viral 
infection [30]. 
5.  RV-specific T cells have a Th1 signature, as judged by their capacity to secrete the Th1-
associated cytokine IFN-gamma. 
Together, these findings confirm the existence of circulating central memory Tfh cells directed 
against conserved RV epitopes that have the potential to rapidly mobilize upon re-infection with 
RV, and to survive as long-term memory cells.    
 
5.  Measuring RV-Specific Serum Antibodies:  Antigen binding radioimmunoassays to 
measure IgG specific for RV-16 VP1 and VP2 have been developed and IgA assays are in 
progress.  This assay can be used to detect a rise in the levels of capsid protein-specific 
antibodies following experimental infection with RV (data not shown).  These findings establish 
the feasibility of tracking serum antibodies specific for viral antigens that are targeted by CD4+ T 
cells of interest.  Such antibodies may be highly relevant to cross-protection. 
 
6.  High-Dimensional Immunophenotyping by Mass Cytometry: T cells will be analyzed in 
the context of a broad array of cell types in order to construct a comprehensive picture of 
adaptive immunity to RV.  This will be accomplished by a systems biology approach using mass 
cytometry (CyTOF).  This technology, which allows simultaneous analysis of at least 45 immune 
markers, allows us to capture data on multiple cell types.  These include, but are not limited to, 

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Th1, Th2, Th17 cells, T follicular helper cells, CD8+ T cells, NKT cells, gamma delta T cells, and 
various B-cell populations. Mass cytometry provides a powerful discovery-based experimental 
tool that will generate large amounts of data that cannot be predicted based on current 
knowledge.  The Woodfolk lab has optimized procedures for performing mass cytometry.   
 
Summary:  We have demonstrated the ability to identify and characterize RV-specific 
CD4+ T cells, to measure RV capsid protein-specific IgG antibodies, and to apply mass 
cytometry to an experimental RV infection in humans.  We are now poised to explore how 
memory T cells specific for conserved RV epitopes mediate protective responses to RV. 
 
Rationale 
 
The experimental RV challenge model has been used for many years to study the pathogenesis 
and treatment of RV infection. In this model, seronegative volunteers are challenged by 
intranasal inoculation of a safety tested RV. The challenge model is ideal for the purposes of 
this trial since the CD4+ cells of the volunteers can be carefully characterized at baseline before 
infection with a known RV serotype, followed over the course of the infection and during 
convalescence, and then reassessed following re-challenge with either the same virus or a 
different serotype.  This longitudinal study allows us to precisely track the dynamic kinetics of 
adaptive immunity to RV in a time-controlled fashion.  Such a study would not be feasible in a 
design that used natural RV infections owing to the complex kinetics of anti-viral responses, and 
the inability to determine at what time point subjects were naturally infected.  These barriers 
would preclude accurate assessment of different phases of the adaptive response to RV. 
 
Our group is one of only a handful in the world that is performing RV challenge studies in 
humans.  A key differentiating feature of our study as compared with current models involving 
experimental inoculation with respiratory viruses in humans, is that we propose a sequential 
challenge with two different RV strains with known infectivity characteristics in the challenge 
model (RV-16 and RV-39) in order to establish proof-of concept that conserved RV peptide 
epitopes are cross-protective.  We hypothesize that Teff and Tfh cells directed against 
conserved RV epitopes expand upon RV exposure, and a minority of these cells persist as 
stable cross-reactive memory populations capable of displaying lineage-specific protective 
functions upon re-infection with related or unrelated strains of RV.  For the first time, we will use 
novel MHCII tetramers displaying RV epitopes to enumerate, phenotype and isolate RV-specific 
CD4+ T cells generated during the course of primary and secondary infections.  In conjunction, 
by applying single-cell high dimensional immunophenotyping (mass cytometry), we will capture 
data on multiple cell types in order to shed light on cellular networks, including T-cell/B-cell 
interactions, which operate during primary and secondary RV infections.  This project capitalizes 
on novel analytical tools (RV peptide/MHCII tetramers), technologies (mass cytometry and 
single-cell gene expression platforms) and computational platforms that have been developed 
and implemented by our group.  It leverages synergistic partnerships among experts in T-cell 
immunology, virology, biochemistry, and single-cell systems biology in order to address 
fundamental aspects of T cell-mediated RV protection.  Additionally, we will gain new insight 
into cellular and molecular components of the adaptive response to RV that extend beyond T 
cells.  Our findings are expected to impact human health by informing vaccine design through 
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RV challenge pools that have been developed under Good Manufacturing Practice conditions 
and are used under appropriate Investigational New Drug Applications (INDs; IND 12934 for 
RV39 and 15162 for RV16) will be used to conduct this study of RV immunity.  Volunteers will 
be challenged by intranasal inoculation via nasal drops with 20-100 TCID50 of the challenge 
virus.  This dose is selected empirically and is known to produce reproducible infection rates of 
80-90% in challenged volunteers. Healthy volunteers are selected for this study to minimize 
potential adverse events (AEs) associated with experimental infection. 
 
Potential Risks and Benefits 
 Potential Risks 
The participants in this study should expect to have a common cold and may experience nasal 
congestion, runny nose, sneezing, cough, sore throat, malaise, headache and chills but no 
significant risks to the health of the volunteers are anticipated from participation in this study.  
 
Experience with the study model suggests that adverse outcomes requiring intervention (otitis 
media, bronchospasm or sinusitis) are rare. The virus pools used for the challenge in this study 
have been thoroughly safety tested and have been approved for investigational use by the Food 
and Drug Administration (FDA; IND 12934 for RV39 and IND 15162 for RV16). AEs will be 
reported to the UVA Human Investigations Committee for review. 
 
RV infection has not been associated with any adverse effects on the fetus or newborn infant, 
however, women who are pregnant or nursing should not participate in the study. A pregnancy 
test will be done on all women who decide to participate in the study. All women who participate 
in the study must have a negative pregnancy test and be using an acceptable method of birth 
control as determined by the investigator.  
 
RV infections are associated with asthma attacks in people who have asthma. Individuals who 
have asthma (as evidenced by a history of asthma, a history of wheezing, recurrent severe 
bronchitis or coughing associated with colds) will be excluded from the study.  
 
The viruses which will be used for the study are RVs which were isolated from volunteers in 
previous studies. The donor volunteers were tested for human immunodeficiency virus (HIV; 
acquired immunodeficiency syndrome [AIDS]), hepatitis C and hepatitis B infection, and found 
to be negative. After isolation, the virus was cultured two times in the laboratory and has been 
tested for the presence of other pathogens which may cause infections in humans. The 
production and safety testing of the pools has been reviewed by the FDA and the pools have 
been granted INDs for experimental RV studies in human volunteers. 
 
 Known Potential Benefits   
An understanding of the pathogenesis of RV associated illness in humans is of substantial 
potential benefit to society.  There are no benefits to the individual volunteers in this study. 
 

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3 
OBJECTIVES 
 
Study Objectives 
 Primary Objectives 
3.1.1.1 
To assess the relationship between T-cell immunity induced by 
infection with the rhinovirus serotype RV-16, and the host response to homologous 
infection with RV-16 or the host response to heterologous infection with RV-39. 
 Exploratory Objectives 
3.1.2.1 
The major objective of the study is to perform mechanistic 
studies that apply state-of-the art experimental technologies to identify, enumerate, 
and monitor a broad array of known and novel cell types during challenge with RV-16 
and re-challenge with RV-16 or RV-39 in order to identify hallmarks of cross-
protection.  The technologies applied include, but are not limited to, MHCII tetramer 
staining, mass cytometry, and single-cell gene expression profiling.  Exploratory 
objectives include, , the following:  (1) Identify new molecular signatures of RV-
specific T cells.  (2)Evaluate the quality and quantity of T-cell responses.  (3) Identify, 
enumerate and monitor a broad array of immune cell types (e.g., B-cell populations, 
CD8+ T-cells, gamma delta T-cells, NKT cells, etc.), and novel cell types.  (4) Test 
multiple relationships between immune cell parameters and infection, serum 
antibodies, and symptoms (illness), at each sampling time point during RV challenge 
and re-challenge. Additional objectives may be explored based upon the results of 
the above mechanistic studies; a protocol amendment will be submitted documenting 
the additional objectives. 
 
Study Outcome Measures 
 Primary Outcome Measures 
Correlation between the number of pre-existing RV-16-specific T cells at Week 
15, and infection after homologous re-challenge with RV-16 or infection after 
heterologous re-challenge with RV-39 at Week 15.   
 
Numbers of pre-existing RV-specific T cells at Week 15 will be determined by 
MHCII tetramer staining followed by flow cytometry analysis.  Infection will be 
determined by isolation of virus in culture.  Infection will be defined as one 
positive RV culture obtained in the 5-day time interval after virus re-challenge at 
Week 15. 

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 Exploratory Outcome Measures   
Exploratory outcome measures include the following: 
• 
Tetramer staining and mass cytometry to immunophenotype RV-specific 
T cells. 
• 
Fluorescent-based flow cytometry and mass cytometry to identify, 
enumerate, and monitor immune cell types. 
• 
Microarray or single-cell analytical platforms to profile gene expression of 
immune cells. 
• 
Multiplex cytokine assays to identify cytokines in nasal lavage fluid. 
• 
Antigen binding assays and microtiter neutralization assays will be used 
to assess serum antibodies. Overall illness will be measured by frequency 
and severity of symptoms that are solicited and/or expressed by the 
subjects during follow-up for challenge and rechallenge.  
 
Alternative techniques may be considered, but studies will be limited to addressing 
the stated exploratory objectives only. 
 
 
 
 
 

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4 
STUDY DESIGN 
 
Description of the Study 
 
This is a mechanistic study to assess the T-cell response to RV infection using the experimental 
rhinovirus challenge model.  All subjects will receive a primary challenge with RV-16 and after 
15 weeks, will be re-challenged with either RV-16 or RV-39.  The DSMB will review the data and 
provide recommendations before the re-challenge. The model will be used to assess the 
correlation between T-cell responses and infection following challenge with homologous or 
heterologous RV strains. This study is designed to provide a detailed characterization of the T-
cell responses to RV infection, to determine whether cross-reacting T-cell responses occur, and 
to assess how the magnitude of T-cell responses induced by primary challenge with RV-16 
relates to infection following re-challenge with RV-16 or RV-39.  The timing of the sample 
collections and the re-challenge is empiric and is designed to correspond with the expected 
timing of effector and memory adaptive T-cell responses to primary and secondary RV 
challenge. 
 
 
The investigational material in this study consists of the virus challenge pools.  Although these 
pools are used under appropriate INDs, these pools are not in a drug/product development 
pathway and will never be advanced to a New Drug Application (NDA).  Under these 
circumstances, the “phase of the trial” has no relevance or meaning.  The size of this trial is 
comparable to a phase II trial in a drug development process. 
 
The investigational material is being used as a research tool and is not under study in this 
protocol.  We do not plan any changes in dosing or administration. 
 
Volunteers 
 
Approximately 750 healthy, young adult volunteers will be recruited for participation in the study. 
These volunteers will be tested for the presence of serum neutralizing antibody to RV type 39 
and RV type 16. Volunteer subjects with an antibody titer ≤1:2 to both virus serotypes (~120 
volunteers) will be eligible for participation in the challenge trial. Volunteers who are eligible after 
antibody screening and who agree to participate will complete a brief medical history 
questionnaire and will have pulse, respiratory rate, body temperature, and blood pressure 
measured.  All female subjects will have a urine pregnancy screen. 
 
Volunteers in this study will be challenged with two different RV serotypes. Volunteers will be 
free of neutralizing antibody to both serotypes at the start of the study. Two weeks prior to virus 
challenge, specimens will be collected for  phenotyping of T cells and other immune cell types 
(PBMC specimens); measurement of both neutralizing and capsid-specific antibodies (serum); 
and assessment of cytokines, quantitative RV culture, and viral PCR (nasal lavage). Two weeks 
later, all volunteers will have these baseline studies repeated and will then be challenged with 
RV16 (day 0).  After challenge, the volunteers will return to the study site daily for 5 days for 
collection of nasal lavage specimens for cytokine assays and quantitative RV culture, and for 

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assessment of common cold symptoms. Specimens will be collected 1, 2, 4 and 9 weeks after 
challenge for antibody analyses, and 1 and 4 weeks after challenge for T cell analyses. 
Approximately 15 weeks after the original challenge, all subjects will be assigned to two groups 
of approximately 35 subjects each. One group will be re-challenged with RV16 and the other 
group will be challenged with RV-39. The specimen collection after the second challenge will be 
the same as that after the first challenge.  Each volunteer will participate in study activities 
intermittently over a total period of approximately 32 weeks.  The study is designed to be 
conducted within the school calendar for the UVA which is the primary population for 
recruitment of volunteers. 
 
The study will be done in three cohorts over 3 years.  Approximately 250 volunteers will be 
screened for each cohort.  The feasibility of this enrollment is demonstrated by the fact that our 
most recent large challenge study in 2013 screened 789 volunteers for three cohorts in 1 year.  
Based on our experience with our study population, we would expect that screening of 250 
volunteers will result in 30-40 volunteers who are seronegative to both virus serotypes, 
appropriately HLA matched to the available tetramers and meet the other inclusion/exclusion 
criteria for the study.  In the first 2 years, approximately 25 subjects/year will be included in the 
first challenge with RV16.  The sample size included in the final cohort will be adjusted based on 
attrition in the first two cohorts to ensure that approximately 70 subjects are included in the 
second challenge with either RV-16 or RV-39. Based on extensive experience with the model, 
we expect limited attrition (~10%) once volunteers have qualified and been inoculated with virus 
in the first challenge.   
 

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5 
STUDY ENROLLMENT AND WITHDRAWAL 
 
Selection of the Study Population 
 
Participants in the experimental RV challenge studies at the UVA are largely drawn from the 
University community.  These studies are widely known among the students and recruitment is 
primarily via an established database of individuals who have asked to be notified when a study 
will occur.  Other recruitment activities include mass emails to the student population and 
“sidewalk chalk” advertising. 
 
Recruitment of eligible volunteers will not be a limitation of this study.  Our study site at the UVA 
has recently completed a separate study in which almost 800 volunteers were screened for 
participation in a challenge study over a 1-year period.  The target of approximately 750 
subjects screened over 3 years is well within the capacity of our site. 
 
In this study, subjects will first be screened for neutralizing antibody to the two study viruses 
under an existing Institutional Review Board (UVA IRB #9948)) approved screening protocol.  
Volunteers who are eligible to participate by serology (~15%) will then be contacted by email or 
telephone and offered the opportunity to participate in the challenge studies. 
 
Given the complexity of the study schedule, we will plan to enroll approximately one-third of the 
study subjects each year for 3 years.  The initial challenge will be done during the fall of each 
year and the second challenge will be done in the winter/spring.  This schedule acknowledges 
the realities of the school calendar and will maximize retention of enrolled subjects.  Subjects 
receive monetary compensation for participation and subject retention is excellent (in a recent 
study of 150 subjects, no subjects withdrew or were lost to follow-up).  The planned enrollment 
over 3 years will allow variation of the cohort size in years 2 and 3 to ensure that the required 
sample size is available for analysis.  
 
There are no gender or minority restrictions in this study.  An age restriction is included in the 
study to provide greater homogeneity in the study population.  In previous studies of this type at 
the UVA, approximately 70% of the subjects who participated have been white (72% of the 
University student body from which volunteer subjects are primarily drawn are white, not 
Hispanic) and approximately 59% of the participants have been female.  The average age of the 
volunteers in these studies is generally in the early twenties.  No gender or racial effects are 
expected for the analyses in this study.  Pregnant women are excluded from the study because 
of the intentional challenge with RV that is a part of the study.  Children are excluded from the 
study because the intentional challenge with RV cannot be ethically done in children.  
 
Subject Inclusion Criteria 
 
Subjects must meet all of the inclusion criteria in order to be eligible to participate in the study: 
1. Are males and non-pregnant females, 18-40 years of age, inclusive. 
2. Provide written informed consent prior to initiation of any study procedures. 

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3. Are able to understand and comply with planned study procedures and be available for all 
study visits. 
4. Subject must have a serum neutralizing antibody titer of ≤1:2 to RV type 39 and RV type 16. 
5. Female subjects* must be using an effective birth control method‡ for at least 30 days prior 
to study enrollment and through 30 days post-rechallenge. 
*for females who are not surgically sterile or post-menopausal 
‡§Includes, but is not limited to, non-male sexual relationships, abstinence from sexual intercourse with a male 
partner, monogamous relationship with a vasectomized partner, male condoms with the use of applied 
spermicide, intrauterine devices, NuvaRing®, and licensed hormonal methods such as implants, injectables 
or oral contraceptives (“the pill”). Method of contraception will be captured on the appropriate data collection 
form. 
 
6. Total IgE <200 IU/mL. 
7. Hemoglobin ≥ 11 gm/dL for females and ≥ 13 gm/dL for males 
 Inclusion at Re-challenge 
Subject must have participated in the first challenge and meet the following inclusion criteria to 
be eligible to participate in the re-challenge: 
 
 
 1.  Are males and non-pregnant females, 18-41 years of age, inclusive. 
2. Are able to understand and comply with planned study procedures and be available 
for all study visits. 
3. Female subjects* must be using an effective birth control method‡ for at least 30 
days prior to study enrollment and for the remainder of the study. 
*for females who are not surgically sterile or post-menopausal 
‡§Includes, but is not limited to, non-male sexual relationships, abstinence from sexual intercourse 
with a male partner, monogamous relationship with a vasectomized partner, male condoms with the 
use of applied spermicide, intrauterine devices, NuvaRing®, and licensed hormonal methods such 
as implants, injectables or oral contraceptives (“the pill”). Method of contraception will be captured 
on the appropriate data collection form. 
 
 
Subject Exclusion Criteria 
 Exclusion at enrollment 
Subjects meeting any of the exclusion criteria at baseline will be excluded from study 
participation: 
 
1. Any abnormalities of the upper respiratory tract that might interfere with the procedures and 
assessments in the study 
2. Any chronic upper respiratory illness  
3. Any chronic lower respiratory disease or any acute lower respiratory illness in the 1 month 
prior to the challenge  
4. Any bleeding tendency by history. 
5. History of hypertension or currently on anti-hypertensive  medication. 
6. History of angina or other cardiac disease. 

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7. Any medical condition that in the opinion of the Investigator is cause for exclusion from the 
study. 
8. Use of statins 
9. Regular use of tobacco in the last 6 months (i.e., more than 2 days out of 7) or inability to 
refrain from smoking during the study. 
10. Participation in any other clinical drug trial in the month prior to the study. 
11. Household contacts/room mates who are under protective precautions due to 
immunosuppression 
 Exclusion at Challenge 
1. Any upper respiratory infection or allergic rhinitis in the 2 weeks prior to challenge. 
2. Use of any common cold therapies in the 2 weeks prior to challenge (see list below*)  
* for example: non-steroidal anti-inflammatory medications, benedryl and other 
antihistamines, Sudafed, pseudoephedrine, phenylephrine, oxymetazoline, 
inhaled/intranasal steroids, leukotrienes, or any bronchodilators, (such as short acting b-
2-agonists, long-acting b2-agonists, anticholinergics). 
3. Female subjects* with a positive urine pregnancy screen on day of challenge 
 *for females who are not surgically sterile or post-menopausalExclusion at Re-
challenge 
To be considered eligible to participate in the Re-challenge, subjects must meet the eligibility 
criteria as listed above in both Inclusion and Exclusion Criteria. In addition, they must not have 
any of the following Exclusion criteria: 
 
1. Subject experienced an infection-related adverse event (e.g. bronchospasm, otitis media, 
sinusitis, fever (T>100.5º F)) following prior challenge. 
2. Female subjects with a positive urine pregnancy screen on day of rechallenge 
 
Treatment Assignment Procedures 
 
 Randomization Procedures 
All volunteers will be challenged with RV16 at the first challenge.  Randomization of subjects for 
challenge with either RV16 or RV39 at the second challenge will be done using GraphPad® 
software with randomization blocks of ten.  The challenge pool used will be recorded on source 
documents and transferred to the data collection form (DCF). 
 Masking Procedures 
This study does not involve any blinding or masking procedures.  Volunteers will not be told 
which virus they are receiving for the second challenge.  The identity of the virus inoculated for 
the second challenge is of no clinical significance and this information will not be shared with the 
subjects.  

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 Reasons for Withdrawal 
Withdrawal Criteria 
Subjects may be withdrawn from the study (i.e., from any further study procedure but not from 
analyses) for the following reasons:  
• 
At their own request or at the request of their legally authorized representative. 
• 
If the volunteer fails to comply with study procedures. 
• 
If they are lost to follow-up. 
 
In all cases, the reason for and date of withdrawal must be recorded in the DCF. The subject 
must be followed up to establish whether the reason was an AE, and, if so, this must be 
reported. 
 
The investigator or appointed representative must make every effort to contact subject lost to 
follow-up. Attempts (3 attempts) to contact such subject, and the method of contact, must be 
documented in the subject’s records (e.g., dates and times of attempted contact). 
 
Any subject may be withdrawn from the study at the discretion of the investigator. The subject is 
also free to terminate participation at any time.  
Subjects who meet exclusion criteria for the re-challenge will not be followed beyond the week 
15 study visit unless they have an adverse event that must be followed by protocol. 
 Handling of Withdrawals 
Withdrawn Subject Data Collection 
The principal investigator (PI) and/or other investigator involved in the study will document the 
reason for the subject withdrawal on the termination page of the DCF as follows:  
• 
Lost to follow-up:  subjects who leave the study without notification, or do not attend 
the study visit or cannot be contacted by phone. Intensive efforts should be made to 
locate and recall them if possible and to determine their health status at a minimum. 
• 
AE:  an AE event form must be completed. 
• 
Adverse laboratory event: this reason should be stated when a laboratory value is 
interpreted as a clinically significant abnormal value not explained by a laboratory 
error, or being not a known or abnormal value commonly observed in this type of 
population.  
• 
Deviation from protocol. 
• 
Consent withdrawn. 
• 
Other:  if the above mentioned reasons are not applicable, then the reason will be 
specified. 
 
Subjects who withdraw from the study during the symptomatic portion of the virus challenge will 
be re-contacted approximately 14 days after the challenge to document resolution of the viral 
illness.  Subjects who withdraw at other times in the study will not require any study follow-up.  
Attempts to contact subjects who are lost to follow-up will be as described in section 5.4.3. 

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 Termination of Study  
Although the study Sponsor has every intention of completing the study, it reserves the right to 
terminate the study at any time for clinical or administrative reasons. Reasons for termination 
include, but are not limited to, study closure due to Data and Safety Monitoring Board (DSMB) 
review and recommendation, and at the discretion of DMID. 

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6 
INVESTIGATIONAL PRODUCT 
 
Investigational Product Description 
 Acquisition 
The challenge viruses used for this study are in the possession of the sponsor-investigator (Dr. 
Turner).   
 Formulation, Packaging, and Labeling 
The virus pools consist of clarified cell culture supernatants of Eagle’s minimal essential media 
supplemented with 10% fetal bovine serum.  The pools are packaged in cryovials labeled to 
identify the pool and lot number, and are stored at -60 °C. 
 Product Storage and Stability 
Vials of the RV pool are stored at -60 °C until thawed for use for the challenge.  The vials are 
diluted in lactated ringer’s solution for human use to produce the desired dilution for the human 
challenge.  The diluted virus is prepared on the day of challenge and is kept cool on wet ice until 
the inoculation of volunteers is competed. The stability of the virus is documented by back 
titration of the diluted inoculum pool to demonstrate that the expected virus titer was delivered. 
 
The virus pools are stored at -60 °C or below.  The challenge pools are stable under these 
conditions for an indefinite period of time measured in decades. 
 
Dosage, Preparation and Administration of Investigational 
Product 
 Challenge and Re-challenge with RV 
The challenge viruses to be used in this study are human RVs A16 and A39.  Both of these 
challenge pools are used under FDA regulation (IND 12934 for RV39 and IND 15162 for RV16).  
Both pools have a concentration of 3.9 X 104 TCID50/mL.  The virus stock is diluted into Lactated 
Ringer’s (LR) solution for human parenteral administration to provide a final concentration of 20-
100 TCID50/mL for the inoculation.   Following a nasal wash, each subject will be given the 
challenge virus by intranasal drops.  The virus is administered in a volume of 0.25 mL/nostril 
with a calibrated pipette.  The 0.25 mL inoculum is administered twice in each nostril 1-3 
minutes apart to provide the total virus inoculum. The lot number of the LR solution will be 
recorded and the virus challenge material will be back-titrated to document the continued 
potency of the challenge pool.   
 Modification of Investigational Product for a Participant 
 

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There will be no modification of dose in this study.  Only one dose of virus is delivered for each 
challenge. 
 
Accountability Procedures for the Investigational 
Product(s) 
 
This study is being conducted by the sponsor-investigator.  The investigational product will be 
under the control of the sponsor-investigator at all times. A dispensing log will be maintained to 
document administration of the challenge virus to the individual subjects. The investigator will 
administer the challenge virus. There will be no shipping or distribution of the investigational 
product.  
 
Assessment of Subject Compliance with Investigational 
Product  
 
Compliance is assured since the product is administered to the volunteer by the investigator. 
 
Concomitant Medications/Treatments 
 
Volunteers who use any common cold therapies in the 2 weeks prior to the study (see list 
below*) will be excluded from study participation. 
* non-steroidal anti-inflammatory medications, benedryl and other antihistamines, 
Sudafed, pseudoephedrine, phenylephrine, oxymetazoline, inhaled/intranasal steroids, 
leukotrienes, any bronchodilators (such as short acting b-2-agonists, long-acting b2-
agonists, anticholinergics), or dietary supplements. 
 
In the 5 days after each RV challenge the volunteers will be asked to refrain from the use 
of common cold therapies (see list below*) to avoid interference with assessment of the 
endpoints of the study. * non-steroidal anti-inflammatory medications, benedryl and other 
antihistamines, Sudafed, pseudoephedrine, phenylephrine, oxymetazoline, 
inhaled/intranasal steroids, leukotrienes, or any bronchodilators, (such as short acting b-
2-agonists, long-acting b2-agonists, anticholinergics). 
 
 
All medications used by the volunteers during the study (Day -14 through week 15 post second 
RV challenge) will be recorded to assist with assessment of compliance and to permit 
assessment of the symptoms and symptom scoring (See Section 8.2.2). 
 
 
 

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7 
STUDY SCHEDULE 
 
Study Schedule 
 
Each challenge cohort will be conducted on an identical schedule of events.  Volunteer 
interactions in each cohort are illustrated in the Study Schedule Table in Appendix A 
 
 
Screening  
 Screening and Enrollment Visit (Study day -14 to -7, study visit 1)  
• 
Review study and obtain informed consent (volunteers who consented are provided 
with a copy of the signed consent) 
• 
Assessment of inclusion/exclusion criteria 
• 
Recording of demographic data to include date of birth, gender, race and ethnic 
origin 
• 
Complete and review medical history questionnaire 
• 
Vital signs to include pulse, respiratory rate, blood pressure, temperature 
• 
Blood specimen for HLA typing, hemoglobin, total IgE, and RV serology (neutralizing 
antibody and capsid protein-specific IgG and IgA) (30 ml) 
• 
Blood specimen for baseline PBMC collection (90 ml) 
• 
Recording of concomitant medications. 
 
First Virus Challenge 
 Study day 0 (study visit 2) 
• 
Baseline symptom assessment  
• 
Review of interval medical history 
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for acute serology and PBMC collection (105 ml) 
• 
Nasal lavage for respiratory virus PCR, and cytokines  
• 
Urine pregnancy screen for females who are not surgically sterile or post-
menopausal (results must be known prior to the RV Challenge) Challenge with RV  
 Study days 1-5 (study visits 3-7) 
• 
Daily symptom assessment  
• 
Assessment of subjective feeling of a cold (day 5 only) 
• 
Daily assessment of AEs 
• 
Daily assessment of concomitant medications 
• 
Daily nasal lavage for RV qPCR, quantitative RV culture, and cytokines 

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 Study day 7 (study visit 8)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for PBMC collection and RV serology (105 ml) 
 Study day 14 (study visit 9)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood collection for RV serology (15 ml) 
 Study day 28 ± 2 (study visit 10)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for PBMC collection and RV serology (105 ml) 
 Study day 63 ± 2, Week 9 (study visit 11)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
 Blood collection for RV serology (15 ml) 
 15 Weeks after Virus Challenge (study visit 12)   
 
(Note that this visit coincides with Study day 0 for the second virus challenge.) 
 
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for RV serology and PBMC collection (105 ml) 
 
Second Virus Challenge 
 Study day RC0 (study visit RC 1)   
• 
Baseline symptom assessment 
• 
Review of interval medical history 
• 
Urine pregnancy screen for females who are not surgically sterile or post-
menopausal 
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Nasal lavage for respiratory virus PCR, and cytokines 
• 
Challenge with RV  

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 Study days RC1-RC5 (study visits RC 2-6) 
• 
Daily symptom assessment 
• 
Assessment of subjective feeling of a cold (day 5 only) 
• 
Daily assessment of AEs 
• 
Daily reassessment of concomitant medications 
• 
Daily nasal lavage for RV qPCR, quantitative RV culture and cytokines 
 Study day RC7 (study visit RC 7)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for PBMC collection and RV serology (105 ml) 
 Study day RC14 (study visit RC 8)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood collection for RV serology (15 ml) 
 Study day RC28 ± 2 (study visit RC 9)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for PBMC collection and RV serology (105 ml) 
 Study day RC63 ± 2, RC Week 9 (study visit RC 10)   
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for RV serology (15 ml) 
 15 Weeks after Second Virus Challenge (study visit RC 11) 
• 
Review of AEs 
• 
Review of concomitant medications 
• 
Blood draw for PBMC collection and RV serology (105 ml) 
 
Follow-up  
 
Volunteers will be followed for 15 weeks after each virus challenge as described in sections 7.2 
and 7.3. 
 
Final Study Visit 
 
The final study visit will occur at week 15 after the second virus challenge.  The procedures 
done at this visit are described in section 7.3.7.  We will not collect AEs beyond this visit.  

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Early Termination Visit 
 
If a volunteer withdraws or is removed from the study due to an AE as described in section 9.1, 
we will continue to follow the subject until the appropriate resolution of the AE.  Early termination 
due to withdrawal of consent will require no further follow-up or procedures. However, in the 
event of early termination due to an AE or any other cause, the subject may be asked whether 
they consent to continue the remaining study procedures. 
 
Unscheduled Visit  
 
 If an unscheduled visit occurs, it will be documented in the notes section of the DCF. 

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8 
STUDY PROCEDURES/EVALUATIONS 
 
Clinical Evaluations 
 Medical history 
A medical history will be obtained at the time of the enrollment visit.  The history is obtained via 
questionnaire in an interactive interview with a member of the study staff who is a health care 
professional.  The information is recorded on a structured source document to be reviewed by 
the PI for eligibility prior to enrollment.  The medical history will include the use of prohibited 
medications in the 2 weeks prior to enrollment.  Medical records will not be reviewed. 
 
 Vital Signs 
Vital signs will be done at the enrollment visit. 
 Concomitant medications  
Volunteers will be asked to report all medication use during an interactive interview with the 
study staff.  This information will be collected at each study visit and will be used to assist with 
assessment of compliance and to permit assessment of the symptoms, symptom scoring, and   
study analyses. Use of cold medications could impact eligibility (see Section 5.3.1 and Section 
6.5), and will impact symptom scoring (see Section 8.2.2).  
 
 
Study Procedures 
 Nasal Lavage  
Nasal lavage will be collected for quantitative viral cultures, viral PCR and qPCR, and for 
determination of cytokine concentrations.  These specimens are collected by instillation of 5 mL 
of sterile 0.9% saline into each nostril.  This wash is then immediately expelled into a waxed 
paper cup and kept chilled until processed.  Lavage for viral culture is diluted 3:1 in 4X 
concentrated viral collection broth and then frozen at -80 °C until cultured for detection of RV.  
Lavage for cytokine assays is stored undiluted at -80 °C.   
 Symptom Scoring  
Symptom assessment will not be the primary outcome variable for any of the experiments in this 
proposal.  Symptom assessment will be done as part of the secondary analysis of the 
correlation between different T-cell subsets and symptomatic illness following homologous or 
heterologous re-challenge.   
 
Volunteers will be asked to refrain from the use of common cold therapies during the 5 days 
after each RV challenge. Symptom scoring is done using a standardized method [31, 32].  Each 

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subject is asked in an interactive interview to assess the severity of the symptoms of rhinorrhea, 
nasal obstruction, cough, sore throat, sneezing, headache, malaise and chilliness as follows: 
 
• 
Absent (0 points) 
 
• 
Mild (1 point) Barely noticeable, does not interfere with daily activity. 
  
• 
Moderate (2 points) Noticeable, but does not prevent daily activity. 
  
• 
Severe (3 points) Interferes or prevents daily activity; or the volunteer used the cold 
medications listed below* to treat cold symptoms, 
* non-steroidal anti-inflammatory medications, benedryl and other antihistamines, 
Sudafed, pseudoephedrine, phenylephrine, oxymetazoline. 
Or any use of bronchodilators, leukotrienes, or inhaled/intranasal steroids.  
 
The symptom scores will be recorded prior to the nasal wash procedure on Study Days 0-5.  
 
Symptom scores will be collected on Days 0-5, however only the scores on Days 1-5 will be 
used to calculate the Total Symptom Score (TSS). 
 
 Assessment of infection and illness 
 
Volunteers who have challenge virus detected in nasal lavage by culture or PCR on any of the 
Study Days 1-5 after virus challenge and/or have a four-fold rise in neutralizing antibody titer to 
the study virus are considered infected.  Volunteers who have a positive culture for the 
challenge virus on any of the Study Days 1-5 after virus challenge are defined as “virus 
shedders” and the time from Day 0 to the first positive virus culture is defined as the “time to 
virus shedding”. 
 
The total symptom score will be the sum of the symptom scores for all symptoms on Study Days 
1-5.  Infected volunteers who have a total symptom score of at least 6 and either at least 3 days 
of rhinorrhea or the subjective impression that they have had a cold will be defined as having a 
symptomatic infection. 
 
Laboratory Evaluations 
 Virology   
Detection of virus in the Study day 0 nasal lavage is done by a multiplex PCR (Luminex xTAG® 
Respiratory Viral Panel [Austin, TX]) in the Clinical Laboratory of the UVA Medical Center. 
 

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RV cultures will be done by routine methods [33].  Nasal lavage collected on Study days 1-5 are 
cultured for RV by inoculation into two tubes of human embryonic lung fibroblast cells (either 
MRC-5 or WI-38) and incubation on roller drums at 33 °C for 14 days.  RV is identified by the 
development of typical cytopathic effect.  One isolate from each subject who sheds virus is 
confirmed as the challenge RV serotype by a neutralization assay with HRV type 39 or type 16 
antiserum.  Subjects who have at least one positive culture for the challenge virus or who have 
at least a four-fold rise in antibody titer to RV type 39 are considered infected. Previous studies 
have established that the challenge inoculum is not re-isolated from uninfected volunteers, thus 
a single positive culture is evidence of virus replication in the upper respiratory tract [34].  Serial 
10-fold dilutions of the nasal lavage will be cultured in microtiter plates for semi-quantitative 
determination of viral shedding.  Titers are calculated using the method of Karber. 
 
We will use a qPCR assay that has been previously described by other members of our 
research group [35].  Briefly, RNA is extracted using QIAamp RNA isolation kits (Qiagen, 
Crawley, UK) and cDNA is generated. The cDNA is amplified using primers and probes specific 
for conserved regions of RV and detected by qPCR (RV forward 5′-
GGCCCTGAATGTGGCTAA-3′; RV reverse 5′-ATCCCCGCAATTGCTCGTTAC-3′; probe 5′-
FAM/ CTTGCAGCCAATGCA-BHQ-3′) (Integrated DNA Technologies, Coralville, IA).  Virus 
quantity is determined by comparison to a standard curve. 
 Serology 
Sera are tested for neutralizing antibody to RV16 and RV39 by a standard microtiter assay [33].  
In addition to neutralizing antibodies, specific IgG and IgA antibodies directed against RV-16 
and RV-39 capsid proteins, VP1 and VP2, will be measured in serum by antigen binding 
radioimmunoassay.   
 Other Laboratory Evaluations 
HLA-DRB1 typing will be performed on whole blood DNA specimens obtained on subjects who 
meet criteria for RV challenge in order to select the relevant MHCII/RV peptide tetramer for T-
cell phenotyping studies.   
 
 Special Assays or Procedures 
At specified time points, PBMC specimens will be collected in order to perform high-dimensional 
immunophenotyping of circulating T cells and other immune cell types by mass cytometry.  In 
addition, gene expression profiling of discrete T-cell subtypes will be performed using standard 
microarrays or single-cell analytical platforms (BioMark, Fluidigm).  
 
Screening lab studies (Hgb and IgE) and the respiratory virus PCR will be done in the Clinical 
Laboratory of the UVA Medical Center.  Specimens will be shipped to the laboratory using 
standard hospital procedures. 
 
For details of laboratory evaluations, please refer to the Manual of Procedures (MOP), which 
contains the following standard operating procedures (SOPs): 
 
 

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1.  Collection of whole blood for serum 
2.  Microtiter screening neutralization assay 
3.  RV isolation in cell culture 
4.  Quantitative RV cultures 
5.  RV typing neutralization assay 
6.  qPCR for RV 
7.  Serum IgG and IgA binding assays 
8.  DNA Isolation and HLA typing from whole blood. 
9.  PBMC isolation and cryopreservation 
10.  PBMC specimen thawing 
11.  Staining PBMCs with tetramers and antibodies  
12.  Analysis of samples by multi-color flow cytometry 
13.  Staining PBMCs for mass cytometry analysis (protocol 1) 
14.  Staining PBMCs for mass cytometry analysis (protocol 2) 
15.  Analysis of samples by mass cytometry 
16.  Single-cell gene expression profiling 
17.  Single-cell gene expression profiling (Fluidigm) 
18.  Gene expression profiling using standard microarrays 
19.  Multiplex cytokine assay for cytokines in nasal lavage fluid 
 Specimen Preparation, Handling, and Shipping 
8.3.5.1 
Instructions for Specimen Preparation, Handling, and 
Storage 
All specimen collection and processing will be done in the research laboratories of the PIs using 
established procedures documented in the laboratory manuals of the respective laboratories 
(see MOP for details).  Specimens will be stored at appropriate temperatures in monitored 
freezers.  Consent for future use of the specimens will be obtained.  All specimens will be 
identified by study number, date, and specimen type, and linked to the DCF and study 
database.  
 
 Specimen Shipment 
DNA specimens may be shipped for HLA typing services to external sites (listed in the MOP).  
Additionally, assay plates containing cDNA specimens may be shipped for single-cell gene 
expression profiling (see MOP for details). 
 

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9 
ASSESSMENT OF SAFETY 
 
Methods and Timing for Assessing, Recording, and 
Analyzing Adverse Events and Serious Adverse Events 
 Adverse Event Definition  
ICH E6 defines an AE as any untoward medical occurrence in a patient or clinical investigation 
subject who was administered a pharmaceutical product, regardless of its causal relationship to 
the study treatment. FDA defines an AE as any untoward medical occurrence associated with the 
use of a drug in humans, whether or not considered drug related. 
 
An AE may be an unfavorable and unintended sign, symptom, syndrome, or illness that develops 
or worsens during the clinical study. Clinically relevant abnormal results of diagnostic procedures 
including abnormal laboratory findings (e.g., requiring unscheduled diagnostic procedures or 
treatment measures, or resulting in withdrawal from the study) are considered to be AEs. Any 
medical condition that is present at the time that the subject is screened will be considered as 
baseline and not reported as an AE. However, worsening of pre-existing medical condition will be 
recorded as an AE.   
 
For the purposes of this study using experimental RV infection, physical signs and symptoms 
that are expected to be associated with the common cold and occur in the seven days after 
virus challenge should not be reported as AEs. The following physical signs and symptoms are 
expected to be associated with the common cold: nasal obstruction (nasal stuffiness), coryza 
(runny nose), sore throat, cough, sneezing, headache, chills, and malaise. If these symptoms 
are worsening beyond seven days after virus challenge they will be recorded as AEs. All other 
untoward medical occurrences during the study will be recorded as adverse events (such as 
fever, bronchospasm, asthma, otitis media, bronchitis and/or sinusitis). 
 Serious Adverse Event Definition 
A Serious Adverse Event (SAE) or Serious Adverse Drug Reaction (SADR) is any Adverse 
Event (AE) that meets at least one of the following criteria: 
1.  Death. 
2. Life-threatening adverse event;*   
3. Inpatient hospitalization or prolongation of an existing hospitalization (see below). 
4. Persistent or significant disability or incapacity or substantial disruption of the ability 
to conduct normal life function (see below). 
5. Is a congenital anomaly or birth defect. 
6. Important medical events ** 
 
 
*Life-threatening refers to immediate risk of death as the event occurred per the reporter. A life-
threatening experience does not include an experience that had it occurred in a more severe 
form might have caused death, but as it actually occurred, did not create an immediate risk of 

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death. For example, hepatitis that resolved without evidence of hepatic failure would not be 
considered life-threatening, even though hepatitis of a more severe nature can be fatal. 
Similarly, an allergic reaction resulting in angioedema of the face would not be life-threatening, 
even though angioedema of the larynx, allergic bronchospasm, or anaphylaxis can be fatal. 
 
**Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered SAEs when, based on appropriate medical judgment, they 
may jeopardize the subject and may require medical or surgical intervention to prevent one of 
the outcomes listed in this definition. Examples of such events include allergic bronchospasm 
requiring intensive treatment in an emergency room or at home, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency or drug 
abuse. 
 
 
Hospitalization is official admission to a hospital. Hospitalization or prolongation of a 
hospitalization constitutes criteria for an AE to be serious; however, it is not in itself considered 
a SAE. In absence of an AE, a hospitalization or prolongation of a hospitalization should not be 
reported as a SAE. This is the case in the following situations: 
 
• 
The hospitalization or prolongation of hospitalization is needed for a procedure 
required by the protocol.  
• 
The hospitalization or prolongation of hospitalization is part of a routine procedure 
followed by the center (e.g., stent removal after surgery). This should be recorded in 
the study file. 
 
In addition, hospitalization, for a pre-existing condition that has not worsened, does not 
constitute an SAE. 
 
Disability is defined as a substantial disruption in a person’s ability to conduct normal life 
functions. 
 
If there is any doubt about whether the information constitutes a SAE, the information is treated 
as a SAE. 
 
 Assessment and Reporting of Adverse Events: 
9.1.3.1 
Recording of Adverse Events 
All AEs should be captured on the appropriate DCF. All AEs occurring while on study must be 
documented appropriately regardless of relationship, severity or seriousness criteria. All AEs will 
be followed to adequate resolution or stabilization. All AEs must be graded for severity and 
relationship to study product. 
 
All volunteers will be specifically asked about any unexpected events at each study visit.  All 
volunteers will be instructed to contact the study site if they experience any unexpected 
symptoms during the time they are in the study (from day 0 - day 63 for each challenge cohort).  
At the final study visit (day 63 for each challenge cohort), volunteers will be asked if they have 

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had any unusual symptoms or problems during the study.  All positive responses will be 
recorded.  Medically significant responses will be followed until the symptom/problem is 
resolved or judged unrelated to the study and the subject has been referred for appropriate 
care. 
9.1.3.2 
Severity 
The severity of AEs will be graded on a three–point scale as described below as assessed by 
the volunteer and reported in detail as indicated on the DCF.  
 
• 
Mild - Barely noticeable, does not interfere with daily activity, 
• 
Moderate - Noticeable, but does not prevent daily activity, 
• 
Severe - Interferes or prevents daily activity 
Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed.  AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
 
9.1.3.3 
Relationship to Study Intervention 
 All AEs must be graded for severity and relationship to study intervention. The investigator’s 
assessment of an AE's relationship to study is part of the documentation process, but it is not a 
factor in determining what is or is not reported in this study. If there is any doubt as to whether a 
clinical observation is an AE, the event should be reported. The relationship to study must be 
assessed for AEs using the terms: related or not related. In a clinical trial, the study product must 
always be suspect. To help assess, the following guidelines are used:  
 
• 
Related: There is a reasonable possibility that the study caused the adverse event. 
Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study product and the adverse event. All causality 
relationship of probably not related, possibly related, probably related, and 
definitely related will be considered related regardless of form of documentation.  
• 
Not Related: The event can be readily explained by other factors such as the 
subject’s underlying medical condition, concomitant therapy, or accident, and no 
temporal relationship exists between the study drug and the event.  
9.1.3.4 
Type and Duration of Follow-up of Subjects after Adverse 
Events 
Adverse events requiring medical intervention will be followed until the symptom/problem is 
resolved or the subject is considered stable.  Subjects who experience AEs judged unrelated to 
the study will be referred for appropriate care as needed. 
 Assessment and Reporting of Serious Adverse Events 
Any SAE occurring in a subject after Day 0 in each challenge cohort until Day 28 of the cohort 
must be reported. Any AE should be recorded on the appropriate DCF. 
 

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Unexpected SAEs that are fatal or life-threatening must be filed as soon as possible with the 
Ethics Committee as soon as possible but not later than 7 calendar days after first knowledge, 
followed by as complete a report as possible within 8 additional calendar days.  
 
Unexpected SAEs that are not fatal or life-threatening must be filed as soon as possible but no 
later than 15 calendar days after first knowledge. 
 
AEs will be followed until resolution even if this extends beyond the study-reporting period.  
Resolution of an AE is defined as the return to pretreatment status or stabilization of the 
condition with the expectation that it will remain chronic.  
All SAEs will be: 
• 
recorded on the appropriate SAE DCF 
• 
followed through resolution by a study clinician 
• 
reviewed and evaluated by a study clinician 
 
Any AE that meets a protocol-defined serious criterion must be submitted immediately 
(within 24 hours of site awareness) on an SAE form to the DMID Pharmacovigilance 
Group, at the following address:  
 
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX Phone Number: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
 
Other supporting documentation of the event may be requested by the DMID 
Pharmacovigilance Group and should be provided as soon as possible.  
 
The DMID Medical Monitor and Clinical Protocol Manager will be notified of the SAE by the 
DMID Pharmacovigilance Group.  The DMID Medical Monitor will review and assess the SAE 
for regulatory reporting and potential impact on study subject safety and protocol conduct.  
 
At any time after completion of the study, if the investigator becomes aware of an SAE that is 
suspected to be related to study product, the investigator will report the event to the DMID 
Pharmacovigilance Group.  
 Reporting Plan for Serious Adverse Events, Unanticipated Problems, 
Protocol Deviations and Data Breaches 
AEs will be reported to the UVA Human Investigations Committee for review as follows. 
 

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Type of Event 
To whom will it 
be reported: 
Time Frame 
for Reporting 
How reported? 
Any internal event 
resulting in death that 
is deemed DEFINITELY 
related to (caused by) 
study participation 
IRB-HSR 
Medical Monitor 
Within 24 
hours 
IRB Online and phone call 
 
www.irb.virginia.edu/ 
 
Internal, Serious, 
Unexpected adverse 
event  
 
  
IRB-HSR 
Medical Monitor 
Within 7 
calendar days 
from the time 
the study team 
received 
knowledge of 
the event. 
IRB Online 
 
www.irb.virginia.edu/ 
 
Unanticipated 
Problems that are not 
adverse events or 
protocol violations  
This would include a 
Data Breach.   
IRB-HSR 
 Medical Monitor 
 
Within 7 
calendar days 
from the time 
the study team 
received 
knowledge of 
the event.  
Unanticipated Problem report form.  
 
http://www.virginia.edu/vprgs/irb/HSR_
docs/Forms/Reporting_Requirements-
Unanticipated_Problems.doc ) 
Protocol 
Violations/Noncomplia
nce 
The IRB-HSR only 
requires that MAJOR 
violations be reported.  
IRB-HSR 
Medical Monitor 
DMID 
 
Within 7 
calendar days 
from the time 
the study team 
received 
knowledge of 
the event.  
Protocol Violation, Noncompliance and 
Enrollment Exception Reporting Form 
 
http://www.virginia.edu/vprgs/irb/hsr_for
ms.html 
 
Data Breach  
The UVA 
Corporate 
Compliance and 
Privacy Office 
 
Medical Monitor 
 
 
 
ITC:  if breach 
involves  
electronic data  
 
 
 
 
UVA Police if 
breach includes 
such things as 
stolen 
computers.  
As soon as 
possible and 
no later than 
24 hours from 
the time the 
incident is 
identified. 
 
As soon as 
possible and 
no later than 
24 hours from 
the time the 
incident is 
identified. 
 
IMMEDIATELY  
 
 
UVA Corporate Compliance and 
Privacy Office Phone:  924-9741 
 
 
 
 
ITC:  Information Security Incident 
Reporting procedure,  
http://www.itc.virginia.edu/security/repo
rting.html 
 
 
 
 
 
Phone:  (434) 924-7166 
 

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UVA PI HELD IND 
Life-threatening and/or 
fatal unexpected 
events related or 
possibly related to the 
use of the 
investigational agent. 
FDA 
Within 7 
calendar days 
of the study 
team learning 
of the event 
Form FDA 3500A (MedWatch) or 
narrative 
Serious, unexpected 
and related or possibly 
related adverse events 
FDA 
Within 15 
calendar days 
after the study 
team receives 
knowledge of 
the event 
Form FDA 3500A (MedWatch) or 
narrative 
All adverse events 
FDA 
Annually 
IND annual report 
 
 
 Reporting of Pregnancy 
Any pregnancy occurring between the screening and final visits for each cohort will be recorded 
and reported to the IRB, DMID and the Data Safety Monitoring Board (DSMB). No further study 
inoculations will be administered to pregnant subjects, but all study-mandated blood samples 
will be obtained and the subject will continue in follow-up for safety events. Pregnancies will be 
followed to pregnancy outcome pending the subject’s permission. 
 
Methods and Timing for Assessment of the Performance of 
the Human Rhinovirus Challenge Model  
 Historical performance of the challenge model 
The experimental RV challenge model is a well-established model that has been used for the 
study of the pathogenesis and treatment of rhinovirus infections since the 1960s.  There have 
been a number of variations on the model but the proposed procedures and evaluations in this 
protocol (described in section 8.2 above) and have been used by the PI for 35 years.  Under 
these conditions, the expected infection rate for volunteers in the study is 85-90%.  Mean 
symptom scores for the RV39 used in the study have typically been in the range of 14-18 with 
standard deviations of ± 13.  The mean total symptom scores for the RV16 was 20 ± 10 in the 
initial evaluation of the challenge pool. 
 
The investigators are not aware of any previous rhinovirus challenge study that was 
discontinued because of unexpected risk to the volunteers in the study.  In light of this  
experience, it is unlikely that events that would require stopping the study would occur.   
 

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 Evaluation of viral infection 
Infection performance will be monitored and assess in two ways: 
 
9.2.2.1 
Back titration of the challenge pool 
After each challenge a portion of the challenge inoculum is back titrated in cell culture to assure 
that viable virus is present at the approximate titer anticipated.  The accuracy of the quantitative 
culture method is generally accepted to be ± 0.5 log10 TCID50/ml.  Failure to detect virus in the 
back titration or detection of greater than 1 log10 TCID50/ml over the intended challenge 
inoculum will be reported to the Medical Monitor and DSMB. 
 
9.2.2.2 
Infection rate 
Evaluation of viral infection to assess the performance of the model will be limited to the initial 
RV16 challenge in each cohort.  The purpose of the study is to evaluate the effect of RV16 
induced immunity on subsequent homotypic and heterotypic challenges so any change in the 
expected characteristics of the model will be more likely an effect of the experimental design 
than a change in the performance of the model.  If the infection rate in the first challenge of any 
cohort is less than 75% this will be reported to the Medical Monitor and DSMB.  Note that a 
decreased infection rate would potentially impact the data analysis and benefit of the study but 
would not pose a direct increased risk to the volunteers. 
 Evaluation of symptoms 
Evaluation of symptoms to assess the performance of the model will be limited to the initial 
RV16 challenge in each cohort.  The purpose of the study is to evaluate the effect of RV16 
induced immunity on subsequent homotypic and heterotypic challenges so any change in the 
expected characteristics of the model in the re-challenged cohorts will be more likely an effect of 
the experimental design than a change in the performance of the model.  Should the initial 
challenge result in an average total symptom score of <5 this will be reported to the Medical 
Monitor and the DSMB.  Note that a decreased symptom burden would potentially impact parts 
of the data analysis and the benefit of the study but would not pose a direct increased risk to the 
volunteers. 
 
Given the historical mean and standard deviation of the total symptom scores in the model, it 
would be an unexpected event if more than 10% of the volunteers in any cohort (initial challenge 
or re-challenge) had a total symptom score >40.  Should this occur we will report the event to 
the Medical Monitor and stop the study pending DSMB review. 
 Halting Rules 
Each study cohort will be challenged with virus over one to two days so application of halting 
rules cannot be done during the cohort.  The following halting rules will be applied and require 
DSMB review before proceeding to subsequent challenges. 

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9.2.4.1 
More than 10% of volunteers in any cohort with a total 
symptom score >40 
9.2.4.2 
Any subject experiences type 1 hypersensitivity immediately 
after receiving the study product 
9.2.4.3 
Any SAE judged by the investigator to be related to the 
challenge virus infection. 
 
 
Safety Oversight (ISM plus DSMB) 
 Independent Safety Monitor (ISM) 
The ISM is a physician with relevant expertise whose primary responsibility is to provide 
independent safety monitoring in a timely fashion.  The ISM will review SAEs and other AEs as 
needed and provide an independent assessment to DMID.  UVA will have an ISM with 
experience in infectious diseases or internal medicine. 
 Data and Safety Monitoring Board (DSMB) 
This clinical trial will utilize a DSMB, which will be comprised of an independent group of experts 
with experience with early phase clinical research studies.  The primary responsibility of the 
DSMB is to monitor subject safety.  The DSMB is external to DMID and composed of at least 
three voting members.  The DSMB will consist of members with appropriate expertise to 
contribute to the interpretation of the data from this trial.  Committee activities will be delineated 
in a DSMB charter that will delineate membership, responsibilities, and the scope and frequency 
of data reviews.  The DSMB will operate on a conflict-free basis independently of the study 
team. DMID, the DSMB, the ISM or the PI may convene ad hoc meetings of the DSMB 
according to protocol criteria or if there are concerns that arise during the study.  The DSMB will 
have access to unblinded data during its closed session, if applicable.  After its assessment, the 
DSMB will recommend continuation, modification, or termination of the clinical trial. 
 
The DSMB will conduct a review of all available safety data after each cohort has completed the 
day 8 study visit (7 days following the first study inoculation).  The review will also include all 
available safety data on second inoculations accrued through the time of data freeze for the 
DSMB meeting and the DSMB will recommend continued enrollment of the remaining subjects, 
modification, or discontinuation of enrollment.   
 
If any of the halting rules are met following the first or second study challenge, the study will not 
proceed with the remaining enrollment or study challenge without a review by and 
recommendation from the DSMB to proceed. 
 
Upon completion of this review and receipt of the advice of the DSMB, DMID will determine if 
study entry or study dosing should be interrupted, or if study entry and study dosing may 
continue according to the protocol.   
A final review of Safety data will be conducted after the study is completed. This review may be 
performed electronically if the DSMB Chair agrees. 

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 Safety Monitoring 
This study is not blinded and involves an intervention that is well-characterized and has an 
extensive safety record.  The investigational material for this study is the challenge pools that 
will be used for the RV infections.  The previous studies that have been done with these pools, 
as well as the extensive experience with the RV challenge model, in general, mitigate concern 
about the safety of the model.  
 
 
Safety will be monitored during the challenge studies by the PI.  Results of the challenge studies 
and collection of AEs will be reviewed by the DSMB as described above. 
 

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10 
CLINICAL MONITORING 
 Site Monitoring Plan 
 
Under the U01 mechanism, site monitoring is the responsibility of the investigators. To ensure 
that human subject protection, study procedures, laboratory procedures, study intervention 
administration, and data collection processes are of high quality and meet sponsor, CGP/ICH, 
and regulatory guidelines, and that the study is conducted in accordance with the protocol and 
sponsor SOPs, and protocol specific MOP, the study will be monitored by an external, 
independent entity, the UVA School of Medicine Clinical Trials Office 
(https://research.med.virginia.edu/clinicalresearch/research-resources/offices-supporting-
clinical-research/clinical-trials-office/available-services/).   
 
In brief, site visits will be made at standard intervals as defined by the Site Monitoring Plan. 
Monitoring visits will include, but are not limited to, review of regulatory files, accountability 
records, case report forms, informed consent forms, medical and laboratory reports, and 
protocol and GCP compliance. Study monitors will meet with investigators to discuss any 
problems and actions to be taken and document visit findings and discussions. See detailed 
protocol monitoring plan for details. The details of the site monitoring process for this study are 
given in a separate document (Protocol Monitoring Plan). This Monitoring process is separate 
and independent from the internal Quality Management Process to be conducted by the PI as 
described in Section 12 of this protocol. 
 

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11 
STATISTICAL CONSIDERATIONS 
 Study Hypotheses 
 
The overall hypothesis that will be addressed by this proposal is that Th and Tfh cells directed 
against conserved RV epitopes expand upon RV exposure and some of these cells persist as 
stable cross-reactive memory populations capable of displaying lineage-specific protective 
functions upon re-infection with related or unrelated strains of RV.  The IP is not the object of 
this study and there are no hypotheses related to the study product. 
 Study Outcome Measures 
This study is a mechanistic study, and thus, the conventional framework of primary and 
secondary outcome measures used in standard clinical trials does not apply.   
 Primary Objectives 
To assess the relationship between T-cell immunity induced by infection with the rhinovirus 
serotype RV-16, and the host response to homologous infection with RV-16 or the host 
response to heterologous infection with RV-39. 
 Exploratory Objectives 
The major objective of the study is to perform mechanistic studies that apply state-of-the art 
experimental technologies to identify, enumerate, and monitor a broad array of known and novel 
cell types during challenge with RV-16 and re-challenge with RV-16 or RV-39 in order to identify 
hallmarks of cross-protection.  The technologies applied include, but are not limited to, MHCII 
tetramer staining, mass cytometry, and single-cell gene expression profiling.  Owing to the large 
amounts of data that will be generated and the exploratory nature of the study, outcomes cannot 
be predicted. Exploratory objectives include, but are not limited to, the following:  (1) Identify 
new molecular signatures of RV-specific T cells.  (2) Evaluate the quality and quantity of T-cell 
responses.  (3) Identify, enumerate and monitor a broad array of immune cell types (e.g., B-cell 
populations, CD8+ T-cells, gamma delta T-cells, NKT cells, etc.), and novel cell types.  (4) Test 
multiple relationships between immune cell parameters and infection, serum antibodies, and 
symptoms (illness), at each sampling time point during RV challenge and re-challenge. 
 Primary Outcome Measure  
Correlation between the number of pre-existing RV-16-specific T cells at Week 15, and infection 
after homologous re-challenge with RV-16 or infection after heterologous re-challenge with RV-
39 at Week 15.  With regard to measurement transformation, in our past characterization of T-
cell responses to RV-16 and RV-39, we have found that the measurement distribution of many 
of the aforementioned outcome measures (e.g., T-cell response, viral load) exhibit prominent 
right-tailed skewness, which depending upon whether a parametric or a non-parametric analysis 
approach is utilized to analyze the data, has required either rescaling the outcome data to a 
logarithmic scale (e.g., log10 scale), or rescaling the data to a rank scale, respectively. 

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 Sample Size Considerations 
 Sample Size Calculation Summary  
The number of subjects enrolled for initial challenge with RV16 (80) is determined based on 
power analyses related to the primary outcome measure in Aim 2.  In Aim 2, our sample size is 
designed to address the hypothesis that high frequencies of pre-existing RV-16-specific 
memory T cells directed against conserved epitopes protect against RV-39 infection.  Our 
primary objective is to test the relationship between the frequency of RV-16-specific 
memory T cells at 15 weeks and viral infection in response to re-challenge with RV-16 or 
RV-39.  In order to do this, we must first establish that there is a scientifically meaningful 
reduction in the RV infection rate for secondary versus primary RV challenge.  A sample size of 
35 volunteers per re-challenge will provide a two-sample test of equal proportions with 85% 
statistical power (with a one-sided  α=0.025 type I error rate) to detect a reduction of as small as 
28% between the infection rate following primary challenge and the infection rate following 
secondary challenges.   
 
If we have 35 volunteers per re-challenge, and if the underlying bivariate correlation between 
the frequency of RV-16-specific memory T cells at 15 weeks and viral infection in response to 
secondary challenge with RV-16 or RV-39 is at least 0.46 units in absolute magnitude - which if 
true, would be considered a scientifically meaningful association - we expect to have at least 
0.80 statistical power to reject the null hypothesis that there is no correlation between the 
frequency of RV-16-specific memory T cells at 15 weeks and viral infection in response to 
secondary challenge with RV-16 or RV-39.   
 Power Calculation Details 
The “TWOSAMPLEFREQ” statement of the SAS version 9.4 (SAS Institute Inc., Cary, NC) 
“POWER Procedure” was used to conduct the first portion of the power analysis.  As user input, 
the infection rates for the primary and secondary RV challenges were specified as 0.90 and 
0.65, respectively.  A zero difference in the infection rates of the primary and secondary RV 
challenges was specified as the null difference and the alterative difference was specified as a 
0.25 unit reduction (i.e., 0.90 - 0.65) in the secondary RV challenge infection rate compared to 
the primary challenge infection rate.  In accordance with the alternative hypothesis “less than” 
direction, an α=0.025 one-sided type I error rate was specified, and the group sample sizes 
were specified as n=80 for the primary RV challenge and n=35 for the secondary RV challenge.  
Based on this set of input parameters, a sample size of 35 volunteers per re-challenge should 
provide a statistical test with at least 85% power (with a one-sided α=0.025 type I error rate) to 
detect a reduction of as small as 28% (i.e., [(0.90-0.65)]/0.90 x 100%) between the infection rate 
following primary challenge and the infection rate following secondary challenge.  
 
The “ONECORR” statement of the SAS version 9.4 “POWER PROCEDURE” was used to 
conduct the second portion of the power analysis.  As user input, the null correlation was 
specified to be zero and the alternative correlation was specified to be 0.46.  The test statistic 
was specified as the “Fishers z statistic”, and the two-sided type I error rate (α) was specified as 
α=0.05.  Based on this set of input variables, the power to detect a correlation of 0.46 in 
absolute magnitude is 0.81.  

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The rationale for the number of subjects screened is as follows:   
• 
750 volunteers will be screened for antibody to provide approximately 120 
volunteers who are seronegative to both RV-16 and RV-39. 
• 
Of the 120 resulting seronegative volunteers, approximately 96 will have a 
matched RV tetramer available for the T-cell studies. 
• 
80 eligible subjects (after review of other inclusion/exclusion criteria) will be 
challenged with RV-16, approximately 72 (90%) will be infected. 
• 
70 subjects (35 per virus) will be rechallenged with either RV-16 or RV-39. 
 
Note that volunteers who have a positive respiratory virus panel (PCR for respiratory syncytial 
virus [RSV], influenza, parainfluenza, metapneumovirus, rhino/enterovirus, and adenovirus) 
from nasal lavage on the day of RV challenge, or who shed a RV other than the challenge virus 
in nasal lavage over the course of the study, will be excluded from the analysis. 
 
Owing to the powerful, yet exploratory, nature of mass cytometry experiments, we anticipate 
that a much smaller sample size will be necessary to discover novel cell types induced by RV 
exposure that are biologically relevant.  Our proposed work is at the forefront of science in this 
regard.  As an example, recent work describing longitudinal monitoring of Tfh cells during HIV 
infection in a primate model using mass cytometry was published in Science Translational 
Medicine with a sample size of 8 [36]. 
 Planned Interim Analyses  
There are no planned interim analyses in this study. 
 Safety Review  
There are no specific safety outcome measures in this study.  Reported AEs will be reviewed 
after each challenge cohort.  Unexpected or serious events judged to be related to the virus 
challenge will be reviewed with the ISM, the DSMB and DMID before the next cohort is 
challenged. 
 Immunogenicity or Efficacy Review 
Immunogenicity or efficacy are not relevant to this study. 
 Analysis Plan 
 General Statistical Plan   
General statistical support related to the primary outcome measure, and many of the 
parameters assessed under exploratory objectives will be provided by Mr. James Patrie, MS 
(Pstat® Accredited Senior Biostatistician, Department of Public Health Sciences).  Analyses 
performed by Mr. Patrie will include, but are not limited to:  (a) comparing infection rates 
resulting from primary challenge with RV-16 and re-challenge with RV-16 or RV-39, (b) 

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comparing RV-specific T-cell numbers before and after primary RV-16 challenge, (c) examining 
correlations between specific T-cell numbers and quantitative shedding as assessed by culture 
and qPCR as well as the time to virus shedding, (d) comparing clinical outcomes (e.g., cytokine 
response, and symptom scores) between primary RV challenge and RV re-challenge, and (e) 
examining correlations between RV specific T-cell numbers and clinical outcomes. 
 Primary Outcome Analyses (Correlation between the number of pre-
existing RV-16 specific T-cells (15 wks.) and infection following re-
challenge with RV-39 as assessed by virus shedding in nasal lavage) 
Infection rates at primary challenge and the infection rates at secondary challenge will be 
analyzed by way of a binomial generalized estimating equation (GEE) model [37].  The GEE 
model response variable will be a binary indicator variable that will be assigned the value 1 if the 
volunteer is deemed infected on RV challenge and the value 0 will be assigned otherwise.  The 
GEE model predictor variable will be 3 level classification variable.  One level will differentiate 
the primary RV-16 challenge outcome data from the secondary RV challenge outcome data, a 
second level will identify the secondary RV-16 challenge outcome data, and the third level will 
identify the secondary RV-39 challenge outcome data.  Since we expect that approximately 
90% of the volunteers who undergo the primary RV-16 challenge will be eligible to undergo 
either RV-16 or RV-39 re-challenge, we cannot legitimately assume that the primary challenge 
infection status and re-challenge infection status of the volunteers who undergo re-challenge 
are statistically independent.  To account for possible intra-subject measurement correlation, the 
components of the GEE model variance and covariance matrix will be estimated by the Huber 
and White sandwich estimator [38, 39].    
 
With regard to hypothesis testing, the GEE version of the Wald test will be used to test whether 
the log-odds for infection is the same for the primary RV-16 challenge and the RV-16/RV-39 re-
challenge.  The Wald test will also be utilized to test whether the log-odds of infection is the 
same for the RV-16 and RV-39 re-challenges.  For the primary challenge versus re-challenge 
comparisons, a 0.025 one-side decision rule will be utilized as the null hypothesis rejection 
criterion, while for the RV-39 re-challenge versus RV-16 re-challenge comparison, a two-sided 
0.05 decision rule will be utilized as the null hypothesis rejection criterion.  With regard to odds 
ratio confidence interval construction, the GEE version of Wald confidence interval will be 
utilized to estimate the lower and upper limits of the 95% confidence interval. The relationship 
between the number of pre-existing RV-16 specific T-cells (15 wks.) and infection following re-
challenge with RV-39, as assessed by virus shedding in nasal lavage, will be quantitatively 
expressed in terms of the Pearson Product-Moment correlation coefficient if the bivariate normal 
assumption is deemed creditable based on the observed data, or quantitatively expressed in 
terms of the Spearman Rank correlation coefficient if the bivariate normal assumption is not 
supported by the observed data.  In either case, the pivotal quantity for the null hypothesis test 
that the true correlation coefficient is equal to zero will be the Fishers z-statistic, and a two-side 
p≤0.05 decision rule will be utilized as the null hypothesis rejection criterion.  Confidence 
interval construction for estimating the underlying correlation coefficient will be based on the 
Fishers z-transformation method [40].    

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 Potential analyses for exploratory endpoints 
Infection rates at primary challenge and the infection rates at secondary challenge will be 
analyzed by way of a binomial generalized estimating equation (GEE) model [37].  The GEE 
model response variable will be a binary indicator variable that will be assigned the value 1 if the 
volunteer is deemed infected on RV challenge and the value 0 will be assigned otherwise.  The 
GEE model predictor variable will be 3 level classification variable.  One level will differentiate 
the primary RV-16 challenge outcome data from the secondary RV challenge outcome data, a 
second level will identify the secondary RV-16 challenge outcome data, and the third level will 
identify the secondary RV-39 challenge outcome data.  Since we expect that approximately 
90% of the volunteers who undergo the primary RV-16 challenge will be eligible to undergo 
either RV-16 or RV-39 re-challenge, we cannot legitimately assume that the primary challenge 
infection status and re-challenge infection status of the volunteers who undergo re-challenge 
are statistically independent.  To account for possible intra-subject measurement correlation, the 
components of the GEE model variance and covariance matrix will be estimated by the Huber 
and White sandwich estimator [38, 39].    
 
With regard to hypothesis testing, the GEE version of the Wald test will be used to test whether 
the log-odds for infection is the same for the primary RV-16 challenge and the RV-16/RV-39 re-
challenge.  The Wald test will also be utilized to test whether the log-odds of infection is the 
same for the RV-16 and RV-39 re-challenges.  For the primary challenge versus re-challenge 
comparisons, a 0.025 one-side decision rule will be utilized as the null hypothesis rejection 
criterion, while for the RV-39 re-challenge versus RV-16 re-challenge comparison, a two-sided 
0.05 decision rule will be utilized as the null hypothesis rejection criterion.  With regard to odds 
ratio confidence interval construction, the GEE version of Wald confidence interval will be 
utilized to estimate the lower and upper limits of the 95% confidence interval. We will examine 
potential relationships between RV-16 specific Tfh/Teff cells and post-RV-16/RV-39 challenge 
infection as assessed by viral load.  Bivariate relationships will be quantified either by the 
Pearson Product Moment correlation coefficient or by the Spearman Rank correlation 
coefficient.  In either case, the pivotal quantity for the null hypothesis test that the correlation 
coefficient is equal to zero will be the Fishers z-statistic, and a Bonferroni corrected two-sided 
p≤0.05/c decision rule will be used as the null hypothesis rejection criterion, where the “c” 
represents the total number of null hypothesis tests.   Correlation coefficient confidence interval 
construction will be based on the Fishers z-transformation method.  As part of the correlation 
analysis, we will also compare the correlation coefficients for RV-16 specific Tfh/Teff cells and 
post-RV-16 challenge viral load relationships to the correlation coefficients for the RV-16 
specific Tfh/Teff cells and post-RV-39 challenge viral load relationships by way of distribution-
free random permutation tests [41], where virus re-challenge type (i.e., RV-16 or RV-39) will be 
the variable utilized to generate the pseudo null permutation distribution. A Bonferroni corrected 
two-sided p≤0.05/c decision rule will be used as the null hypothesis rejection criterion, where the 
“c” represents the total number of null hypothesis tests. Cox Proportional Hazards regression 
will be use examine multivariate relationships between the pre-challenge RV specific T-cell 
frequencies and time to RV-16 virus shedding.  
 
We will examine potential relationships between the levels of RV-16- and RV-39-specific 
antibodies and illness (i.e., total symptom score on day of peak symptom) following heterotypic 
re-challenge with RV-39, as well as potential relationships between the levels of RV-16- and 
RV-39-specific antibodies and illness following homotypic re-challenge with RV-16.  Bivariate 

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relationships will be quantified either by the Pearson Product-Moment correlation coefficient or 
by the Spearman Rank correlation coefficient. In either case, the pivotal quantity for the null 
hypothesis test that the true correlation coefficient is equal to zero will be the Fishers z-statistic, 
and a Bonferroni corrected two-sided p≤0.05/c decision rule will be implemented as the null 
hypothesis rejection rule, where the c represents the total number of null hypothesis tested.  
Correlation coefficient confidence interval construction will be based on the Fishers z-
transformation method.  As part of analysis, we will also compare the correlation coefficients for 
the relationships between RV-16- and RV-39-specific antibodies and illness following 
heterotypic re-challenge with RV-39 to the correlation coefficients for the relationships between 
RV-16- and RV-39-specific antibodies and illness following homotypic re-challenge with RV-16 
by way of distribution free random permutation tests [40], where virus re-challenge (i.e., 
heterotypic or homotypic) will be the variable utilized to generate the pseudo null permutation 
distribution. A Bonferroni corrected two-sided p≤0.05/c decision rule will be used as the null 
hypothesis rejection criterion, where the “c” represents the total number of null hypothesis tests. 
 
Pre and post-RV-16-specific Tfh/Teff cells frequencies and pre- and post-challenge RV-16- and 
RV-39-specific antibody frequencies will be analyzed via linear mixed effects models. For each 
outcome variable, the linear mixed model will be specified to include a single fixed-effect, which 
will identify the outcome assessment time (i.e., pre- or post-RV-16 challenge), and a single 
random effect that will identify the subject.  Based on our prior work, we anticipate that the 
majority of these outcomes variables will be have to be analyzed on the log10 scale to reduce 
the impact of right tailed distribution skewness, and hence, the pre- to post-change in the 
response will be quantified as a ratio (post:pre) of geometric means. The pivotal quantity for 
hypothesis testing will be the “linear-contrast of means” t-statistic, and a two-sided p≤0.05 
decision rule will be utilized as the rejection criterion for testing the null hypothesis that there is 
no underlying pre- to post-challenge change in the mean/geometric mean of outcome 
distribution. 
 
The longitudinal clinical outcome data such as viral load, cytokine concentration, and cold 
symptom scores, that will be collected on the day of RV inoculation (i.e., day 0) and on post-
challenge days 1, 2, 3, 4, and 5 will be analyzed via linear mixed models (e.g., cytokine 
response) and via generalized estimating equation models (e.g., total daily symptom score).  
The linear mixed models and generalized estimating equation models will be specified so that 
the longitudinal response profiles of the secondary challenges can be compared with the 
longitudinal response profiles of the primary challenge.  With regard to hypothesis testing, all 
statistical tests will be two-sided and a Bonferroni corrected p≤0.05/c decision rule will be 
implemented as the null hypothesis rejection criterion, where c denotes the number of between-
challenge comparisons.   
 Mass Cytometry Data Analysis  
Mass cytometry data will be analyzed using Cytobank tools [http://www.cytobank.org] [42] 
including new packages (SPADE, viSNE, Wanderlust) [43-47], as well as “classic” tools (biaxial 
plots, principal component analysis, heat maps). Statistical and bioinformatics support for high-
dimensional immunophenotyping data will be provided by Dr. Irish’s lab under a subcontract 
agreement, in conjunction with the UVA Flow Cytometry Core.  New and emerging 
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The UVA Bioinformatics Core will provide bioinformatics support for gene expression data sets 
and for integrative analytics aimed at combining proteomic and transcriptomic data sets.     
 
High content single cell data analysis and associated bioinformatics. Team members at UVA 
and Vanderbilt University will analyze mass cytometry experiment data using appropriate 
computational tools, including recently development tools for cytometry (viz. SPADE, viSNE, 
Wanderlust) and classic tools (viz. biaxial plots, principal component analysis, heat maps).  Dr. 
Irish’s lab has developed several tools to automatically identify enriched features of cell 
populations in mass cytometry data.  Dr. Irish’s lab is an expert in the bioinformatics and 
statistics of connecting surface marker and signaling features of cells to other immune 
parameters (e.g., antibodies) and clinical features of patient populations [48-51].  Dr. Irish also 
co-created the Cytobank bioinformatics cloud computing platform for flow cytometry data in 
order to analyze the data for these studies and has led the integration of key informatics tools 
(SPADE, viSNE, heat maps) into the web interface of Cytobank.  
 
Mass cytometry experiment design and instrumental tracking and optimization. In order to 
ensure uniformity within the mass cytometry data set, the Woodfolk lab will perform all mass 
cytometry experiments.  Dr. Irish’s lab will work together with UVA team members and the UVA 
Flow Cytometry Core to monitor the quality, signal to noise, and signal stability of the mass 
cytometry (CyTOF) data generated. Personnel in the Woodfolk and Irish labs, as well as in the 
UVA Core, have all received advanced training in the operation of the instrument and are 
experienced in connecting mass cytometry experiment analysis, instrument troubleshooting, 
and experiment design. These collaborations have been in place for over 18 months.  A variety 
of quality control (QC) measures are in place to minimize variability among samples and to limit 
“batch” effects that may arise from running multiple samples on different days.  These include 
running all specimens harvested from the same subject on the same day, and using a barcoding 
system to maximize sample throughput on the CyTOF and minimize variability in staining 
among specimens.  QC measures encompass mass cytometry panel design, antibody 
conjugation and titration, and instrument tracking controls. These controls, which are 
conceptually the same as those used for tracking fluorescent cytometers, are evidenced by 
production of data at the same or higher level of quality by the UVA CyTOF instrument as 
compared with the Vanderbilt CyTOF.  
 
 Gene Expression Data Analysis  
To ensure the reliability of the data generated in single-cell analyses, validated primers and 
SOPs have been provided by the Roederer lab (NIH/NIAID) [52].  Microarray data will be 
analyzed by the UVA Bioinformatics Core using a combination of custom-built and open-source 
application-specific software in the R statistical computing environment (www.r-project.org), 
including limma packages from Bioconductor.  Data generated using the Biomark platform will 
be analyzed using application-specific tools implemented in the SINGuLAR package developed 
by Fluidigm in the R statistical computing environment [53, 54].  Hierarchical cluster analysis of 
the full set of gene expression results will be performed and cross-platform concordance of 
transcriptomic versus proteomic data will be analyzed as a QC check through collaborative 
efforts between the Woodfolk lab and UVA Bioinformatics and Flow Cytometry Cores.  As data 
becomes available, the UVA Bioinformatics Core will also work in conjunction with other 

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components of the scientific team to develop integrative analytical pipelines that link gene 
expression profiles with immune parameters, infection, and symptoms.   

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12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
The study site will maintain appropriate research records for this trial, in compliance with 
Section 4.9 of ICH E6 GCP, and regulatory and institutional requirements for the protection of 
confidentiality of subjects. The site will permit authorized representatives of DMID and 
regulatory agencies to examine (and when required by applicable law, to copy) clinical records 
for the purposes of quality assurance (QA) reviews, audits and evaluation of the study safety 
and progress. All data collected directly from the volunteers will be collected on paper DCFs. If a 
subject is withdrawn from the study the principal investigator (PI) and/or other investigator 
involved in the study will document the reason for the subject withdrawal on the termination 
page of the DCF. Data that result from the laboratory procedures will be collected as laboratory 
reports/source documents. 
 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. 
 
All data collection forms should be completed in a neat, legible manner to ensure accurate 
interpretation of data.  Black or blue ink is required to ensure clarity of reproduced copies.  
When making a change or correction, cross out the original entry with a single line and initial 
and date the change.  Do not erase, overwrite, or use correction fluid or tape on the original. 
Entries into the DCF will be attributed to the staff that completed them with a signature 
and date. 
 
Data reported in the DCF should be consistent with the source documents or any discrepancies 
should be explained. 
 
 

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QUALITY CONTROL AND QUALITY ASSURANCE 
This is a single-site study and all human subject activity will be conducted under the supervision 
of Ronald Turner, MD, one of the protocol PIs.  All clinical protocol related data will be recorded 
on appropriate source documents or directly into the paper DCF.  Entries that are transcribed 
from source documents will be audited by the study coordinator or her designee. The process 
and tools for QC and QA are detailed in the associated Clinical Quality Management Plan. A 
final audit of the DCFs will be conducted by the study monitor. After the study monitor has 
reviewed the original DCFs, data will be entered into the database for subsequent analysis.  
 
An independent internal audit is provided by the Post-Approval Monitoring Program that is 
conducted by the IRB at the UVA.  This program is a monitoring program that addresses 
regulatory compliance review rather than data integrity. 
 
The investigational site is responsible for conducting routine quality assurance (QA) and quality 
control (QC) activities to internally monitor study progress and protocol compliance.  The 
Principal Investigator will provide direct access to all source data/documents, and reports for the 
purpose of monitoring and auditing, and inspection by local and regulatory authorities. The 
Principal Investigator will ensure all study personnel are appropriately trained and applicable 
documentations are maintained on site. The process and tools for QC and QA are detailed in 
the associated Clinical Quality Management Plan.   
 
Clinical site monitors will verify that the clinical trial is conducted and data are generated, 
documented (recorded), and reported in compliance with the protocol, Good Clinical Practice, 
and the applicable regulatory requirements. An independent regulatory compliance audit is 
conducted by the IRB at the UVA.  
 
Quality control procedures will be implemented by the database which will generate reports to 
ensure data quality, accuracy and completeness and audit of data entered.  Any missing data or 
data anomalies will be communicated to the investigative site for timely clarification/resolution.  
 
 

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14 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
 Ethical Standard 
 
The investigator will ensure that this study is conducted in full conformity with the principles set 
forth in The Belmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research of the US National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46, 21 CFR 
Part 50, 21 CFR Part 56 and/or the ICH E6; 62 Federal Regulations 25691 (1997).   
 Good Clinical Practice 
 
The investigators will assure that this study is conducted in full conformity with GCP.  All study 
personnel who are involved with the clinical study will have documentation of up-to-date training 
in HSP and/or GCP through the UVA IRB (Collaborative Institutional Training Initiative training). 
 Institutional Review Board 
 
This study will be conducted under the authority of the UVA IRB for Health Sciences Research. 
 
Federalwide Assurance (FWA) #: 00006183  
Exp. Date:  June 13, 2018 
 
IRB for HSR (IRB-HSR) Registered with Office for Human Research Protections 
(OHRP) and FDA 
IRB#:  00000447  
Expires August 6, 2015 
 
Physical Address:  
UVA IRB-HSR  
One Morton Drive  
Suite 400, Box 5  
Charlottesville, VA 22903 
 
The IRB will provide review of the informed consent documents, recruitment materials, protocol, 
amendments to the consent documents or protocol, safety reports, deviation report and 
continuing reviews. Before they are placed into use, the study protocol and informed consent 
documents will be reviewed and approved by the UVA IRB. Any amendments to the protocol or 
consent materials will first be reviewed and accepted by DMID, then reviewed and approved by 
the UVA IRB before such changes are placed into use.   
 
Collaborating institutions will not receive identifiable private information about specific 
volunteers and therefore are not conducting human subjects research as defined under the 
common rule, and will not be required to obtain IRB approval for the study. 

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 Informed Consent Process 
 
The site principal investigator will choose subjects in accordance with the eligibility criteria 
detailed in Section 5. Before any study procedures are performed, subjects must sign an 
informed consent form and subjects must provide consent as appropriate for age that complies 
with the requirements of 21 CFR Part 50 and 45 CFR 46 and the local IRB. 
Informed consent is a process that is initiated prior to the individuals agreeing to participate in a 
trial and continuing throughout the individual’s trial participation. Before any study procedures 
are performed, including pre-screening of subjects for eligibility, subjects will receive a 
comprehensive explanation of the proposed study procedures and study interventions/products, 
including the nature and risks of this trial, alternate therapies, any known AEs, the 
investigational status of the components, and the other elements that are part of obtaining 
proper informed consent. Subjects will also receive a detailed explanation of the proposed use 
and disclosure of their protected health information, including specifically their serum samples. 
Subjects will be allowed sufficient time to consider participation in the trial, after having the 
nature and risks of the trial explained to them, and have the opportunity to discuss the trial with 
their family, friends or legally authorized representative or think about it prior to agreeing to 
participate. 
Informed consent forms describing in detail the study interventions/products, study procedures, 
risks and possible benefits are given to the subjects. The informed consent form must not 
include any exculpatory statements. Informed consent forms will be IRB-approved and the 
subject will be asked to read and review the appropriate document. Upon reviewing the 
appropriate document, the site principal investigator (or designee) will explain the research 
study to the subject and answer any questions that may arise. Subjects must sign the informed 
consent form, and written documentation of the informed consent process is required prior to 
starting any study procedures/interventions being done specifically for the trial, including 
administering study product. 
DMID will provide the site principal investigator, in writing, any new information that significantly 
impacts the subjects' risk of receiving the investigational product. This new information will be 
communicated by the site principal investigator to subjects who consent to participate in the trial 
in accordance with IRB requirements. The informed consent document will be updated and 
subjects will be re-consented, if necessary. 
Study personnel may employ IRB-approved recruitment efforts prior to obtaining the subjects 
consent; however, before any study procedures are performed to determine protocol eligibility 
an informed consent form must be signed. Subjects will be given a copy of all informed consent 
forms that they sign. 
By signing the informed consent form, the subject agrees to complete all evaluations required 
by the trial, unless the subject withdraws voluntarily, or is withdrawn or terminated from the trial 
for any reason. 
 

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Volunteers for the screening blood draw will be recruited from the UVA community by posted, 
newspaper, or email advertisements or by contacting volunteers who have added their contact 
information to an IRB approved recruitment database (IRB-HSR #13653).  All advertising 
material will be reviewed and approved by the IRB.  Volunteers who express interest will be 
asked to come to the study site where they will be given a consent form (IRB #9948) to read 
and all questions will be answered by study staff.  Volunteers will be provided with a brief 
description of the various studies under recruitment that will be potentially available for 
participation. Volunteers who wish to participate will have their blood drawn and tested for 
serum neutralizing antibodies to the study virus.   
 
Volunteers who are seronegative will be contacted and offered the opportunity to participate in 
this clinical trial.  Those who express interest will be emailed a copy of the consent form for this 
study and a time for the enrollment visit will be scheduled.  At the enrollment visit, the consent 
form will be reviewed with the volunteer and all questions answered.  Volunteers who wish to 
participate will provide written affirmation of their consent prior to any study procedures.  All 
volunteers who consent to participation will be given a copy of the signed consent form. 
 Exclusion of Women, Minorities, and Children (Special 
Populations) 
 Participation of Women  
There are no gender restrictions for participation in these studies. Experience from previous 
studies using the human experimental RV infection model at the UVA suggests that about 59% 
of subjects who participate will be female.  This gender distribution reflects the current gender 
distribution of undergraduates at UVA (56% female).  Pregnant women are excluded from the 
study due to the intentional challenge with RV that is a part of the study. 
 Participation of Minorities  
There are no racial or ethnic restrictions in this study.  In previous studies using this 
experimental model at UVA, approximately 83% of volunteers have identified as white, 5% 
Black or African-American, and 10% Asian.  The UVA undergraduate population is 
approximately 72% White, 6% African-American, and 12% Asian.  Approximately 6% of the 
undergraduate students identify themselves as Hispanic or Latino.   
 Participation of Children 
Children are excluded from the study because the intentional challenge with RV cannot be 
ethically done in children. 
 Subject Confidentiality 
 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s) and their agents.  This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participating subjects. 

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The study protocol, documentation, data, and all other information generated will be held in 
strict confidence.  No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.  
 
The study monitor or 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 (office, clinic, or hospital) and pharmacy records for the subjects in this study.  The 
clinical study site will permit access to such records.  
 
Health Insurance Portability and Accountability Act (HIPAA) identifiers will be collected in the 
source documentation and recorded for purposes of payment of volunteers. Identifying 
information will be retained in a secure location by the clinical study site. Study records will be 
maintained behind two locked doors, and computer records will be password protected.  All 
volunteers will be assigned a study number and laboratory specimens will be identified only by 
study number, type of specimen, study visit number and date of collection.  The electronic 
database will be coded by study number and will contain no HIPAA identifiers. 
 Study Discontinuation 
 
This study does not involve a therapeutic intervention.  If the study is discontinued during the 
symptomatic portion of the virus challenge, challenged subjects will be re-contacted 
approximately 14 days after the challenge to document resolution of the viral illness.  If the 
study is discontinued at other times in the study subjects will not require any study follow-up.  
Attempts to contact subjects who are lost to follow-up will be as described in section 5.4.3. 
 Future Use of Stored Specimens 
 
This is a mechanistic study and there is an expectation that there may be unexpected results 
that will lead to further evaluation that cannot be predicted a priori.  We will seek consent from 
the volunteers to use stored specimens for unanticipated studies that are related to the overall 
objectives of the protocol. No genetic testing will be performed on specimens stored for future 
use.   
 
Subjects will be asked for permission to keep any remaining specimens for possible use in 
future research studies.  All samples are de-identified at the clinical site prior to transfer to the 
laboratory.  Samples will be stored in the investigator’s laboratory in -70 C freezers located in 
laboratory areas that are accessible only to laboratory personnel.  These specimens may be 
shared with other investigators at other institutions.  The samples will not be sold or used 
directly for production of any commercial product.  Each sample will be labeled with a study 
number, date, and specimen type.  Within two years of study completion the study specimens 
will be de-linked from subject identifiers. 
 
There are no benefits to subjects in the collection, storage, and subsequent research use of 
specimens.  Reports about future research done with a subject’s samples will not be kept in 

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their health records.  Subjects can decide if they want their samples to be used for future 
research or have their samples destroyed at the end of the study.  A subject’s decision can be 
changed at any time by notifying the study doctors or nurses in writing.  However, if a subject 
consents to future use and some of their blood has already been used for research purposes, 
the information from that research may still be used. Participant consent for stored specimens 
will be part of the study Informed Consent Form (ICF) which will be asked for during the 
enrollment visit. 
 
 

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15 
DATA HANDLING AND RECORD KEEPING 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported.  All source documents should be completed in a neat, legible 
manner to ensure accurate interpretation of data.  Black ink is required to ensure clarity of 
reproduced copies.  When making changes or corrections, cross out the original entry with a 
single line, and initial and date the change.  DO NOT ERASE, OVERWRITE, OR USE 
CORRECTION FLUID OR TAPE ON THE ORIGINAL. 
 
Copies of the DCF will be provided for use as source documents and maintained for recording 
data for each subject enrolled in the study.  Data reported in the DCF derived from source 
documents should be consistent with the source documents or the discrepancies should be 
explained. 
 
DMID and/or it designee will provide guidance to investigators on making corrections to the 
source documents and DCF. 
 Data Management Responsibilities 
 
All source documents and laboratory reports must be reviewed by the clinical team and data 
entry staff, who will ensure that they are accurate and complete.  AEs must be graded, 
assessed for severity and causality, and reviewed by the site PI. 
 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site PI.  During the study, the investigator must maintain complete and accurate 
documentation for the study. 
 
Source documentation and DCF completion will be the responsibility of the study coordinator 
and designated study staff.  The PI will have overall responsibility for the study data. 
 Data Capture Methods 
 
Primary data will be collected on DCFs.   
 
The symptom score data for this study will be entered into an existing challenge model 
database created in FoxPro.  These data will be merged with laboratory assay data as required 
for the mechanistic analyses proposed. 
 Types of Data 
 
The data collected for this study by the clinical site will include demographic, safety, virology, 
serology, and symptom score data.  The mechanistic data from the assays supported by the 
clinical trial will be collected by the individual laboratories and merged with the clinical data for 
analysis. 

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 Timing/Reports 
 
The AEs will be reviewed on an ongoing basis by the study investigator and a report will be 
made to the ISM/DSMB at the end of each challenge cohort. Reports for the DSMB will be 
prepared for the DSMB according to a schedule determined at the first convened DSMB 
meeting.  All data will be stored and manipulated as coded data.  The data analysis for the 
outcomes defined in this study will occur after all clinical interventions are complete.  There is no 
planned interim analysis.  This is a mechanistic study and the study endpoints will be evaluated 
using experimental laboratory procedures. It is anticipated that the performance of laboratory 
analyses will be an iterative process with the results of initial analyses informing the 
performance of subsequent analyses.  A final study report will be produced when all analyses 
proposed in the study are complete. 
 Study Records Retention 
 
Study records and reports including, but not limited to, eDCFs, source documents, informed 
consent forms, and study drug disposition records shall be maintained for 2 years after a 
marketing application is approved for the drug for the indication for which it is being 
investigated; or, if no application is to be filed or if the application is not approved for the drug, 
until 2 years after the investigation is discontinued and the FDA has been notified. The 
participating site must contact DMID for authorization prior to the destruction of any study 
records. Informed consent forms for future use will be maintained as long as the sample exists. 
 Protocol Deviations 
 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or MOP 
requirements.  The noncompliance may be either on the part of the subject, the investigator, or 
the study site staff.  As a result of deviations, corrective actions are to be developed by the site 
and implemented promptly.  
 
These practices are consistent with ICH E6: 
4.5 
Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
5.1 
Quality Assurance and Quality Control, section 5.1.1 
5.20 
Noncompliance, sections 5.20.1, and 5.20.2. 
 
It is the responsibility of the site to use continuous vigilance to identify and report deviations 
within 5 working days of identification of the protocol deviation, or within 5 working days of the 
scheduled protocol-required activity.   
 
Protocol deviations will be recorded in the source documents and  via the UVA IRB-HSR 
protocol violation form placed in the regulatory binder.  Protocol deviations will be reviewed by 
the PI who is also the sponsor of the IND.  Any protocol deviation meeting the criteria to be 
reported to the UVA IRB-HSR must also be reported to DMID in the same time-frame. 
 

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16 
PUBLICATION POLICY 
It is anticipated that the results of this study will be published in scientific journals. This study 
has been registered with clinicaltrials.gov. Any publications resulting from this proposal will be 
made publicly available as required by NIH policy. 
All investigators funded by the NIH must submit or have submitted for them to the 
National Library of Medicine’s PubMed Central (http://www.ncbi.nlm.nih.gov/pmc/) an 
electronic version of their final, peer-reviewed manuscripts upon acceptance for 
publication, to be made publicly available no later than 12 months after the official date 
of publication. The NIH Public Access Policy ensures the public has access to the 
published results of NIH funded research. It requires investigators to submit final peer-
reviewed journal manuscripts that arise from NIH funds to the digital archive PubMed 
Central upon acceptance for publication. Further, the policy stipulates that these papers 
must be accessible to the public on PubMed Central no later than 12 months after 
publication. 
Refer to: 
•  NIH Public Access Policy, http://publicaccess.nih.gov/ 
•  NIH OER Grants and Funding, http://grants.nih.gov/grants/oer.htm 
Following completion of this trial, the lead principal investigator is expected to publish 
the results of this research in a scientific journal. The International Committee of 
Medical Journal Editor (ICMJE) member journals have adopted a trials-registration 
policy as a condition for publication. This policy requires that all clinical trials be 
registered in a public trials registry, such as ClinicalTrials.gov* (http://clinicaltrials.gov/), 
which is sponsored by the National Library of Medicine. Other biomedical journals are 
considering adopting similar policies. 
The ICMJE defines a clinical trial as any research project that prospectively assigns 
human subjects to intervention or comparison groups to study the cause-and-effect 
relationship between a medical intervention and a health outcome. Studies designed for 
other purposes, such as to study pharmacokinetics or major toxicity (e.g., Phase I 
trials), would be exempt from this policy. 
It is the responsibility of DMID to register this trial in an acceptable registry. Any clinical 
trial starting enrollment after 01 July 2005 must be registered on or before subject 
enrollment. For trials that began enrollment prior to this date, the ICMJE member 
journals will require registration by 13 September 2005, before considering the results of 
the trial for publication. 
For trials in which DMID is not the IND/IDE sponsor, or there is no IND/IDE, and DMID 
does not provide data management services, it is the responsibility of the investigator to 

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register the trial and post results in compliance with Public Law 110-85, the Food and 
Drug Administration Amendments Act of 2007 (FDAAA). 
Refer to: 
•  Public Law 110-85, Section 801, Clinical Trial Databases 
*Journal Citation: De Angelis C, Drazen JM, Frizelle FA, Haug C, Hoey J, Horton R, et 
al. Clinical trial registration: a statement from the International Committee of Medical 
Journal Editors. N Engl J Med. 2004; 351:1250-1. 
 
 
 

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34. 
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Irish JM. Beyond the age of cellular discovery. Nat Immunol 2014; 15(12): 1095-7. 
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Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM. Cytometry by time-of-flight 
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Qiu P, Simonds EF, Bendall SC, et al. Extracting a cellular hierarchy from high-
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Myklebust JH, Irish JM, Brody J, et al. High PD-1 expression and suppressed cytokine 
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52. 
Dominguez MH, Chattopadhyay PK, Ma S, et al. Highly multiplexed quantitation of gene 
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53. 
Buganim Y, Faddah DA, Cheng AW, et al. Single-cell expression analyses during 
cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 2012; 
150(6): 1209-22. 
54. 
Flatz L, Roychoudhuri R, Honda M, et al. Single-cell gene-expression profiling reveals 
qualitatively distinct CD8 T cells elicited by different gene-based vaccines. Proc Natl 
Acad Sci U S A 2011; 108(14): 5724-9. 
 
 

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APPENDIX A: SCHEDULE OF EVENTS 
 
First Challenge 
 
Day 
-14 
Day 
01 
Days 
1-5 
Day 
7 
Day 
14 
Day 
28 
Week 
9 
Week 
151 
Re-Challenge= RC 
RC 
Day  
01 
RC 
Days 
1-5 
RC 
Day 
7 
RC 
Day 
14 
RC 
Day 
28 
RC 
Week 
9 
RC 
Week 
15’ 
Informed consent2 
x 
 
 
 
 
 
 
 
Medical history questionnaire 
x 
 
 
 
 
 
 
 
Urine pregnancy screen 3 
 
x 
 
 
 
 
 
 
HLA typing 
x 
 
 
 
 
 
 
 
Hemoglobin 
x 
 
 
 
 
 
 
 
Serum IgE 
x 
 
 
 
 
 
 
 
Serum RV-specific antibody 
x 
x 
 
x 
x 
x 
x 
x 
Nasal lavage for multiplex PCR 
 
x 
 
 
 
 
 
 
RV challenge 
 
x 
 
 
 
 
 
 
Nasal lavage for cytokines 
 
x 
x 
 
 
 
 
 
Nasal lavage for quantitative RV culture 
 
 
x 
 
 
 
 
 
Nasal lavage for RV qPCR 
 
 
x 
 
 
 
 
 
Symptom Score 
 
x 
x 
 
 
 
 
 
Blood collection for PBMC isolation (90cc) 
x 
x 
 
x 
 
x 
 
x 
Adverse event evaluation 
 
x 
x 
x 
x 
x 
x 
x 
Review of concomitant medication use 
x 
x 
x 
x 
x 
x 
x 
x 
1Note that the Week 15 visit is the final specimen collection for the primary challenge and Day 0 for the 
re-challenge.  The first challenge will be with RV16 and the second challenge will be with either RV16 or 
RV39.  The complete study involves two virus challenges with sample collection over a total of 32 weeks. 
2Volunteers will be screened for neutralizing antibody to the challenge virus strains under a separate 
antibody screening protocol (UVA HSR-IRB #9948) 2-4 weeks prior to enrollment in this protocol.  Only 
seronegative volunteers will be asked to participate in this protocol. 
3 A urine pregnancy screen is required before each challenge (Study Day 0 and Study Day RC 0)