The Effect of Inflammation and Damage to Lymph Node Structures on Durable Protective Immunity Following Yellow Fever Vaccination
Phase: PHASE1
Conditions: Yellow Fever
Sponsor: University of Minnesota

The effect of inflammation and damage to lymph node structures on 
durable protective immunity following yellow fever vaccination 
Protocol Number: JC3318 
National Clinical Trial (NCT) Identified Number:  NCT04269265 
Principal Investigator: Timothy Schacker, MD 
Funded by: National Institute of Allergy and Infectious Diseases 
Version Number:  2.0 
14 August 2023 
 
 

   
 
 
ii 
Summary of Changes from Previous Version: 
Version 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
1.2 
1.1, 2.1, 8.2, 
10.1.5  
Clarified endpoints, corrected inclusion criteria, 
corrected PI contact phone number 
Clarification and correction  
1.3 
1.1, 1.2, 1.3, 2.1, 
2.2, 2.3.1, 4.1, 
5.1, 8.2, 8.3.3.1, 
10.1.10, 10.2 
Updated vaccine information to Stamaril, added 
pregnancy test to indicated visits, removed all 
mention of colon and rectal biopsies, improved 
formatting for ease of reading, corrected 
inclusion criteria, updated AE grading scale, 
clarified reporting requirements, added key 
roles, addition of rectal swabs 
Stamaril is the available 
yellow fever vaccine, 
correction and update of visit 
procedures, overall effort to 
make protocol consistent, up 
to date, and clear 
1.4 
1.3, 2.3.3., 7.2, 
8.3.4, 8.3.6, 
10.1.4, 10.1.9.1, 
10.1.9.2 
Added Joshua Rhein at UMN as a Co-I; Medical 
history will be taken at screening and not 
baseline; Addressed comments from Ugandan 
Scientific Council Review: update title to include 
“yellow fever”, update assessment of risk and 
benefits, JCRC has insurance to cover 
participants with study procedure 
complications, samples will be destroyed if a 
participant is discontinued from the study, all 
SAEs in Minnesota will also be reported to the 
Ugandan IRB, materials will not be stored for 
future research, additional information on data 
management, records will be kept at JCRC for a 
minimum of 5 years 
This version reflects revisions 
to the protocol as requested 
by the Ugandan Scientific 
Council Review.   
1.5 
1.2, 1.3, 2.3.1, 
5.1, 5.2, 5.3, 
6.2.1, 7.2, 8.2, 
8.3.6, 8.4.2, 
10.1.8.1 
Added AE assessment and COVID-19 screenings; 
modified the schedule of stool collection and 
rectal swab for the microbiome study; corrected 
the schema to match the schedule of activities; 
moved all Day 1 procedures expect the yfv to 
baseline; changed I/E criteria from >18 years to 
≥18 years; added pregnancy within the first 28 
days as an exclusion criteria; added pregnancy 
tests to baseline and month 18 (prior to 
leukapheresis); added Fairview Uptown and 
Park Nicollet Travel Clinics as vaccine 
administration sites; updated the reporting and 
Adding changes to match 
responses to JCRC IRB 
application as well as to 
specify the Minnesota details.  

   
 
 
iii 
storage to match the JCRC IRB application; 
removed “AHC services” for data management  
1.6 
1.2, 1.3, 2.3.1, 
8.2, 10.1.5 
Added NCT to cover page; Removed rectal 
swabs and stool collection at day 12 and week 
3; Added stool collection at day 10 and week 4 
and included storage of adipose tissue; Updated 
the safety oversight from DSMB to two ISM – 
one for each site; Added YF-VAX to the list of yf 
vaccines as it is available again; added JCRC 
investigator names per IRB comments 
 
1.7 
1.2, 1.3, 8.2 
Changed the screening period to Day -42 to 1 
and the baseline period to Day -35 to 1; Large 
volume blood draw (60 cc) will be done if a 
participant is not eligible for leukapheresis; 
corrections to section 8.2 regarding stool 
collection to match the schedule of activities  
 
1.8 
1.2, 1.3, 4.1, 8.2,  
Moved 2nd leukapheresis from month 18 to 
month 6 and extended the month 6 timepoint 
 
1.9 
1.2, 1.3, 8.2, 
10.1.6,  
Per Ugandan National Council, the JCRC ICFs will 
be reviewed on site by the JCRC internal 
monitor; remove collection of adipose tissues 
(baseline and week 3) 
 
2.0 
10.1.8.2 
We have added the following to "section 
10.1.8.2 Study records retention" of the 
protocol: "HIPAA authorizations are to be 
retained for 6 years from the date of its creation 
or the date when it was last in effect, whichever 
is later." 
Modification submitted after 
QA Human Research Audit. 

Key Roles and Contact Information 
 
Timothy Schacker, M.D., Principal Investigator 
University of Minnesota 
420 Delaware Street SE 
Minneapolis, MN 55455 
612-624-9955 
Schac008@umn.edu 
 
Cissy Kityo, M.D., Ph.D., Co-Investigator 
Joint Clinical Research Centre 
Plot 101 Lubowa Estates, off Entebbe Road 
P.O. Box 10005 
Kampala, Uganda 
ckityo@jcrc.org.ug 
 
Daniel Douek, M.D., Ph.D., Collaborator 
National Institute of Allergy and Infectious Disease (NIAID) 
Vaccine Research Center 
Human Immunology Section 
Building 40, Room 3504 
MSC 3022 
Bethesda, MD 20898 
ddouek@mail.nih.gov 
 
Cavan Reilly, Ph.D., Biostatistician 
University of Minnesota 
2221 University Avenue SE 
Minneapolis, MN 55414 
612-624-9644 
cavanr@biostat.umn.edu 
 
Erika Helgeson, Ph.D., Biostatistician 
University of Minnesota 
2221 University Avenue SE 
Minneapolis, MN 55414 
612-626-1166 
helge@umn.edu 
 
Joshua Rhein, M.D., Co-Investigator 
University of Minnesota 
420 Delaware St SE 
Minneapolis, MN 55455 
612-624-9996 
rhei0005@umn.edu 
 
Henry Mugerwa, M.D., Ph.D., Co-Investigator  

   
 
 
v 
Joint Clinical Research Centre 
Plot 101 Lubowa Estates, off Entebbe Road 
P.O. Box 10005 
Kampala, Uganda 
hmugerwa@jcrc.org.ug 
Francis Ssali, M.D., Co-PI, DED 
Joint Clinical Research Centre 
Plot 101 Lubowa Estates, off Entebbe Road 
P.O. Box 10005 
Kampala, Uganda 
Stella Namuddu, M.D., Co-Investigator 
Joint Clinical Research Centre 
Plot 101 Lubowa Estates, off Entebbe Road 
P.O. Box 10005 
Kampala, Uganda 
Mary Nakibuuka, M.D., Co-Investigator 
Joint Clinical Research Centre 
Plot 101 Lubowa Estates, off Entebbe Road 
P.O. Box 10005 
Kampala, Uganda 
 

  
 
 
ii 
Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................. 1 
1 
PROTOCOL SUMMARY ............................................................................................................................... 1 
1.1 
Synopsis ................................................................................................................................................ 1 
1.2 
Schema ................................................................................................................................................. 3 
1.3 
Schedule of Activities (SoA) ............................................................................................................... 1 
2 
INTRODUCTION ............................................................................................................................................ 2 
2.1 
Study Rationale .................................................................................................................................... 2 
2.2 
Background ........................................................................................................................................... 3 
2.3 
Risk/Benefit Assessment .................................................................................................................. 12 
2.3.1 
Known Potential Risks ................................................................................................... 12 
2.3.2 
Known Potential Benefits .............................................................................................. 13 
2.3.3 
Assessment of Potential Risks and Benefits .............................................................. 13 
3 
OBJECTIVES AND ENDPOINTS .............................................................................................................. 13 
4 
STUDY DESIGN ........................................................................................................................................... 14 
4.1 
Overall Design .................................................................................................................................... 14 
4.2 
Scientific Rationale for Study Design .............................................................................................. 15 
4.3 
Justification for Dose ......................................................................................................................... 15 
4.4 
End of Study Definition ..................................................................................................................... 15 
5 
STUDY POPULATION ................................................................................................................................ 15 
5.1 
Inclusion Criteria ................................................................................................................................ 15 
5.2 
Exclusion Criteria ............................................................................................................................... 15 
5.3 
Lifestyle Considerations .................................................................................................................... 16 
5.4 
Screen Failures .................................................................................................................................. 16 
5.5 
Strategies for Recruitment and Retention ...................................................................................... 16 
6 
STUDY INTERVENTION ............................................................................................................................ 16 
6.1 
Study Intervention(s) Administration ............................................................................................... 16 
6.1.1 
Study Intervention Description ..................................................................................... 16 
6.1.2 
Dosing and Administration ............................................................................................ 17 
6.2 
Preparation/Handling/Storage/Accountability ................................................................................ 17 
6.2.1 
Acquisition and accountability ...................................................................................... 17 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ................................................ 17 
6.2.3 
Product Storage and Stability ....................................................................................... 17 
6.2.4 
Preparation ...................................................................................................................... 17 
6.3 
Measures to Minimize Bias: Randomization and Blinding ........................................................... 18 
6.4 
Study Intervention Compliance ........................................................................................................ 18 
6.5 
Concomitant Therapy ........................................................................................................................ 18 
6.5.1 
Rescue Medicine ............................................................................................................ 18 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .................................................................................................................... 18 
7.1 
Discontinuation of Study Intervention ............................................................................................. 18 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................................... 18 
7.3 
Lost to Follow-Up ............................................................................................................................... 19 
8 
STUDY ASSESSMENTS AND PROCEDURES ...................................................................................... 19 
8.1 
Efficacy Assessments ....................................................................................................................... 19 
8.2 
Safety and Other Assessments ....................................................................................................... 19 
8.3 
Adverse Events and Serious Adverse Events ............................................................................... 22 

  
 
 
iii 
8.3.1 
Definition of Adverse Events (AE) ............................................................................... 22 
8.3.2 
Definition of Serious Adverse Events (SAE) .............................................................. 22 
8.3.3 
Classification of an Adverse Event .............................................................................. 22 
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 23 
8.3.5 
Adverse Event Reporting .............................................................................................. 24 
8.3.6 
Serious Adverse Event Reporting ............................................................................... 24 
8.3.7 
Reporting Events to Participants ................................................................................. 24 
8.3.8 
Events of Special Interest ............................................................................................. 24 
8.3.9 
Reporting of Pregnancy ................................................................................................ 24 
8.4 
Unanticipated Problems .................................................................................................................... 24 
8.4.1 
Definition of Unanticipated Problems (UP) ................................................................. 24 
8.4.2 
Unanticipated Problem Reporting ................................................................................ 24 
8.4.3 
Reporting Unanticipated Problems to Participants ................................................... 25 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 25 
9.1 
Statistical Hypotheses ....................................................................................................................... 25 
9.2 
Sample Size Determination .............................................................................................................. 26 
9.3 
Populations for Analyses .................................................................................................................. 26 
9.4 
Statistical Analyses ............................................................................................................................ 26 
9.4.1 
General Approach .......................................................................................................... 26 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) ............................................................. 27 
9.4.3 
Analysis of the Secondary Endpoint(s) ....................................................................... 27 
9.4.4 
Safety Analyses .............................................................................................................. 28 
9.4.5 
Baseline Descriptive Statistics ..................................................................................... 28 
9.4.6 
Planned Interim Analyses ............................................................................................. 28 
9.4.7 
Sub-Group Analyses ..................................................................................................... 28 
9.4.8 
Tabulation of Individual participant Data .................................................................... 28 
9.4.9 
Exploratory Analyses ..................................................................................................... 28 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 29 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ........................................................... 29 
10.1.1 
Informed Consent Process ........................................................................................... 29 
10.1.2 
Study Discontinuation and Closure ............................................................................. 29 
10.1.3 
Confidentiality and Privacy ........................................................................................... 30 
10.1.4 
Key Roles and Study Governance .............................................................................. 30 
10.1.5 
Safety Oversight ............................................................................................................. 31 
10.1.6 
Clinical Monitoring .......................................................................................................... 31 
10.1.7 
Quality Assurance and Quality Control ....................................................................... 32 
10.1.8 
Data Handling and Record Keeping ............................................................................ 32 
10.1.9 
Protocol Deviations ........................................................................................................ 33 
10.1.10 
Publication and Data Sharing Policy ........................................................................... 33 
10.1.11 
Conflict of Interest Policy .............................................................................................. 34 
10.2 
Abbreviations ...................................................................................................................................... 34 
11 
REFERENCES ............................................................................................................................................. 36 
 

Yellow Fever Vaccine Study 
Version 2.0 
 
14 August 2023 
 
 
1 
STATEMENT OF COMPLIANCE 
 
The trial will be carried out in accordance with International Conference on Harmonization Good 
Clinical Practice (ICH GCP) and 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 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
 
1 
 
PROTOCOL SUMMARY 
 
1.1 
SYNOPSIS  
Title: 
The effect of inflammation and damage to lymph node structures on 
durable protective immunity following yellow fever vaccination 
Study Description: 
Our hypothesis is that infections other than HIV can cause lymph node 
(LN) inflammation and collagen damage to the fibroblastic reticular cell 
network (FRCn), which will lead to CD4 T cell depletion and impaired 
vaccine responses. This protocol will study yellow fever vaccine (YFV) in 
two cohorts of people, one from Uganda and the other from Minnesota 
where we collect lymphoid tissues (LT) and peripheral blood monocytes 
(PBMCs) before and after vaccination using a new technique to catalog 
infectious burden of the individual, determine the relationship between 
IA, Infections, and immune response.  
Objectives: 
 
Primary Objective:  To determine the relationship between endemic 
infections, immune activation (IA), T cell zone (TZ) fibrosis with loss of the 
fibroblastic reticular cell network (FRCn), CD4 and CD8 T cell subsets and 
the magnitude and durability of neutralizing antibody response to YFV in a 
cohort shown to have elevated IA, a damaged FRCn, and pan T cell 
depletion and a cohort that does not. 
Endpoints: 
Primary Endpoints: 
1) Peak titer of neutralizing antibody to yellow fever vaccination 

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2 
 
Secondary Endpoints:  
1. Measures of T cell zone fibrosis (collagen and desmin) in 
lymphatic tissue 
2. Measures of past and present infection (pathseq, 
molecular analyses of helminth infection, serum antibody 
studies) in lymphatic tissue 
3. Measures of immune activation (Ki67+ and CD69 + cells) in 
lymphatic tissue 
4. Measures of systemic and lymphatic inflammation (plasma 
cytokines, frequency of TGFß+ and TNF+ cells in lymphoid 
tissues) in lymphatic tissue 
5. Histologic analyses of lymphoid tissues for T follicular 
helper cells, changes in B cell populations, measures of 
BAFF+ cells, changes to B cell follicles. 
6. Size and change to the frequency of yellow fever antigen 
specific CD4+ T cell population in lymphatic tissues. 
7. Changes in CD8 T cell number and function in lymphatic 
tissues measured with transcriptomic analyses and 
standard functional assays (stimulation assays). 
Study Population: 
This study will include two cohorts of healthy adults in Kampala, Uganda 
and in Minnesota, USA. The cohort in Uganda will be 30 adults (15 men 
and 15 women) and the cohort in Minnesota will be 16 adults (8 men and 
8 women). 
Phase: 
N/A 
Description of 
Sites/Facilities Enrolling 
Participants: 
Participants in Minnesota will be recruited and enrolled at the University 
of Minnesota, Minneapolis, MN. Study visits will occur in the University of 
Minnesota Health Clinical Research Unit. 
Participants in Kampala, Uganda will be recruited and enrolled at the Joint 
Clinical Research Center. 
Description of Study 
Intervention: 
Yellow fever vaccine (YFV) is a live-attenuated virus vaccine. It is 
administered in a single subcutaneous injection of 0.5mL of reconstituted 
vaccine. 
Study Duration: 
60 months 
Participant Duration: 
18 months 
 
 

Yellow Fever Vaccine Study 
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3 
1.2 
SCHEMA 
 
 
 
 
 
Day -35 to Day 1 
(Baseline) 
Day 1 

YFV Administration 
Day 3 to Day 17 
(Seen Every 2- 3 days) 
 

YF Neutralizing Antibody 

PBMC/Plasma storage 

Blood draw (Day 17) 

Stool collection (Day 10) 
Week 3 

LN Tissue bx  

T Cell Panel 

Luminex 

PBMC/plasma storage 
Weeks 4 – 10 
(Seen Every 2 Weeks) 

YF Neutralizing Antibody 
(week 8) 

PBMC/Plasma storage 

Stool collection (week 4) 
 
Week 12 

Stool and urine collection 

HIV antibody 

Luminex 

PBMC/Plasma storage 
 
Months 6 - 18 
Seen Every 3 Months 

stool and urine collection  

Luminex (month 12) 

PBMC/Plasma storage 

HIV antibody 
Month 6 

Leukapheresis 
Month 18- Final Visit 

Stool and urine collection 

HIV antibody 

YF Neutralizing Antibody 

Luminex 

PBMC/Plasma storage 
 
Study N= 46 
Day -42 to Day 1 
(Screening) 
• 
Leukapheresis 
• 
LN Tissue bx 
• 
Luminex 
• 
Stool and urine collection 
• 
PBMC/plasma storage 
• 
T Cell Panel 

Yellow Fever Vaccine Study 
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1 
1.3 
SCHEDULE OF ACTIVITIES (SOA) 
 
 
Procedures 
Screening 
Day -42 to 1 
Baseline 
Day -35 to 1 
Day 1 
Day 3 (+/- 1 day) 
Day 5 (+/- 1 day) 
Day 7 (+/-1 day) 
Day 10 (+/-1 day) 
Day  12 (+/-1 day) 
Day 14 (+/-1 day) 
Day 17 (+/-1 day) 
Week 3 (+/-1 day) 
Week 4 (+/- 3 days) 
Week 6 (+/- 3 days) 
Week 8 (+/- 3 days) 
Week 10 (+/- 3 days) 
Week 12 (+/- 3 days) 
Month 6 (+/- 14 days) 
Month 9 (+/- 7 days) 
Month 12 (+/- 7 days) 
Month 15 (+/- 7 days) 
Final Study Visit  
Month 18 (+/- 7 days)  
Informed 
consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical exam 
(including 
height and 
weight) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine 
Collection 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
 
X 
Stool 
Collection 
 
X 5,6 
 
 
 
 
X5 
 
 
 
 
X5 
 
 
 
X6 
X5 
 
X5,6 
 
X5,6 
CBCD/Plts 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chemistry 
Panel3 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
T Cell Panel 
X 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
PT/PTT 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV antibody 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
 
X 
YF Neutralizing 
Ab 
X 
 
 
X 
 
X 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
X 
Luminex 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
X 
 
 
X 
 
X 
LN Biopsy  
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
YF Vaccine 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Leukapheresis8 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
PBMC/Plasma 
Storage 
X1 
X 
 
X 
X 
X 
X 
X 
X 
X4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum or urine 
pregnancy 
test2 
X 
X 
 
 
 
X 
 
 
X 
 
X 
X 
 
 
 
 
X 
 
 
 
 
COVID-19 
screening7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AE Assessment 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
1. 
Plasma to screen for absence of yellow fever antibodies 
2. 
For females of childbearing potential 
3. 
Chemistry Panel to include Albumin, ALT, AST, Alk. Phosphate, Bilirubin (total), Calcium, Chloride, CO2, Creatinine, Glucose, Potassium, 
Protein (total), Sodium, Urea Nitrogen 
4. 
This blood draw will be 60 cc for research 
5. 
Samples will go to the Klatt lab 
6. 
Samples will go to the Nutman lab 
7. 
Verbal screening related to symptoms and recent exposures (see SOP for details)  
8. 
Large volume blood draw (60 cc) will be done if a participant is not eligible for leukapheresis  
 

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2 
 
INTRODUCTION 
 
2.1 
STUDY RATIONALE  
Vaccine responses are known to vary geographically. Bacillus Calmette-Guerin (BCG) for tuberculosis 
(TB) is perhaps the best known example of a vaccine where efficacy rates vary according to geography, 
with better responses occurring in northern latitudes (1). Rotavirus vaccine is another where sustained, 
neutralizing antibody titers are achieved after a single dose in the U.S. and Europe but in developing 
countries protection is absent or short-lived, even after multiple doses (2-4). Polio and cholera, are also 
associated with poor antibody responses in developing countries (5-11) and it was recently shown that 
yellow fever vaccine (YFV) was associated with low levels of neutralizing antibodies in Uganda compared 
to Switzerland with heightened immune activation (IA) in Ugandans found to be an important factor 
limiting vaccine responses (12). 
Reasons for these geographic differences in vaccine response are unknown, but one possible 
explanation is that local environmental conditions or endemic infections affect immune function. HIV is 
an obvious example where infection decreases immune function because of loss of CD4 T cells. There 
are multiple mechanisms that contribute to CD4 T cell loss in HIV infection, however the major one is 
from virus replication in lymph nodes (LN) causing an inflammatory reaction, mediated by increased 
expression of TGFß from T-regulatory cells(13), that leads to collagen formation in the parafollicular T 
cell zone (TZ)(14). This fibrosis replaces the fibroblastic reticular cell network (FRCn), a structure that is 
vital to normal immune function. Loss of the FRCn leads to depletion of IL-7(15), a cytokine made by the 
FRCn, and important for T cell homeostasis. As collagen increases, IL-7 levels decrease and T cell 
apoptosis increases (15, 16). It is unknown if this process of LN fibrosis leading to lower CD4 counts 
occurs with other infections, however in large population-based studies of HIV negative people in East 
Africa, the average CD4 T cell count is significantly less than in Northern European populations. In 
Uganda, the mean CD4 T cell count is 754 cells/mm3 for men and 894 cells/mm3 for women (17) and in 
Ethiopia the average for the entire population is 667 cells/mm3 but in Denmark it is 1067 cells/mm3 
(18). There are similar studies from other parts of the world that show the same trend of higher CD4 T 
counts the further north or more developed the country (19-21). 
Our hypothesis is that infections other than HIV can cause LN inflammation and collagen damage to the 
FRCn which will lead to CD4 T cell depletion and impaired vaccine responses. This hypothesis is 
supported by our preliminary data documenting high levels of collagen in the LN TZ of HIV negative 
Ugandans (compared to the U.S.) with concomitant loss of the FRCn, the magnitude of which correlates 
to peak yellow fever neutralizing antibody titer after vaccination. We believe that infections common in 
the developing world (e.g., helminths, TB, Malaria, and Salmonella) can cause LN inflammation that 
leads to chronic immune activation (IA) and collagen deposition in the FRCn with subsequent loss of T 
cells, (including T follicular helper cells). The loss of the FRCn impairs humoral responses to vaccination. 
We propose a direct test of this hypothesis by studying yellow fever vaccine (YFV) in two cohorts of 
people, one from Uganda and the other from Minnesota where we collect lymphoid tissues (LT) (i.e., 
lymph node biopsies), and PBMCs before and after vaccination and using a new technique to catalog 
infectious burden of the individual, determine the relationship between IA, Infections, and immune 
response. We will build on our previous work and study larger cohorts of people with more frequent 
collection of samples. 

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3 
A central question in vaccine research is why vaccine efficacy rates vary with good protection in some 
parts of the world and poor protection in others. For BCG, there is a clear geographic difference in 
efficacy with vaccination in northern latitudes achieving greater efficacy rates (1). Our preliminary data 
suggest one possible explanation is that inflammation from chronic endemic infections damages LN 
structures that are required for robust immune responses. We have previously demonstrated that HIV 
replication in LN causes inflammation that directly leads to CD4 T cell depletion by a process of fibrosis 
replacing the FRCn of the TZ. The FRCn produces IL-7, which is a primary homeostatic mechanism 
supporting LT CD4 and CD8 T cells but the FRCn is also important for B cell function and follicle 
formation. Loss of the FRCn directly leads to increased apoptosis of T cells, and all of their subsets (15, 
16), decreased B Cell Activating Factor (BAFF), fewer follicles, and decreased responses to vaccination 
(22). As described below we now have evidence to suggest that other infections may cause LN fibrosis 
leading to decreased CD4 T cells which, in turn, may limit vaccine responses. This model is described in 
Fig.1. Demonstration that this process occurs with infections beyond HIV and that it limits vaccine 
efficacy is highly significant and would suggest that alternative vaccine constructs or strategies may be 
necessary to improve vaccine efficacy rates, especially for infections like HIV, Malaria, and TB where the 
greatest burden of infection is in the developing world. 
 
2.2 
BACKGROUND  
We have previously demonstrated an indirect and significant relationship between TZ fibrosis and CD4 T 
cell loss by a mechanism of FRCn damage (14-16, 23). This finding, along with the observation of lower T 
cell counts in developing countries (17-21) raised the possibility that infections other than HIV might 
also cause LN inflammation and fibrosis. To explore this possibility, we studied 3 groups of HIV negative 
people (Table 1). Group 1 included 30 people from Kampala, Uganda (16 women, median CD4 874 
cells/µl) for assessment of baseline measures of inflammatory cytokines and LN fibrosis. A subset of 20 
were then given YFV with follow-up LN biopsy 10-14 days after vaccination, a time-point when germinal 
center formation is at its peak (24, 25) and we then obtained blood samples over 14 months of follow-
up to measure the titer and duration of YF neutralizing antibodies. Group 2 consisted of 10 HIV-negative 
Fig. 1: Proposed mechanism for LN inflammation and 
TZ fibrosis leading to impaired vaccine responses. 

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4 
people from Minnesota, USA (3 women, median CD4 953 cells/µl) who did not receive YFV but where 
we did have LN samples to analyze TZ architecture concurrently with Group 1. Group 3 consisted of 10 
HIV uninfected participants of a study (26) at Emory University in Atlanta, GA (4 women, CD4 
unavailable) where they did receive YFV and where plasma samples from baseline before and again 2 
weeks after vaccination were available, allowing direct comparison of antibody titers to Group 1. We 
also used previously published (23) LN collagen data from HIV+ people from the US as positive controls.  
Group 
Site 
N 
YFV 
Samples 
Duration FU 
1 
Uganda 
30 
Subset of 20 
LN & blood before and after 
14 months 
2 
Minnesota 
10 
No 
LN 
1 time-point 
3 
Emory University 
10 
Yes 
Blood at baseline and 2 weeks after YFV 
2 weeks 
Table 1: Description of each group, which groups received YFV, and what samples were collected over FU 
 
At baseline (prior to vaccination) HIV-negative Ugandans have elevated levels of immune activation (IA) 
and inflammation: We measured IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-21, IL-22, IL-23, IL-13, MIP-1β, TNF, 
TGFß, and IP10 in plasma samples from Group 1 (Uganda) and Group 3 (Emory) prior to YFV 
administration. We observed significantly higher levels of TGFß, IL-6, IL-4 and IL-21 and MIP-1ß in 
Ugandans (p < 0.001, p = 0.03, p = 0.004, p = 0.001, and p = 0.01 respectively, Fig. 2A, B). There were no 
other statistically significant differences detected. The increase in TGFß in Ugandans is interesting as it is 
the pro-inflammatory cytokine we have previously associated with TZ fibrosis (13) and may be an 
important mechanism for LN fibrosis in this population as well. We used immunohistochemistry (IHC) 
and quantitative image analysis (QIA) to stain for Ki67+ cells in LN as a measure of IA. Ugandans had 
significantly greater frequency of Ki67+ cells (p = 0.0019, Fig. 2C-E). Collectively these data demonstrate 
increased IA and inflammation in HIV negative Ugandans, especially in LN.  
Fig. 2: Increased IA in HIV negative Ugandans. Increased levels of TGFß 
(A), IL-6 (B), and Ki67+ cells in LN (C) in Ugandans. LN sections stained 
with Ki67 from U.S. (D) and Ugandan I. 

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There is increased collagen and decreased CD4 T cells in HIV negative Ugandan LN. We used trichrome 
staining and QIA to determine the amount of collagen in the TZ in Groups 1 & 2 (Fig. 3 A,B). In Group 1 
the mean area of the TZ with collagen was 12.4% (S.D. 3.4%) whereas in Group 2 it was 4.3% (s.d. 2.9%). 
The amount of TZ collagen in HIV negative Ugandans was similar to the HIV+ people from Minnesota 
where the mean amount of TZ fibrosis was 12.9% (s.d. 7.4%). The regression analysis, after log 
transformation of the percent TZ area, showed a significantly increased amount of TZ collagen in Group 
1 compared to Group 2 (p<0.0001, Fig. 3C). We measured the size of the CD4 population in Group 1 and 
found the expected (14) significant inverse correlation between TZ collagen and TZ CD4 T cells 
(p=0.0322, Fig. 3D). 
 
 
Fig. 3: Increased TZ 
fibrosis in Ugandans. 
Trichrome stain from a 
U.S. participant (A) and 
Ugandan (B)) shows 
increased collagen (blue 
fibers) in the Ugandan 
LN, similar to HIV+ 
people in the U.S. (C). 
There is the expected 
inverse relationship 
between TZ collagen and 
the size of the LN CD4 T 
cell population in Group 
1 (D). 
 
 
We next measured the FRCn by staining tissues with an antibody against Desmin. In Fig. 4A we show a 
representative section of the TZ of a LN from the Minnesota group where the Desmin positive TZ area 
was 22.6% whereas in the Ugandan Group it was 9.5% (range 5.1% - 19.4%, Fig. 4B-C).  As expected, 
there was a direct and significant correlation between the log-transformed area of the TZ that was 
Desmin positive and the log-transformed size of the TZ CD4 T cell population (p< 0.0001, Fig. 4D).  
 

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Fig. 4: The FRCn is depleted in Ugandans. We used QIA to identify TZ Desmin 
(brown) in HIV negative people in the U.S. (A) and Uganda (B and C) and then used 
quantitative image analysis to compare the amount of Desmin in the section to the size 
of the CD4 T cell population in the LN (D) showing the significant and direct relationship. 
 
In summary, people in Uganda have increased IA, inflammation, TZ collagen, damage to the FRCn, and 
depletion of CD4 T cells. We next gave Group 1 YFV to determine if development of antibodies 
correlated to any measures of inflammatory damage to the LN. 
 
There were few follicles prior to YFV and no changes in B cell follicles after YFV. We obtained an 
additional inguinal LN from 20 people in Group 1 and then administered YFV in the thigh opposite the 
one just biopsied. Ten to fourteen days later we obtained another LN from the same side the vaccine 
was given. We measured B cell populations, frequency and location of germinal centers, and frequency 
of Ki67+ cells before and after vaccination. We found few primary or secondary follicles (germinal 
centers) before vaccination with no significant increase after vaccination. In 63% of participants we saw 
no follicle formation and in only 3 of the participants did we see an increase in B cell follicles. The mean 
area of the follicles with B cells prior to vaccination was 25.5% (range 15.7-39.4%) whereas after 
vaccination it was 24.1% (range 14.0-34.1%, p=0.34). There was a significant increase in the frequency of 
Ki67+ cells (p=0.002).  
 
YFV antibody responses were blunted and of short duration. Titers of neutralizing antibodies to YF were 
measured by plaque reduction assay (PRA). Initially we compared the week 2 samples between Group 1 
and Group 3 using a PRA assay with a lower limit of detection of 10 plaque forming units (pfu) and found 
only a few of the Ugandans had detectable antibodies whereas most of Group 3 had detectable 
antibodies (p < 0.0001, Fig. 5A). We point out that we only had access to plasma samples from Group 3 

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at the 2 time-points (i.e., D0 and W2) and LN biopsies were not done in that study, so our comparative 
analysis of antibody formation and decay between these two groups is limited. However, as described 
below there are historical controls that can be used with Group 1 to make relevant comparisons about 
peak titer and decay rate(12). We next studied the longitudinally collected plasma samples from Group 
1 using a PRA assay with a limit of detection of 100 pfu. We switched to a PRA with a higher threshold of 
detection titer because the lab performing the first analysis was no longer available to us. We did not 
duplicate the analysis between Group 1 and 3 as there was limited sample available. Peak titer was seen 
at the week 3 time-point with a 1.3 log range and a median decrease of 0.9 log by week 60 (Fig. 5B).   
 
Fig. 5: Yellow fever antibody titers. Titers of neutralizing antibody from Groups 1 & 3 are 
compared using an assay with a lower limit of detection of 20 pfu (A). B. decay of antibodies 
through Month 14. C correlation of Desmin to peak antibody titer. 
 
These response rates and decay characteristics are similar to those reported by Muyanja in a similar 
group of Ugandans (12) (and personal communication L. Trautmann, 2017). Given the variable response 
in antibody formation and the anatomic changes we found in Ugandan LN, we looked for an anatomic 
correlate of poor vaccine response and found that measures of the FRCn correlated to the peak 
antibody titer; the more FRCn, the higher the peak titer of antibodies (p = 0.02, Fig. 5C). 
 
T Follicular Helper (Tfh) cells are depleted in HIV-negative Ugandans and do not increase with YFV. The 
striking fact that along with the diminished antibody response, we did not see an increase in follicles or 
B cell numbers we thought it possible that T Follicular helper cell (Tfh) populations might be diminished 
(as part of the overall CD4 T cell decrease) as they are required for normal B cell function and germinal 
center formation(27). We used a multiplexed confocal imaging assay allowing for the simultaneous 
detection of 6 markers in LN before and after vaccination with antibodies directed against CD20 for B 
cells, CD8, Ki67, and PD1 and CD57 for Tfh cells (28-30). We observed 3 distinct histologic patterns in 
these tissues. After vaccination only 3/20 had lack of Ki67 staining (Participant 1688, Fig. 6K,L). 
Ugandans exhibited a skewed cytokine response to YFV vaccination. We next measured IL-1ß, IL-4, IL-6, 
IL-10, IL-17A, IL-21, IL-22, IL-23, IL-13, MIP-1β, TNF, and TGFß in the plasma 2 weeks after YFV for 
comparison to the baseline measures discussed below (Table 2). In Group 3 (Emory) there was no 
change in any of the analytes between baseline and week 2 after vaccination other than IL-13, but we 
did measure significant changes in several cytokines in Group 1. There was a significant increase in IL-10, 
IL-17A, IL-21, IL-22, IL-23, IL-1ß, CXCL13, and IP10 in Ugandans after YFV (p > 0.01 for all). Levels of TGFß 
decreased significantly (p = 0.002) and there was a trend for decrease in TNF levels (p=0.065). The 
elevated levels of TGFß and IL-6 in the Ugandans prior to vaccination suggest prior exposures to 
extracellular pathogens or infections that promote IL-17 responses as evidenced by an increase in IL17A 
in the Ugandans after vaccination. The results of a lasso regression analysis show that IL6 and IL1b at 
Day 0 and IL22, TGFß and IL6 at W2 were predictive of yellow fever antibody titers at W3. Only IL1b was 
negatively correlated, the others were positively correlated.  

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Fig. 6: B cell follicles are diminished with a paucity of Tfh cells in B cell follicles in Ugandans. This figure shows LN analyses from 
3 different participants before vaccination and again at week 2 after vaccination. Participant 1996 has recognizable follicles pre-
vaccination with maturing secondary follicles after vaccination (red arrows). The presence of PD-1 staining cells in the secondary follicle 
(green and blue staining cells in the secondary follicle appear yellow) is expected vaccination (D). Participant 1682 had fewer, more 
poorly formed follicles Day 0 and a lack of recognizable secondary follicles with vaccination. PD-1 staining cells are not inside of the 
follicle structure. Participant 1688 had no recognizable follicles prior to vaccination and no response to vaccination. 
 
 
–p - values 
 
D 0 
Uganda 
W 2 
Uganda 
D 0 
US 
W 2 
US 
Uganda 
D0-W2 
US 
D0-
W2 
Between 
Countries 
D0 
Between 
Countries 
W2 
TGFß 17768.7 11480.8 2464.6 
2763 
0.002 
0.608 
<0.001 
<0.001 
IL-13 
17.4 
22.7 
10.2 
8.5 
0.107 
0.013 
0.083 
0.002 
IL-10 
4.1 
9 
3.8 
5.2 
<0.001 
0.063 
0.646 
0.179 
IL-17A 
33.6 
51.1 
35 
37.3 
0.004 
0.31 
0.889 
0.053 
IL-4 
3.2 
7 
0.8 
0.7 
0.051 
0.701 
0.004 
<0.001 
IL-6 
1.4 
1.6 
0.9 
1 
0.1 
0.646 
0.033 
0.001 
IL-21 
1.5 
3.3 
<0.001 <0.001 
0.006 
1.0 
0.001 
<0.001 
IL-22 
0.5 
3.2 
<0.001 <0.001 
0.022 
1.0 
0.362 
0.008 

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IL-23 
0.1 
2.3 
0.6 
0.6 
<0.001 
0.781 
0.588 
0.012 
IL-1ß 
1.2 
3.2 
1.6 
0.6 
0.005 
0.351 
0.69 
0.004 
TNF 
63.6 
48.3 
65.7 
56.9 
0.065 
0.81 
0.811 
0.324 
CXCL13 
30.1 
37.9 
22.7 
24.5 
0.006 
0.489 
0.451 
0.085 
IP10 
265 
453.7 
208.8 
223.8 
<0.001 
0.107 
0.062 
<0.001 
MIP1ß 
57.6 
49.3 
36.8 
35.4 
0.065 
0.137 
0.01 
0.077 
Table 2: Summary of cytokine data in terms of models based estimates of mean levels. The models controlled 
for age, gender, and measurements over time from the same subject. 
Our hypothesis is that infections cause inflammation and LN fibrosis. A particularly innovative feature of 
this protocol is the ability to characterize the infectious burden of an individual using the new “Pathseq” 
technique developed by Dr. Daniel Douek. This involves deep sequencing of RNA and DNA fragments 
(ranging in size from ~100-1000 bases) in plasma to obtain an overall quantitative and qualitative 
measure of the viral, bacterial, fungal, and parasite microbial burden as well as helminth burden in an 
individual. This approach differs from standard microbiome approaches such as bacterial 16s rDNA 
sequencing or virus purification and sequencing as it is sequence agnostic and takes advantage of the 
fact that circulating nucleic acids are typically highly fragmented to provide information about the 
“circulating microbiome”. Nucleic acids from all microbes, worms and even food are readily detected 
using our PathSeq approach, and likely represent recent history of non-human genomic burden as 
circulating nucleic acids are typically very short-lived.  
The methodology itself is a validated adaptation of our standard cellular RNAseq approach. Peripheral 
blood is collected in EDTA tubes, with one tube from the same batch remaining empty and processed 
identically as a control for contaminating nucleic acids. 1-10 ml plasma is separated and RNA and DNA 
are extracted. For the RNA, double-stranded cDNA is generated using random primers, and is then 
fragmented, end-repaired and dA-tailed. For the DNA, the protocol begins at the fragmentation step. 
Samples are cleaned with SPRI beads, and Illumina adaptors and barcodes ligated. A PCR enrichment is 
performed followed by low-cut purification to remove adaptor dimers.  Libraries are now ready for QC, 
quantification and pooling. The control sample should show no detectable product at this stage and a 
positive signal would imply environmental contamination. Depending on the number of samples pooled, 
sequencing is then performed using Illumina MiSeq or HiSeq4000 instruments in the Douek lab. 
Following pre-processing of the raw sequences, host genomic and transcriptomic derived sequences are 
identified and removed from the dataset.  De novo assembly is then performed on the remaining, non-
host sequences.  The assembled sequences are then aligned against the comprehensive “nt” database 
from NCBI to determine from which organism the assembled sequences were derived.  The original, raw 
sequences are then mapped back to the assembled sequences to quantify the abundance of the 
assembled sequences and then, by proxy, the abundance of the organism from which the assembled 
sequences are derived. We assume the sensitivity of this approach is ~100% as our bio-informatic 
pipeline uses the most up-to-date microbial sequence databases but also corrects for inherent errors in 
these databases due to sequence contamination. In Fig. 7 we show the PathSeq results on serial plasma 
samples collected from one Ugandan HIV+ individual over a 9-month period after starting antiretroviral 
therapy (ART). The y-axis shows fragments per kilobase of transcript per million reads (FPKM) 
normalized for total reads in each sample to allow for comparisons across time-points and subjects. The 
HIV reads become undetectable by 3 months of treatment. Bacteria account for the greatest number of 
reads with proteobacteria (predominantly gram-negative bacteria) being the largest component 
detected, presumably translocating from the gut.  Helminths (both flat and round worms) also 
contributed substantially to the infectious burden. The Apicomplexan reads are predominantly derived 
from Plasmodium falciparum and the ssRNA virus reads are entirely derived from GBV-C virus. 
Importantly, the number of HIV reads and GBV-C reads were confirmed directly by specific qRT-PCR and 

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were completely concordant, providing further validation of this technique to assess the microbial 
burden. Also of importance, the pattern is remarkably stable over a 9-month period, suggesting patterns 
that emerge can be viewed as a chronic pattern of microbial burden. 
Fig 7: Longitudinal analysis of microbial burden in one HIV 
infected persons. Each bar represents the microbial burden at a 
different time-point over 9 months of follow-up. Bacteria make 
up the majority of reads detected and the pattern is remarkably 
stable over time. The blue arrow points to the blue checkered 
band that represents HIV. This individual started ART at that 
time and this band diminishes over time (as would be 
expected). 
In Fig. 8 we show the PathSeq result from 10 Ugandan HIV+ individuals collected over 9 months after 
initiating ART. The y-axis shows relative read frequency. We noted two distinct patterns of infectious 
burden. The plasma of the 6 participants in the upper row of Fig. 8 predominantly contained bacterial 
nucleic acids from the Proteobacteria family whereas in the lower row of 5 participants GBV-C virus and 
helminthic nucleic acids predominated. Each group exhibited a markedly different cytokine profile after 
9 months of ART. Infection with primarily proteobacteria was associated with higher levels of the pro-
inflammatory cytokines IL-6 and TNF, and sCD14 (a marker of bacterial translocation) and inversely 
correlated to IL-17 and MIP1b. In contrast, participants with predominantly helminthic and GBV-C 
nucleic acids had exactly the opposite cytokine profile after 12 months of ART; high levels of IL-17 and 
MIP1b and low levels of IL-6 and sCD14. 

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Fig 8: Two patterns of microbial burden identified over 9 months of FU.  
Pathseq is a tool to provide quantitative information about the chronic infectious burden of an 
individual that we can use to investigate relationships between infections, LN inflammation and fibrosis, 
and vaccine responses. There is evidence that such relationships likely exist. For example, in a recent HIV 
vaccine study in Africa, participants infected with S. mansoni had significantly lower titers of antibodies 
than those who were not (31). Combining Pathseq with our analyses of antibody titers, cytokine profiles 
before and after vaccination, and our analysis of LN architecture will provide important information 
about the role of the local infectious burden as a determinant of immune response to vaccination. 
We show in our preliminary data that chronic IA, inflammation and damage to LN architecture in HIV 
negative Ugandans correlates with neutralizing antibody titers after YFV. The demonstration of 
increased levels of TGFß at baseline along with an IL-10 and IL-17 response after YFV suggests helminthic 
infections may be contributing to abnormal vaccine responses and supports our hypothesis that 
endemic infections may affect immune responses to vaccination. We demonstrate the ability to 
characterize infectious burden, and while this does not define the entire life history of pathogen 
exposure, it is surprisingly stable over time allowing us to use this as a record of infectious exposures. 
We now propose to build on these initial observations by conducting a controlled study with YFV in both 
Uganda and the USA to better understand how endemic infections and environmental factors may 
affect innate and adaptive immune responses to vaccination and better define how these factors might 
inhibit immune mechanisms and components that lead to sustained immunity. Our data raise several 
important questions, including 1) what is causing the generalized IA, 2) how does systemic IA lead to a 
local process of collagen formation in the FRCn, 3) how are antigen specific CD4 and CD8 T cells affected 
by loss of the FRCn, 4) Why are their fewer TFh in Ugandans, 5) does the frequency and function of TFh 
cells in Ugandans affect the magnitude and durability of the vaccine response, 6) does relative T cell 
depletion in this population limit recall responses, and 7) does the mucosal environment, in particular in 
GALT infections from helminths or enteric pathogenic bacteria, affect the magnitude or durability of 
immune responses?  Our hypothesis is that infections other than HIV can cause LN inflammation and 
collagen damage to the FRCn which will lead to CD4 T cell depletion and impaired vaccine responses.  
We will address this hypothesis by giving YFV to a group of otherwise healthy people in Uganda and 
Minnesota and we will systematically collect blood, and LN specimens before and after YFV. Our 
approach is feasible as we have already demonstrated that we can conduct a trial of this nature at the 

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Joint Clinical Research Center in Kampala, Uganda where we have collected LN and blood samples at 
multiple time-points throughout several protocols, including the YFV study described above. In the past 
8 years, we have enrolled over 105 participants in HIV pathogenesis and vaccine studies at the JCRC who 
have contributed 228 LN biopsies. Importantly, we now have capacity to perform leukapheresis at the 
JCRC in Uganda as they recently acquired the equipment and expertise to perform this procedure. We 
have demonstrated the ability to process, maintain, and ship specimens from the Uganda site to 
Minnesota. In Minnesota, we have enrolled 170 HIV+ and 100 HIV- people in multiple pathogenesis and 
interventional trials where we have collected 413 LNs, 1657 PBMC and plasma samples from these 
individuals. We now propose to collect the relevant specimens before and after YFV to apply state-of-
the-art technologies to address fundamental question about how and why vaccine responses are limited 
in some populations. 
 
2.3 
RISK/BENEFIT ASSESSMENT   
 
2.3.1 KNOWN POTENTIAL RISKS  
Risks Associated with Yellow Fever Vaccine 
Common side effects: 
• 
Injection site reaction, including erythema and pain at injection site 
• 
Headache 
• 
Fever 
• 
Myalgia 
• 
Malaise 
 
Rare side effects: 
Severe Allergic Reactions 
Severe allergic reactions (e.g., anaphylaxis) may occur following the use of Stamaril or YF-VAX, even in 
individuals with no prior history of hypersensitivity to the vaccine components. Appropriate medical 
treatment and supervision must be available to manage possible anaphylactic reactions following 
administration of the vaccine. 
 
Yellow fever vaccine-associated viscerotropic disease  
Age greater than 60 years is a risk factor for yellow fever vaccine-associated viscerotropic disease (YEL-
AVD) which may present as non-specific multi-organ system failure or can be similar to fulminant yellow 
fever caused by wild-type yellow fever virus, with liver failure and internal bleeding, leading to death. 
Available evidence suggests that the occurrence of this syndrome may depend upon undefined host 
factors, rather than intrinsic virulence of the yellow fever strain 17D vaccine, based on characterization 
of vaccine viruses isolated from individuals with YEL-AVD. YEL-AVD has been reported to occur only after 
the first dose of yellow fever vaccine; there have been no reports of YEL-AVD following booster dose. 
The decision to vaccinate individuals 60 years of age and older needs to weigh the risks and benefits of 
vaccination and the risk for exposure to yellow fever virus.  
 
Yellow fever vaccine-associated neurotropic disease  
Age greater than 60 years and immunosuppression are risk factors for post-vaccinal encephalitis, also 
known as yellow fever vaccine-associated neurotropic disease (YEL-AND). Almost all cases of YEL-AND 
have been in first-time vaccine recipients. The decision to vaccinate individuals 60 years of age and older 

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and immunosuppressed individuals needs to weigh the risks and benefits of vaccination and the risk for 
exposure to yellow fever virus. 
 
Risks Associated with Protocol Directed Procedures: 
Lymph Node Biopsy 
The risks of the lymph node biopsy are bleeding, infection, seroma, and scarring.  Any participant who 
develops a seroma will have it drained with a syringe in the clinic.  These complications are rare.  All 
post-operative and post-biopsy care will be provided by study nurses. Participants will be monitored for 
a minimum of 4 hours post-surgery prior to being allowed to return home. If complications occur, the 
participant will be seen immediately by a qualified surgeon. 
 
Leukapheresis 
The risks of the apheresis process include bruising and/or bleeding in the arms when needles are placed 
for the apheresis; a reaction to the drug used as an anticoagulant during the procedure, citrate; and loss 
of blood, especially platelets.  Rarely, veins cannot be found to insert the needles a catheter must be 
placed in a large neck vein to collect the cells.  
 
Blood Draw 
Events associated with venipunctures include discomfort, slight bruising, bruising, bleeding, 
lightheadedness, fainting, infection at the venipuncture site, nausea, anxiety and swelling at the 
venipuncture site. 
 
2.3.2 KNOWN POTENTIAL BENEFITS  
There are no anticipated benefits to participants on this study.  
 
There is a potential for societal benefit, as this study could demonstrate the need for alternative vaccine 
constructs and other strategies to improve vaccine efficacy rates. 
 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
There is a low possibility of side effects.   There are no potential benefits besides findings from the 
proposed research could lead to a clearer understanding of the requirements for inducing durable 
protective immunity. 
 
3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
To determine the relationship between 
endemic infections, immune activation 
(IA), T cell zone (TZ) fibrosis with loss of 
the fibroblastic reticular cell network 
(FRCn), CD4 and CD8 T cell subsets and 
the magnitude and durability of 
neutralizing antibody response to YFV 
in a cohort shown to have elevated IA, 
Peak titer of neutralizing antibody to 
yellow fever vaccination 
 
The primary analysis 
endpoint is the peak titer 
of neutralizing antibody. 
We believe that there will 
be significant differences 
in the peak titer and 
durability of titers 
between the two groups. 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
a damaged FRCn, and pan T cell 
depletion and a cohort that does not. 
Secondary 
 
 
To determine predictors of levels of 
neutralizing antibodies 
1. Measures of T cell zone fibrosis 
(collagen and desmin) 
2. Measures of past and present 
infection (pathseq, molecular 
analyses of helminth infection 
and serum antibody studies) 
3. Measures of immune activation 
(Ki67+ and CD69 + cells) 
4. Measures of systemic and 
lymphatic inflammation (plasma 
cytokines, frequency of TGFß+ 
and TNF+ cells in lymphoid 
tissues). 
5. Changes in T cell number and 
function 
We believe the reason 
that titers will be 
different is that 
inflammation from 
endemic infections alters 
the architecture and 
therefore the function of 
the lymphatic tissues 
which results in less 
antibody being made. 
With these secondary 
endpoints we will 
measure these potential 
predictors. 
Tertiary/Exploratory  
 
 
To determine the impact of LN fibrosis 
on specific T cell populations including 
T follicular helper cells and antigen 
specific T cells. 
1. Histologic analyses of lymphoid 
tissues for T follicular helper 
cells and B cells. 
 
2. Size and change to the 
frequency of yellow fever 
antigen specific CD4+ T cell 
population. 
We have shown that 
lymphatic tissue fibrosis 
causes depletion of CD4 
and CD8 + T cells 
however we have never 
examined the impact on 
highly specific subsets. 
 
 
4 
STUDY DESIGN  
 
4.1 
OVERALL DESIGN 
The primary aim of this study is to determine the difference between antibody titers in the two study 
groups and study the relationship between endemic infections, IA, the FRCn, and CD4 and CD8 T cell 
subsets and the magnitude and durability of neutralizing antibody response to YFV in a cohort shown to 
have elevated IA, a damaged FRCn, and pan T cell depletion and a cohort that does not. This is a single 
arm, open-label, two cohort study of healthy adults in Kampala, Uganda and in Minnesota, USA. The 
cohort in Uganda will be 30 adults (15 men and 15women) and the cohort in Minnesota will be 16 adults 
(8 men and 8 women). Everyone will be screened to ensure there are no contraindications to receiving 
YFV (e.g., immunosuppression) or the planned procedures. The inclusion and exclusion criteria are 
discussed in detail in the protocol that is included in the appendix. Participants will have an inguinal LN 
biopsy and leukapheresis prior to YFV and another LN biopsy 3 weeks after the vaccine administration. 
The vaccine will be given in the contralateral thigh from the first LN biopsy so that the second biopsy will 
be from a draining LN. PBMC and plasma will be collected at regular intervals over the 18-month follow-
up period. Leukapheresis will be done again at the month 6 visit. 
 

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4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
Our preliminary data shows that lymph node inflammation and damage to the FRCn correlates to peak 
titer. We also show data to suggest that there is significantly less LN damage from inflammation in 
native Minnesotans (not immigrants—we have not studied that). We think the differences are because 
of exposure to endemic infections. We will therefore use an open-label, non-randomized design to test 
our hypothesis.  
 
4.3 
JUSTIFICATION FOR DOSE 
This is the standard of care dose of yellow fever vaccine. 
 
4.4 
END OF STUDY DEFINITION 
A participant is considered to have completed the study if he or she has completed all phases of the 
study including the last visit or the last scheduled procedure shown in the Schedule of Activities (SoA), 
Section 1.3. 
 
 
5 
STUDY POPULATION 
 
5.1 
INCLUSION CRITERIA 
Study entry is open to adults regardless of race or ethnic background.  While there will be every effort to 
seek out and include minority patients from both genders, the patient population is expected to be no 
different from that of the populations in Minnesota and Kampala, Uganda. 
 
In order to be eligible to participate in this study, an individual must meet all of the following criteria 
1. Age >18 years but < 60 years 
2. No contraindication to Yellow Fever vaccine (immunosuppressed for any reason or on an 
immunosuppressive drug where a live virus vaccine is contraindicated). 
3. If female of childbearing age must agree to contraception for one month following 
administration of the vaccination. 
 
5.2 
EXCLUSION CRITERIA 
An individual who meets any of the following criteria will be excluded from participation in this study 
1. History of yellow fever or previous vaccination for yellow fever 
2. Known bleeding disorder 
3. Prior surgery complicated by clotting abnormality 
4. Psychiatric or behavioral disorder that, in the opinion of the investigator, will make it difficult for 
the participant to complete the study 
5. History of acute hypersensitivity reaction to any component of the vaccine (including gelatin, 
eggs, egg products, or chicken protein). 
6. Thymus disorder associated with abnormal immune function 
7. Immunosuppression from any of the following: HIV infection or AIDS, malignant neoplasms, 
primary immunodeficiencies, transplantation, transplantation, immunosuppressive or 

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immunomodulatory therapy (corticosteroids, alkylating agents, antimetabolites, TNF inhibitors, 
IL-1 blocking agents, monoclonal antibodies targeting immune cells), previous radiation therapy. 
8. Pregnant or breastfeeding at the time of vaccination. 
9. Planning to conceive within 28 days of enrollment and vaccination with the yellow fever vaccine. 
 
 
5.3 
LIFESTYLE CONSIDERATIONS 
N/A 
 
5.4 
SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently eligible to be entered in the study. A minimal set of screen failure information is required 
to ensure transparent reporting of screen failure participants, to meet the Consolidated Standards of 
Reporting Trials (CONSORT) publishing requirements and to respond to queries from regulatory 
authorities. Minimal information includes demography, screen failure details, eligibility criteria, and any 
serious adverse event (SAE). 
 
5.5 
STRATEGIES FOR RECRUITMENT AND RETENTION 
In Minneapolis, Minnesota, participants will be recruited through the use of flyers via email, mail, and 
fax, study website, advertisements and primary care providers making information available to potential 
participants.  In addition, the research team will work with the Fairview Uptown travel clinic to identify 
potential participants based on yellow fever vaccination appointments. In Kampala, Uganda recruiting 
will occur by referral from primary doctors and advertisements.   
 
6 
STUDY INTERVENTION 
 
There are no study interventions and the following information is provided as they are central to the study. 
Stamaril and YF-VAX are the manufacturers and both can be used at either study site.  
 
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
YF-VAX® and Stamaril®, Yellow Fever Vaccine, for subcutaneous use, is prepared by culturing the 17D-
204 strain of yellow fever virus in living avian leukosis virus-free (ALV-free) chicken embryos. The vaccine 
contains sorbitol and gelatin as a stabilizer, is lyophilized, and is hermetically sealed under nitrogen. No 
preservative is added. Each vial of vaccine is supplied with a separate vial of sterile diluent, which 
contains Sodium Chloride Injection USP – without a preservative. Stamaril is formulated to contain not 
less than 4.74 log10 plaque forming units (PFU) per 0.5 mL dose throughout the life of the product. 
Before reconstitution, Stamaril is a pinkish color. After reconstitution, Stamaril is a slight pink-brown 
suspension. The vial stoppers for Stamaril and diluent are not made with natural rubber latex. 
 
YF-VAX and Stamaril are approved for the prevention of yellow fever in persons 9 months of age and 
older. 
 

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Stamaril is an already licensed product in Uganda.  It was approved by the National Drug Authority 
(NDA) under registration / reference number NDA/MAL/HDP/1482.  The vaccine in manufactured by 
SANOFI PASTUER in France and is distributed by Laborex (U) Limited in Uganda.  
 
6.1.2 DOSING AND ADMINISTRATION 
YF-VAX and Stamaril are administered in a single subcutaneous injection of 0.5 mL of reconstituted 
vaccine. 
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
In Minnesota, the vaccine will be administered per the study’s vaccine administration SOP at Park 
Nicollet Travel Clinic in St. Louis Park, MN, Fairview Uptown Travel Clinic in Minneapolis, MN, or any 
travel clinic that is authorized to administer the yellow fever vaccine. The travel clinician will indicate the 
vaccine administration site for study documentation.    
 
In Uganda, the vaccine will be supplied from the commercial pharmacy and administered in the research 
clinic in a manner identical to what is described above in our background discussion. 
 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
YF-VAX® and Stamaril®, Yellow Fever Vaccine, for subcutaneous use, is prepared by culturing the 17D-
204 strain of yellow fever virus in living avian leukosis virus-free (ALV-free) chicken embryos. The vaccine 
contains sorbitol and gelatin as a stabilizer, is lyophilized, and is hermetically sealed under nitrogen. No 
preservative is added. Each vial of vaccine is supplied with a separate vial of sterile diluent, which 
contains Sodium Chloride Injection USP – without a preservative. Stamaril is formulated to contain not 
less than 4.74 log10 plaque forming units (PFU) per 0.5 mL dose throughout the life of the product. 
Before reconstitution, Stamaril is a pinkish color. After reconstitution, Stamaril is a slight pink-brown 
suspension. The vial stoppers for Stamaril and diluent are not made with natural rubber latex. 
 
6.2.3 PRODUCT STORAGE AND STABILITY 
Store at 2° to 8°C (35° to 46°F). DO NOT FREEZE. Do not use vaccine after expiration date. Stamaril does 
not contain a preservative. The following stability information for Stamaril is provided for those 
countries or areas of the world where an adequate cold chain is a problem and inadvertent exposure to 
abnormal temperatures has occurred. Half-life is reduced from approximately 14 days at 35° to 37°C to 
3-4.5 days at 45° to 47°C. 
 
6.2.4 PREPARATION 
Reconstitute the vaccine using only the diluent supplied (0.6 mL vial of Sodium Chloride Injection USP 
for single dose vial of vaccine and 3 mL vial of Sodium Chloride Injection USP for 5 dose vial of vaccine). 
After removing the "flip-off" caps, cleanse the vaccine and diluent vial stoppers with a suitable 
germicide. Do not remove the vial stoppers or metal seals holding them in place. Using aseptic 
technique, use a suitable sterile needle and syringe to withdraw the volume of supplied diluent shown 
on the diluent label and slowly inject the diluent into the vial containing the vaccine. Allow the 
reconstituted vaccine to sit for one to two minutes and then carefully swirl mixture until a uniform 
suspension is achieved. Avoid vigorous shaking as this tends to cause foaming of the suspension. Do not 

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dilute reconstituted vaccine. Use aseptic technique and a separate sterile needle and syringe to 
withdraw each 0.5mL dose from the single dose or multidose vial of reconstituted vaccine. 
 
Before reconstitution, the vaccine is a pinkish color. After reconstitution, the vaccine is a slight pink 
brown suspension. Parenteral drug products should be inspected visually for particulate matter and 
discoloration prior to administration, whenever solution and container permit. If either of these 
conditions exists, do not administer the vaccine. 
 
Administer the single dose of 0.5 mL subcutaneously using a suitable sterile needle. 
 
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
Investigators conducting image analysis will be blinded to which group the tissue they are analyzing is 
from (Uganda vs. Minnesota). 
 
A special numbering system will be created by Drs. Helgeson and Reilly and used for all samples so that 
people conducting other analyses of samples will be blinded to which group the tissue they are 
analyzing is from (Uganda vs. Minnesota). 
 
Only Drs. Reilly and Helgeson will have access to the identification information and will be able to match 
samples to group.  
 
6.4 
STUDY INTERVENTION COMPLIANCE 
This is a single dose of vaccine. Compliance should not be an issue. 
 
6.5 
CONCOMITANT THERAPY 
There are no medication restrictions on this protocol. 
 
6.5.1 RESCUE MEDICINE 
Not applicable. 
 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
N/A 
 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following reasons: 
 
• 
Pregnancy within 28 days of vaccination 
• 
Significant study non-compliance  
• 
If any clinical adverse event (AE), laboratory abnormality, or other medical condition or situation 
occurs such that continued participation in the study would not be in the best interest of the 
participant 

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• 
Acquisition of HIV  
The reason for participant discontinuation or withdrawal from the study will be recorded on a Case 
Report Form (CRF) designed for this purpose. Subjects who sign the informed consent form but do not 
receive the study intervention may be replaced.  Subjects who sign the informed consent form, and 
receive the study intervention, and subsequently withdraw, or are withdrawn or discontinued from the 
study, will not be replaced. 
7.3 
LOST TO FOLLOW-UP 
A participant will be considered lost to follow-up if he or she fails to return for scheduled visits and is 
unable to be contacted by the study site staff.  
 
The following actions must be taken if a participant fails to return to the clinic for a required study visit: 
• 
The site will attempt to contact the participant and reschedule the missed visit and counsel the 
participant on the importance of maintaining the assigned visit schedule and ascertain if the 
participant wishes to and/or should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, 
a certified letter to the participant’s last known mailing address or local equivalent methods). 
These contact attempts should be documented in the participant’s study file.  
• 
Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
EFFICACY ASSESSMENTS  
This study does not contain a therapeutic intervention and therefore there are no efficacy assessments. 
 
8.2 
SAFETY AND OTHER ASSESSMENTS 
 
Screening Procedures (Day -42 to 1) 
Participants will be consented by the investigator or qualified study staff before any procedures take 
place.  
The following screening blood tests will be obtained: 
• 
CBCD 
• 
Chemistry Panel (including Albumin, ALT, AST, Alk. Phosphate, Bilirubin (total), Calcium, Chloride, 
CO2, Creatinine, Glucose, Potassium, Protein (total), Sodium, Urea Nitrogen) 
• 
T Cell Panel 
• 
PT/PTT 
• 
HIV antibody 
• 
Yellow Fever Neutralizing Antibody 
• 
PBMC/Plasma storage (to screen for yellow fever) 
 
We will obtain a detailed medical history to ascertain that potential subjects have not had yellow fever, 
been vaccinated for yellow fever, and have no history of thymus disease. Females of childbearing 

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potential will have a serum or urine pregnancy test.  During the COVID-19 pandemic, participants will be 
screened for COVID-19 symptoms before coming in for their visit.  
 
Baseline Procedures (Day -35 to 1) 
Medical history and concurrent medications ascertained.  Physical examination and vital signs, including 
blood pressure, pulse and weight will be conducted. There will also be an AE assessment.  Leukapheresis 
will be performed.  Before Leukapheresis, females of childbearing potential will have a serum or urine 
pregnancy test.  If a participant is not eligible for leukapheresis based on the apheresis consultation 
appointment or is unable to tolerate leukapheresis, a large volume blood draw (60 cc) will be done 
instead.  Participants will undergo a lymph node biopsy tissue will be stored. Participants will have stool 
and urine collected for a helminth check. Stool will be collected for microbiome study.  
Blood will be collected for: 
• 
T cell panel 
• 
Luminex 
• 
PBMC/plasma storage 
 
During the COVID-19 pandemic, participants will be screened for COVID-19 symptoms before coming in 
for their visits. 
 
Day 1 
Yellow fever vaccine will be administered.  During the COVID-19 pandemic, participants will follow the 
travel clinics’ procedures for screening of COVID-19 symptoms before coming in for their visit. 
 
Days 3 through 14 
Participants will return every 2 to 3 days (see SOEs, Sections 1.2 and 1.3) for blood collection for: 
• 
PBMC/plasma storage 
• 
Yellow fever neutralizing antibody 
 
Stool will be collected on Day 10 for microbiome study.  
 
On Days 7 and 14, females of childbearing potential will have a serum or urine pregnancy test.   
 
During the COVID-19 pandemic, participants will be screened for COVID-19 symptoms before coming in 
for their visits.  There will also be AE assessments.   
 
Day 17 
At Day 17, participants will have a research blood draw of 60 cc. Blood will be collected for yellow fever 
neutralizing antibody. There will also be an AE assessment.  During the COVID-19 pandemic, participants 
will be screened for COVID-19 symptoms before coming in for their visit.  
 
Week 3 
At Week 3, participants will undergo a lymph node biopsy will be stored. Blood will be collected for: 
• 
T cell panel 
• 
Luminex 
• 
PBMC/plasma storage 
 

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Females of childbearing potential will have a serum or urine pregnancy test.  There will also be an AE 
assessment.  During the COVID-19 pandemic, participants will be screened for COVID-19 symptoms 
before coming in for their visit.  
 
Weeks 4 through 10 
Participants will return every 2 weeks (see SOEs, Sections 1.2 and 1.3) for blood draws for: 
• 
PBMC/plasma storage 
• 
Yellow fever neutralizing antibody (week 8 only) 
 
At Week 4, females of childbearing potential will have a serum or urine pregnancy test.  Stool will be 
collected for microbiome study. 
 
There will also be an AE assessment.  During the COVID-19 pandemic, participants will be screened for 
COVID-19 symptoms before coming in for their visit. 
 
Week 12 
Blood will be collected for: 
• 
HIV antibody 
• 
PBMC/plasma storage 
• 
Luminex 
 
Stool and urine will be collected to check for helminths. There will also be an AE assessment.  During the 
COVID-19 pandemic, participants will be screened for COVID-19 symptoms before coming in for their 
visit. 
 
Months 6 through 15 
Participants will return quarterly starting Month 6 through Month 18. 
 
Months 6, 9, 12 and 15 participants will have blood collected for PBMC/plasma storage.   
 
Month 6 will have stool will be collected for microbiome study. During the month 6 timepoint, 
leukapheresis will be performed. Before leukapheresis, females of childbearing potential will have a 
serum or urine pregnancy test.  If a participant is not eligible or unable to tolerate leukapheresis, a large 
volume blood draw (60 cc) will be done.   
 
At the month 12 visit, participants will have stool and urine collected for helminth check. Stool will be 
collected for microbiome study, and blood will be collected for: 
• 
HIV antibody  
• 
Luminex  
• 
PBMC/plasma storage 
 
There will also be an AE assessment.  During the COVID-19 pandemic, participants will be screened for 
COVID-19 symptoms before coming in for their visit. 
 
Month 18 
The month 18 visit will be the final study visit. Stool and urine will be collected for helminth check. Stool 
will be collected for microbiome study. Blood will be collected for: 

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• 
T cell panel 
• 
HIV antibody  
• 
Yellow fever neutralizing antibody 
• 
Luminex  
• 
PBMC/plasma storage  
 
There will also be an AE assessment.  During the COVID-19 pandemic, participants will be screened for 
COVID-19 symptoms before coming in for their visit. 
 
   
8.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
Adverse event means any untoward medical occurrence associated with the use of an intervention in 
humans, whether or not considered intervention-related (21 CFR 312.32 (a)). 
 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of the 
investigator, it results in any of the following outcomes: death, a life-threatening adverse event, 
inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant incapacity 
or substantial disruption of the ability to conduct normal life functions, or a congenital anomaly/birth 
defect. Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they may 
jeopardize the participant and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. Examples of such medical events include allergic bronchospasm 
requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do 
not result in inpatient hospitalization, or the development of drug dependency or drug abuse. 
 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.3.3.1 
SEVERITY OF EVENT 
Toxicity and adverse events will be classified according to Division of AIDS (DAIDS) Table for Grading the 
Severity of Adult and Pediatric Adverse Events, Corrected Version 2.1. A copy of this can be downloaded 
from the DAIDS home page at https://rsc.niaid.nih.gov/sites/default/files/daidsgradingcorrectedv21.pdf 
  
AE/SAE Grading Assignment 
All adverse events will be graded in the following manner: 
• 
Grade 1 (Mild): Events require minimal or no treatment and do not interfere with the 
participant’s daily activities. 
• 
Grade 2 (Moderate): Events result in a low level of inconvenience or concern. Moderate events 
may cause some interference with functioning. 
• 
Grade 3 (Severe): Events interrupt a participant’s usual daily activity and may require systemic 
drug therapy or other treatment. Severe events are usually incapacitating. 

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• 
Grade 4 (Life-threatening): Any adverse drug experience that places the participant, in the view 
of the investigator, at immediate risk of death from the reaction as it occurred (i.e., it does not 
include a reaction that had it occurred in a more severe form, might have caused death). 
• 
Grade 5 (Death) 
 
8.3.3.2 
RELATIONSHIP TO STUDY INTERVENTION 
All adverse events (AEs) must have their relationship to study intervention assessed by the clinician who 
examines and evaluates the participant based on temporal relationship and his/her clinical judgment. 
The degree of certainty about causality will be graded using the categories below. In a clinical trial, the 
study product must always be suspect.  
 
• 
Related – The AE is known to occur with the study intervention, there is a reasonable possibility 
that the study intervention caused the AE, or there is a temporal relationship between the study 
intervention and event. Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study intervention and the AE. 
• 
Not Related – There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study intervention 
and event onset, or an alternate etiology has been established. 
 
8.3.3.3 EXPECTEDNESS  
The principal investigator will be responsible for determining whether an adverse event (AE) is expected 
or unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the event is 
not consistent with the risk information previously described for the study intervention. 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the attention of 
study personnel during study visits and interviews of a study participant presenting for medical care, or 
upon review by a study monitor. 
 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured on the 
appropriate case report form (CRF). Information to be collected includes event description, time of 
onset, clinician’s assessment of severity, relationship to study product (assessed only by those with the 
training and authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs 
occurring while on study must be documented appropriately regardless of relationship. All AEs will be 
followed to adequate resolution. 
 
Any medical condition that is present at the time that the participant is screened will be considered as 
baseline and not reported as an AE. However, if the study participant’s condition deteriorates at any 
time during the study, it will be recorded as an AE.  
 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the event 
at each level of severity to be performed. AEs characterized as intermittent require documentation of 
onset and duration of each episode. 
 
The principal investigator or study staff will record all reportable events with start dates occurring any 
time after informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last 
day of study participation.  At each study visit, the investigator will inquire about the occurrence of 

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AE/SAEs since the last visit.  Events will be followed for outcome information until resolution or 
stabilization. 
 
In Uganda, the sponsor has taken out clinical trials insurance policy to cover compensation for any 
personal injury resulting from participants taking the study medication and/or study procedures, 
provided such personal injury is not due to fault or negligence of the study doctor or his team. 
 
8.3.5 ADVERSE EVENT REPORTING  
Adverse events will be reported to the University of Minnesota Institutional Review Board annually, at 
the time of continuing review, in accordance with their reporting policy. 
 
8.3.6 SERIOUS ADVERSE EVENT REPORTING  
All serious adverse events that meet the University of Minnesota Institutional Review Board’s prompt 
reporting requirements will be reported to the IRB no later than 5 business days.  All SEAs will also be 
reported to the JCRC IRB within 5 business days of awareness.   
 
8.3.7 REPORTING EVENTS TO PARTICIPANTS  
Not applicable. 
 
8.3.8 EVENTS OF SPECIAL INTEREST  
Not applicable. 
 
8.3.9 REPORTING OF PREGNANCY  
Not applicable. 
 
 
8.4 
UNANTICIPATED PROBLEMS 
 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
 
8.4.2  UNANTICIPATED PROBLEM REPORTING  

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The investigator will report unanticipated problems (UPs) to the University of Minnesota Institutional 
Review Board (IRB) and JCRC IRB per the UMN IRB’s and JCRC IRB’s prompt reporting requirements.  
 
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
Not applicable. 
 
9 
STATISTICAL CONSIDERATIONS  
 
9.1 
STATISTICAL HYPOTHESES 
 
• 
Primary Efficacy Endpoint(s):  
 
Null hypothesis: There is no difference in peak yellow fever antibody titer between individuals from the 
US and Uganda. 
Alternative hypothesis: There is a difference in peak yellow antibody titer. 
 
• 
Secondary Efficacy Endpoint(s): 
 
Null hypothesis: There is no difference in baseline LN architecture measures (TZ collagen, FRCn, B cell 
follicles and populations, Tfh cells, and B Cell activating factor) between individuals from the US and 
Uganda. Alternative hypothesis: There are differences in these measures between the two countries.   
 
Null hypothesis: There is no association between baseline measures of LN architecture and rate of 
YFV antibody decay.  Alternative hypothesis: There are associations between these measures and 
rate of antibody decay.  
 
Null hypothesis: Principal components of pathseq data are not associated with country of origin. 
Alternative hypothesis: Some principal components of pathseq data are be associated with country. 
 
Null hypothesis: There are no difference in helminth infection patterns between the US and Uganda. 
Alternative hypothesis: Differences in helminth infections exist between the two countries. 
 
Null hypothesis: Pathseq principal components with strong loadings for infection organisms are not 
associated with baseline measures of LN architecture.  Alternative hypothesis: PC components with 
strong loadings for infectious organisms are associated with baseline LN architecture measures.  
 
Null hypothesis: Presence of helminth infection is not associated with baseline LN architecture 
measures. Alternative hypothesis: Presence of helminth infection is associated with baseline LN 
architecture measures. 
 
Null hypothesis: There is no association between changes in immune function after vaccination and 
measures of LN architecture. Alternative hypothesis: There is an association between change in immune 
function and measures of LN architecture.  
Null hypothesis: There is no association between CD4, CD8 functional variables and CD4 subsets 
and peak YFV antibody titers or rate of antibody decay. Alternative hypothesis: There is an 

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association between CD4, CD8 functional variables and CD4 subsets and peak YFV antibody titers 
and rate of antibody decay. 
Null hypothesis: There is no association between CD4, CD8 functional variables and CD4 subsets 
and peak specific IgM, IgG1, IgG2, IgG3 and IgG4 responses or rate of change. Alternative hypothesis: 
There is an association between CD4, CD8 functional variables and CD4 subsets and peak specific 
IgM, IgG1, IgG2, IgG3 and IgG4 responses or rate of change. 
Null hypothesis: There is no change in immune activation or inflammation measures or CD4 and CD8 
cells after immunization. Alterative hypothesis: There is a change in immune activation or inflammation 
measures or CD4 and CD8 cells after immunization. 
9.2 
SAMPLE SIZE DETERMINATION 
We will enroll 30 individuals from Uganda and 16 individuals from the US. With this sample size our 
study will have more than adequate power to test our primary aim, differences in peak antibody titers 
between countries. Assuming yellow fever antibody titers show a similar range as in our previous 
studies (a standard deviation of 1 log) for both populations we have over 85% power to detect a 
difference in peak antibody titers of 1 log, with a two-sided test with alpha level of 0.05. This 
calculation is based on comparison to a T distribution with 43 degrees of freedom.  
 
Since, in fact, we will be using a Mann-Whitney U test for this comparison we also illustrate that 
this sample size is sufficient using power simulations. For all simulations we assume that the 
Ugandan population will have a mean of 7.89 log titers and approximate standard deviation of 1 
and the Minnesota population has a mean of 8.89 and an approximate standard deviation of 1. One 
thousand iterations were conducted for each simulation scenario. As is illustrated in the table 
below we will have over 84% power for a variety of distributional assumptions 
 
9.3 
POPULATIONS FOR ANALYSES 
For comparisons involving the primary endpoint, YFV titers, we will only include study participants who 
received the YF vaccine. For analysis of secondary endpoints, we will use data from all available study 
participants. 
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
Medians and interquartile ranges will be presented for descriptions of continuous data. 
Percentages will be presented for categorical data.  
Counts of cell frequency and QIA endpoints involving the percent area will be log-transformed and 
linear regression analyses will be conducted where appropriate. If there are no detectable cells or 
there is no staining detected for a large portion of study participants logistic regression modeling 
will be conducted, instead, with presence or absence of the measure being the outcome.  
Distribution 
Uganda parameters 
MN parameters 
Power 
Normal 
µ=7.89, σ=1 
µ=8.89, σ=1 
0.871 
Gamma 
k=62.25, θ=0.13 
k=79.03, θ=0.11 
0.874 
T + shift  
ν= 20, shift=7.89 
ν= 20, shift= 8.89 
0.841 

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Two-sided tests will be conducted for all comparisons. Subjects specific random effects will be 
included in all mixed effects models and permutation p-values will be presented. To control for 
multiple comparisons, a cut-off of alpha=0.01 will be used for determining statistical significance of 
all secondary endpoints. 
Measures which have non-zero coefficients in lasso models will be considered to be associated with 
the outcome. Measures which have non-zero correlation coefficients after applying sparse 
canonical correlation analysis will be considered to be correlated with the outcome measure.  
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
Mann-Whitney U tests will be used to test for differences in peak YFV titer between the US and 
Uganda. Analyses will be stratified by gender to investigate if gender has a major impact on the 
observed differences. No imputation will be conducted if there is missing data. 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
Differences in rate of YFV antibody titer decay will be compared between vaccinated Ugandans and 
Minnesotans using linear mixed effects regression model with measurement time, country, and the 
interaction between measurement time and country as predictors. Baseline LN measures (area with 
collagen, area with desmin staining, frequency and size of B cell follicles, and frequency of Tfh cells) will 
be compared between the two countries using Mann-Whitney U tests.  
 
We will examine the association between vaccine response over time and baseline LN measures using 
linear mixed effects models adjusting for measurement time and gender.  
 
To examine differences in infection patterns between the two countries, we will perform principal 
component analyses on the baseline pathseq data. We will test for differences in infection patterns 
between Uganda and the US populations using logistic regression with country as the outcome and the 
scores for each of the first five principal components as predictors. Differences in helminth infections 
between Ugandans and Minnesotans will be compared using Fisher’s exact test.  
 
We will examine the association between infection history and baseline LN architecture by fitting 
separate linear regression models for each measure of baseline LN architecture with gender and the 5 
pathseq principal component scores as predictors. The association between helminth infections and 
baseline LN architecture measures will be assessed using Mann-Whitney U tests. 
 
To elucidate the role of previous infections on inflammation and activation we will use sparse canonical 
correlation analysis (Reference: Witten, D., Tibshirani, R., & Hastie, T. (2009). A penalized matrix 
decomposition, with applications to sparse principal components and canonical correlation analysis. 
Biostatistics., 10(3), 515-534.) to identify sets of organismal sequences correlated with baseline 
measures of systemic and lymphatic inflammation (plasma cytokines, frequency of TGFß+ and TNF+ cells 
in lymphoid tissues) and immune activation (Ki67+ and CD69 + cells). The association between helminth 
infection and measures of inflammation and activation be assessed using a logistic lasso regression 
model. Measures which have non-zero coefficients in the model will be considered associated with 
helminth infection. 
 

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The association between change in measures of CD8 function (IFN, TNFα, and IL-2) after vaccination 
and measures of LN architecture will be examined by fitting mixed effects linear models using area with 
collagen staining, area with desmin staining, and measurement time and gender as predictors.  
 
The association between immune function after vaccination and measures of inflammation and 
activation will be assessed using separate lasso regression for each measure of immune function 
(antigen specific CD4, CD8 function measures, and CD4 and CD8 subpopulations). Inflammation and 
activation measures which have non-zero coefficients in the model will be considered predictive of 
immune function. 
 
We will examine the impact of measures of immune function on YFV response using separate mixed 
effects linear models with gender, measurement time and measures of immune function (antigen 
specific CD4, CD8 functional variables, or CD4 and CD8 subpopulations) as predictors. Similar models will 
be fit for YF specific IgM, IgG1, IgG2, IgG3 and IgG4 responses. 
 
The impact of vaccination on measures of inflammation and activation be assessed using mixed effects 
repeated measures model with gender, measurement time, and vaccination status as predictors. To 
assess the impact of vaccination on CD4 and CD8 cells in LT similar models will be fit, but only gender 
and vaccination status (measurement taken before or after vaccination) will be used as predictors. 
 
Missing data will not be imputed and a significance level of 0.01 will be used for secondary analyses due 
to the large number of hypotheses to be tested. The results from all of these investigations will be 
reported so that the reader can assess the effect of multiple hypothesis testing. 
 
9.4.4 SAFETY ANALYSES 
Any AEs will be reported annually in a study report that will document AEs and completeness of data. 
This report will include the total number of AEs for each cohort and the total number of participants in 
each cohort that experience an AE.  AEs will be summarized in terms of severity and relatedness and the 
number of participants experiencing SAEs will also be reported for each cohort. A line listing will also be 
included in this report. 
 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
Demographic baseline characteristics will be compared between the MN and Ugandan populations using 
Mann-Whitney U tests (continuous outcomes) or Fisher's exact test (categorical outcomes). 
 
9.4.6 PLANNED INTERIM ANALYSES  
N/A- There is no planned interim analysis. 
 
9.4.7 SUB-GROUP ANALYSES 
In addition to including gender, we will assess how additional clinical factors (such as age and other 
demographic variables) influence immune response to vaccination by including relevant clinical factors 
in to our models. 
 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
Individual participant data will not be listed by measure and time point. 
 
9.4.9 EXPLORATORY ANALYSES 

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The association between change in the CD4 and CD8 T cell populations after vaccination and measures 
of LN architecture will be examined by fitting mixed effects linear models estimating T cells populations 
using area with collagen staining, area with desmin staining, and measurement time and gender as 
predictors.  
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO 
PARTICIPANTS 
A signed consent form will be obtained from the participant. The consent form will describe the purpose 
of the study, the procedures to be followed, and the risks and benefits of participation. A copy of the 
consent form will be given to the participant, and this fact will be documented in the participant’s 
record. 
 
Research staff will ensure that candidates understand all elements of the consent form by addressing 
questions posed during the consent procedure and by asking for verbal confirmation that the candidate 
has no additional questions and verbally understands the purpose of the study, study intervention, basic 
procedures, risks, and that participation is voluntary. Participants will be given as much time as they 
need to read and understand the consent form. 
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continues throughout the individual’s study participation. Consent forms will be Institutional 
Review Board (IRB)-approved and the participant will be asked to read and review the document. The 
investigator will explain the research study to the participant and answer any questions that may arise. 
A verbal explanation will be provided in terms suited to the participant’s comprehension of the 
purposes, procedures, and potential risks of the study and of their rights as research participants.  
Participants will have the opportunity to carefully review the written consent form and ask questions 
prior to signing. The participants should have the opportunity to discuss the study with their family or 
surrogates or think about it prior to agreeing to participate. The participant will sign the informed 
consent document prior to any procedures being done specifically for the study. Participants must be 
informed that participation is voluntary and that they may withdraw from the study at any time, without 
prejudice. A copy of the informed consent document will be given to the participants for their records. 
The informed consent process will be conducted and documented in the source document (including the 
date), and the form signed, before the participant undergoes any study-specific procedures. The rights 
and welfare of the participants will be protected by emphasizing to them that the quality of their 
medical care will not be adversely affected if they decline to participate in this study. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Insufficient compliance to protocol requirements 

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• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
 
10.1.3 CONFIDENTIALITY AND PRIVACY  
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their 
staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover testing of 
biological samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, 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.  
 
All research activities will be conducted in as private a setting as possible. 
 
The study monitor, other authorized representatives of the sponsor, representatives of the Institutional 
Review Board (IRB), or regulatory agencies may inspect all documents and records required to be 
maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) 
and pharmacy records for the participants in this study. The clinical study site will permit access to such 
records. 
 
The study participant’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as 
long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. 
 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be transmitted to and stored at the University of Minnesota. This will not include the participant’s 
contact or identifying information. Rather, individual participants and their research data will be 
identified by a unique study identification number. The study data entry and study management 
systems used by clinical sites and by University of Minnesota research staff will be secured and 
password protected. At the end of the study, all study databases will be de-identified and archived at 
the University of Minnesota. 
 
Certificate of Confidentiality  
To further protect the privacy of study participants, a Certificate of Confidentiality will be issued by the 
National Institutes of Health (NIH).  This certificate protects identifiable research information from 
forced disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, legislative, 
or other proceeding, whether at the federal, state, or local level. By protecting researchers and 
institutions from being compelled to disclose information that would identify research participants, 
Certificates of Confidentiality help achieve the research objectives and promote participation in studies 
by helping assure confidentiality and privacy to participants. 
 
10.1.4 KEY ROLES AND STUDY GOVERNANCE 
 
Principal Investigator 
Timothy W Schacker, MD 
University of Minnesota 

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MMC 250 
516 Delaware Street 
Minneapolis, MN 55455 
Phone: 612-624-9955 
E-Mail: schacker@umn.edu 
 
10.1.5 SAFETY OVERSIGHT 
This study will use local Independent Safety Monitor (ISM) for JCRC and UMN.  The ISMs will be 
independent physicians with relevant expertise and free of conflicts of interest whose primary 
responsibility will be to provide independent monitoring of the study. Participation will be for the 
duration of the study. The role will include ongoing review for clinical data for all participants, and also 
the following items:  
1. Determine frequency of safety review needed throughout the study (individually, if applicable) 
2. Give input on the protocol and procedures  
This role requires both ISMs to review all adverse events and thoroughly investigate any serious and 
unexpected events. Both ISMs will be able to readily access participant records in real time. The study 
team shall notify both ISMs via email of any SAE’s immediately after becoming aware of any such event.  
The ISMs and study team will review the case as soon as possible, and where appropriate examining the 
participant in question. The ISMs will produce a short report on each SAE. 
Every year the study team will provide both ISMs a summary report of all the adverse events.  The ISMs 
will produce a short report on your review of these events. 
After the first five participants are enrolled, the study team will give both ISM a report of all the adverse 
events and participant data.  The ISMs will review the data for any trends or events that may be clinically 
significant, and provide a short one page summary report. If no clinically significant items are found 
after initial review, the study team will then continue to send data to review every one year.  If the ISMs’ 
summary report finds items of clinical significance, the frequency of the medical review will be altered 
as the ISMs see fit.  
 
10.1.6 CLINICAL MONITORING 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of 
the trial is in compliance with the currently approved protocol/amendment(s), with International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory 
requirement(s).  
 
• 
The CTSI clinical trial monitors will conduct on-site monitoring at the University of Minnesota 
and virtual monitoring for JCRC.  The type of monitoring (e.g., on-site, centralized), frequency 
(e.g., early, for initial assessment and training versus throughout the study), and extent (e.g., 
comprehensive (100% data verification) versus targeted or random review of certain data (less 

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than 100% data verification or targeted data verification of endpoint, safety and other key data 
variables)), and the distribution of monitoring reports. 
• 
Per Uganda National Counil for Science and Technology, the JCRC clinical trial monitor will 
monitor all of the JCRC informed consents. 
• 
Clinical trial monitoring will also be conducted by the Ugandan Regulatory Authority. 
 
10.1.7 QUALITY ASSURANCE AND QUALITY CONTROL 
Each clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion by clinical trial monitors.  
 
Quality control (QC) procedures will be implemented beginning with the data entry system and data QC 
checks that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated to the site(s) for clarification/resolution. 
 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is 
conducted and data are generated and biological specimens are collected, documented (recorded), and 
reported in compliance with the protocol, International Conference on Harmonization Good Clinical 
Practice (ICH GCP), and applicable regulatory requirements.  
 
The investigational site will provide direct access to all trial related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities. 
 
10.1.8 DATA HANDLING AND RECORD KEEPING  
 
10.1.8.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. 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.   
 
Hardcopies of the study visit worksheets will be provided for use as source document worksheets for 
recording data for each participant enrolled in the study.  Data recorded in the electronic case report 
form (eCRF) derived from source documents should be consistent with the data recorded on the source 
documents.  Hardcopies of study materials will be kept behind lock and key and only accessible to 
authorized personnel.   
 
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse reactions 
data) and clinical laboratory data will be entered into REDCap, a 21 CFR Part 11-compliant data capture 
system provided by the University of Minnesota. The data system includes password protection and 
internal quality checks, such as automatic range checks, to identify data that appear inconsistent, 
incomplete, or inaccurate. Clinical data will be entered directly from the source documents. The data 
center at University of Minnesota has QA procedures in place that are easily integrated into REDCap. 
These systems can operate on de-identified data and include functionality that goes beyond what can be 
accomplished using REDCap, such as data quality checks that involve data from multiple CRFs. 

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REDCap uses a MySQL database via a secure web interface with data checks used during data entry to 
ensure data quality. REDCap includes a complete suite of features to support HIPAA compliance, 
including a full audit trail, user-based privileges, and integration with the institutional LDAP server. The 
MySQL database and the web server will both be housed on secure servers. Access to the study's data in 
REDCap will be restricted to the members of the study team by username and password. 
 
 
10.1.8.2 STUDY RECORDS RETENTION  
Per NIH requirements, study documents should be retained for a minimum of 3 years from the date of 
Federal Financial Report (FFR) submission. These documents should be retained for a longer period, 
however, if required by local regulations. No records will be destroyed without the written consent of 
the sponsor, if applicable. It is the responsibility of the sponsor to inform the investigator when these 
documents no longer need to be retained. 
HIPAA authorizations are to be retained for 6 years from the date of its creation or the date when it was 
last in effect, whichever is later. 
All study documents at JCRC in Uganda will be stored for a minimum of 5 years per Uganda National 
Council for Science and Technology (UNCST) and National Drug Authority (NDA) guidelines.  
 
10.1.9 PROTOCOL DEVIATIONS  
A protocol deviation is any noncompliance with the clinical trial protocol, International Conference on 
Harmonization Good Clinical Practice (ICH GCP), or Manual of Procedures (MOP) requirements. The 
noncompliance may be either on the part of the participant, 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 GCP:  
• 
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 investigator 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.  All deviations must be addressed in study source documents. 
Protocol deviations must be sent to the reviewing Institutional Review Board (IRB) per their policies. The 
site investigator is responsible for knowing and adhering to the reviewing IRB requirements. Further 
details about the handling of protocol deviations will be included in the MOP. 
 
10.1.10 
PUBLICATION AND DATA SHARING POLICY 
This study will be conducted in accordance with the following publication and data sharing policies and 
regulations: 
 
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has access to the 
published results of NIH funded research. It requires scientists to submit final peer-reviewed journal 
manuscripts that arise from NIH funds to the digital archive PubMed Central upon acceptance for 
publication. 
 

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This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-
reviewed journals.   
 
In addition, this study will comply with the NIH Genomic Data Sharing Policy, which applies to all NIH-
funded research that generates large-scale human or non-human genomic data, as well as the use of these 
data for subsequent research. Large-scale data include genome-wide association studies (GWAS), single 
nucleotide polymorphisms (SNP) arrays, and genome sequence, transcriptomic, epigenomic, and gene 
expression data. 
 
10.1.11 
CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical.  Therefore, any actual conflict of interest of persons who have a role in the design, 
conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, 
persons who have a perceived conflict of interest will be required to have such conflicts managed in a 
way that is appropriate to their participation in the design and conduct of this trial.  The study 
leadership in conjunction with the National Institute of Allergy and Infectious Diseases has established 
policies and procedures for all study group members to disclose all conflicts of interest and will establish 
a mechanism for the management of all reported dualities of interest.   
10.2 ABBREVIATIONS 
 
AE 
Adverse Event 
ANCOVA 
Analysis of Covariance 
BX 
Biopsy 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
FRCn 
Fibroblastic reticular cell network 
GALT 
Gastric Associated Lymphatic Tissue 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 

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HIPAA 
Health Insurance Portability and Accountability Act  
IA 
Immune Activation 
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonization  
ICMJE 
International Committee of Medical Journal Editors 
IHC  
Immunohistochemistry  
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
LN 
Lymph Node 
LT 
Lymphoid Tissue 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PBMCs 
Peripheral Blood Monocytes 
PI 
Principal Investigator 
PRA 
Plaque Reduction Assay 
QA 
Quality Assurance 
QC 
Quality Control 
QIA 
Quantitative Image Analysis 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
Tfh 
T Follicular Helper 
TZ 
T cell Zone 
UP 
Unanticipated Problem 
US 
United States 
YF 
Yellow Fever 
YFV 
Yellow Fever Vaccine 
 
 

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