Human Immune Responses to Yellow Fever Vaccination
Phase: PHASE4
Conditions: Yellow Fever
Sponsor: Emory University

Study Protocol 
 
 
 
Human Immune Responses to Yellow Fever Vaccination 
 
 
Protocol Date: February 15, 2023 
 
 
NCT00694655 
 

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Human Immune Responses to Yellow Fever Vaccination 
(YF Immune Response)
Principal Investigator: 
Srilatha Edupuganti, MD, MPH
Hope Clinic of Emory University
Decatur, GA 30030
Funding: 
NIH, NIAID, Cooperative Centers for Human Immunology, 
(2U19AI057266-16, PI: Ahmed)
Co-Investigators at 
Emory Vaccine Center:     Rafi Ahmed, PhD
Donald McGuire, PhD
Sakshi Malik, PhD
External Laboratory 
Collaborators:
Shane Crotty, PhD
Associate Professor, Division of Vaccine Discovery
La Jolla Institute for Allergy & Immunology (LIAI)
Bali Pulendran, PhD
Professor of Microbiology and Immunology 
Stanford University

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TABLE OF CONTENTS
Page
1
Background Information and Scientific Rationale.................................................................4
1.1
Background Information............................................................................................4
1.2
Rationale for including non-travelers and travelers...................................................6
1.3
Scientific Rationale ...................................................................................................7
1.4
Potential Risks and Benefits .....................................................................................8
2
Objectives.............................................................................................................................9
3
Study Design ........................................................................................................................9
4
Study Enrollment ................................................................................................................10
4.1
Subject Inclusion Criteria ........................................................................................10
4.2
Subject Exclusion Criteria.......................................................................................10
4.3
Randomization Procedures.....................................................................................11
5
Study Intervention...............................................................................................................11
5.1
Study Product Description ......................................................................................11
5.2
Dosage, Preparation and Administration of Study Product.....................................11
5.3
Accountability Procedures for the Study Vaccine ...................................................12
6
Study Procedures ...............................................................................................................12
6.1
Screening Visit........................................................................................................12
6.2
Pre-Vaccination Visit...............................................................................................12
6.3
Enrollment/Vaccination Visit (Day 0).......................................................................12
6.4
Leukapheresis Procedure.......................................................................................13
6.5
Follow-up Visits.......................................................................................................13
6.6
Final Study Visit ......................................................................................................14
7
Safety Assessment.............................................................................................................15
7.1
Adverse Events and Serious Adverse Events ........................................................15
7.2
Assessment of Severity of Adverse Events ............................................................16
7.3
Relationship to Study Intervention ..........................................................................17
8
Laboratory...........................................................................................................................18
8.1
Laboratory Assays or Procedures...........................................................................18
8.2
Specimen Preparation, Handling, and Shipping .....................................................20
9
Statistical Considerations ...................................................................................................20
10
Source Documents and Access to Source Data/Documents .............................................20
11
Quality Control and Quality Assurance...............................................................................20
12
Ethics/Protection of Human Subjects .................................................................................20
13
Data handling and Record Keeping....................................................................................21
14
Publication Policy................................................................................................................21
15
Literature References .........................................................................................................21
16
Appendices.........................................................................................................................23

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PROTOCOL SUMMARY
Title: YFV Immune Responses: Human Immune responses to Yellow Fever Vaccination
Population: Healthy males and females between 18 to 45 years of age
Number of sites: 1
Study Duration: 5 years
Number of subjects: 250 adults, ages 18-45. We may need to screen up to 1000 adults to 
enroll a total of 250 participants due to screen failures) 
Subject Participation Duration: 12 months
Description of Intervention: YF-17D (YF-VAX®;Yellow Fever Vaccine)
Objectives:
Primary:
1.
Isolation and characterization of yellow fever vaccine-specific adaptive immune 
responses: characterize the magnitude and quality of yellow fever virus-specific T cell 
responses, antibody secreting cells and memory B cells.
2.
Determine the signatures of innate immune responses: cytokines, chemokines, dendritic 
cells and microarray analyses on peripheral blood mononuclear cells.
Secondary:
1.
Compare the magnitude and kinetics of T and B cell responses, antibody secreting cells 
and memory B cells at various timepoints. 
2.
Isolation and characterization, and phenotypic analysis of Epstein-Barr virus, 
cytomegalovirus and yellow fever virus-specific CD8 T cells.
Description of Study Design:
Healthy adults (18-45 years of age) with no previous vaccination history for flaviviruses and no 
contraindications will be vaccinated using YF-17D. Serial blood specimens will be collected from 
participants up to 1 year after YF-17D administration. Peripheral blood mononuclear cell 
isolation and sorting of yellow fever virus tetramer-specific population will be performed. 
Subsets of participants will undergo leukapheresis and lymph node fine needle aspirates. 

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1
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE
1.1
Background Information
Yellow fever (YF) is a viral disease that is transmitted to humans through the bite of an infected 
mosquito.  Yellow fever is a life-threatening infection that can result in hepatitis, renal failure and 
coagulation abnormalities, and in severe cases, death1. Yellow fever was a major public health 
threat in the colonial United States in the 18th and 19th centuries.
Yellow fever is endemic in over 40 countries, and approximately 125 countries require proof of 
vaccination for entry by travelers at risk.  An estimated 200,000 cases of yellow fever occur 
annually in South America and Africa, making it an important vaccine-preventable disease 
among travelers to endemic areas2. Yellow fever can be prevented by vaccination with the 
Yellow Fever Vaccine, YF-VAX® (made from the 17D strain of YFV).  Currently, the Centers for 
Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend the 
Yellow Fever Vaccine for people aged 9 months to 59 years who are traveling to or living in a 
yellow fever endemic area2.  
Many flaviviruses, including yellow fever virus (YFV), dengue virus (DEN), West Nile virus, 
Japanese encephalitis virus and tick-borne encephalitis (TBE) virus are important human 
pathogens.  Many flaviviruses are transmitted by hematophagous arthropods (mosquitoes and 
ticks). Indeed, dengue, yellow fever, the TBE virus complex and Japanese encephalitis globally 
represent the most important mosquito-borne diseases causing extensive morbidity and 
mortality around the world1. Even within the United States (US), the dramatic spread of West 
Nile virus infection over the past few years has provided both the public and public health 
authorities with a vivid example of the present (and potential future) threats posed by emerging 
zoonotic infections. 
A very interesting, but unexplained aspect of flavivirus biology is how infection with different 
members of the same virus family can lead to such diverse types of host-virus interactions and 
variable disease outcomes.  For example, YFV infection can be fatal, but if the infected host 
survives, long-term protective immunity is seen1, 3, 4. Alternatively, DEN causes an acute 
infection with associated acute disease manifestations. However, more severe disease 
outcomes are observed following secondary infection with a distinct serologic type of DEN1, 3. 
Understanding why the human immune system can successfully contain one flavivirus infection, 
but not another is both a fascinating scientific enigma in human immunology and a topic with 
substantial practical importance to public health. Given the great global public health threats 
posed by epidemic and emerging flavivirus infections, and the need to define the biological 
basis of successful induction and maintenance of protective immunity by vaccination, 
elucidation of the immunologic mechanisms underlying the generation and maintenance of 
protective immunity to the Yellow Fever Vaccine should be extremely useful. Furthermore, 
definition of the attributes of such a highly effective vaccine should help expedite the 
development and evaluation of new and/or improved vaccines to prevent important prevalent 
and emerging infectious diseases.
YF-17D is considered to be one of the safest and most effective viral vaccines ever developed. 
This vaccine is known to stimulate broad-spectrum immune responses, including cytotoxic T 
cells, and Th1 and Th2 responses, as well as neutralizing antibody titers that can persist for up 
to 30 years, after a single vaccination5, 6.  Despite the great success of this empiric vaccine, 

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there has been relatively little understanding of the mechanisms by which YF-17D induces such 
robust protective immune responses.  In our past and current U19 grant we have begun to 
elucidate the innate and adaptive signatures induced by YF-17D in healthy adults between the 
ages of 18 and 457, 8 and T cell memory responses9-12.  
We hope to apply the best contemporary methods in immunology, genomics, and proteomics to 
characterize in detail a successful immune response to YF-17D.  This characterization should 
identify new immunologic predictors that could serve as surrogates for future vaccine efficacy 
studies.  In addition, these findings could guide development of a safer yellow fever vaccine (or 
the derivation of safer alternative vaccination regimens using the currently available vaccine).
The goal of this study is to use the live attenuated Yellow Fever Vaccine (YF-VAX®, Sanofi-
Pasteur) as a safe and effective model for viral infection to understand human immune 
response to viral antigens.  Yellow fever virus vaccine is the viral infection model that we have 
chosen for the following reasons: 

The yellow fever live, attenuated 17D vaccine strain is one of the most efficacious 
vaccines available and has been in use since the 1930s.  

Since most of the US population is not exposed to YFV, immunization of adult 
participants with the 17D vaccine strain allows for examination of the innate immunity 
and the naïve T and B cell response in humans during primary infection and the 
subsequent development of memory T cells after resolution of the primary infection. 

YF-17D vaccination induces long-term immunity that lasts for decades. By studying how 
the human immune system responds to an effective vaccine such as the Yellow Fever 
Vaccine, we hope to learn more about the normal functioning of the immune system so 
that it might be possible to design new, more effective vaccines to prevent important 
infectious diseases. Therefore, this vaccine can serve as an ideal model to help us 
decipher the properties that make a vaccine effective. 

YF-17D vaccination leads to limited infection without causing the disease.
1.2
Rationale for including non-travelers and travelers
The Yellow Fever Vaccine (YF-17D; YF-VAX®) is a live, attenuated vaccine that has been used 
for prevention of yellow fever since 1937 with over 500 million doses administered2. There is no 
treatment available for yellow fever and the only yellow fever vaccine currently licensed for use 
in the US is YF-VAX®, manufactured by Sanofi Pasteur (Swiftwater, Pennsylvania). According to 
the Advisory Committee on Immunization Practices (ACIP) guidelines, the Yellow Fever Vaccine 
is recommended for persons who are aged 9 months and older who are traveling to or living in 
areas at risk for YFV activity or traveling to a country with an entry requirement for vaccination. 
In some cases, people younger than 9 months who are at increased risk may receive the yellow 
fever vaccine. Laboratory personnel who might be exposed to yellow fever virus or vaccine virus 
may also need YF-VAX®13. We plan to enroll adults between 18 to 45 years of age, who are 
traveling to yellow fever endemic areas, laboratory workers and also those who don’t have 
imminent travel plans.
The Yellow Fever Vaccine is a safe and effective vaccine. Adverse events (AEs) seen with YF-
17D are typically mild and may include headache, myalgias, low-grade fever and discomfort at 
the injection site. These mild AEs occur in about 25% of vaccinees13. There have been rare 
reports of severe AEs. Specifically, the yellow fever vaccine associated neurotropic disease 

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(YF-AND), and yellow fever vaccine-associated viscerotropic disease (YF-AVD). YF-AND may 
include post-vaccinal encephalitis, Guillain–Barré syndrome, and autoimmune disease with 
central or peripheral nervous system involvement. YF-AVD is a disease that clinically resembles 
naturally acquired infection and may include a spectrum of disease severity, from moderate 
illness with focal organ dysfunction to severe disease with overt multiple organ system failure 
and death. The risk factors for YF-AVD are advanced age and thymus dysfunction2, 5, 6, 13.
AEs seen with administration of the Yellow Fever Vaccine are noted on the Yellow Fever 
Vaccine Information Sheet (VIS) published by the CDC. All participants will be required to read 
the most current VIS prior to being vaccinated (https://www.cdc.gov/vaccines/hcp/vis/vis-
statements/yf.html).  A review of AEs reported to the U.S. Vaccine Adverse Event Reporting 
System (VAERS) from 2000 to 2006 was published by the CDC2. It showed that the overall 
reporting rate (for all ages) for anaphylaxis was 1.8 per 100,000 doses. YF-AND was 0.8 per 
100,000 doses and YF-AVD was 0.4 per 100,000 doses.  The reporting rates were higher for 
vaccinees aged > 60 years – 1.8 and 1.4 per 100,000 doses for YF-AND and YF-AVD, 
respectively. In general, all AEs were higher in males than females. Based on the above data, 
and discussions with the Yellow Fever Vaccine experts at the CDC, we feel that the overall risk 
of a serious adverse event (SAE) for participants we plan to recruit between 18 to 45 years old 
will be low (0. 4 to 0.8 cases per 100,000 doses).
This study plans to recruit both travelers and non-travelers to yellow fever endemic areas for 
participation. International travel has been severely restricted in the US due to the COVID-19 
pandemic which began in January 2020 and remains ongoing, significantly shrinking the pool of 
potential traveler participants. We are restricting the maximum age of our participants to 45 
years (age cut off by the CDC is 59 years) and we plan to select healthy adults who meet the 
inclusion/exclusion criteria to minimize any potential yellow fever vaccine-related SAEs (see 
section 4). To meet the planned enrollment goals of this study (funded via the NIH U19 grant 
mechanism), we need to be inclusive of both travelers and non-travelers. It is also worth noting 
that colleagues at other universities who conduct similar studies—since the Yellow Fever 
Vaccine is a model vaccine—use the approach of including non-travelers. Much of the 
recruitment will still focus on travelers since they will have the greatest interest in participation.  
1.3
Scientific Rationale
To better understand the innate and adaptive immune differentiation of CD8 T cells in response 
to a virus, we have used the YF-17D human model previously. In these studies, we:
1.
Have identified a combination of surrogate markers that identifies CD8 effectors7 and a 
YFV-specific CD8 T cell epitope in HLA-A2 specific individuals and characterized it 
extensively8.
2.
Plan to profile YFV-specific CD8 T cells at different stages of the response post-
vaccination.  We plan to identify genes that are differentially expressed in these cells and 
analyze how the pattern of gene expression changes as cells transit from effector and 
memory cell stages. The surrogate markers and the immunodominant epitope thus far 
identified will enable physical separation of this cell population. We also plan to profile 
these cells using microarray technology8.  We plan to determine the magnitude and 
quality of YFV-specific T cell responses and the genetic signatures of effector CD8 T 
cells.

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In addition, during activation of antigen specific T cells in response to YF-17D, CD8 T 
cells dramatically alter their expression of CD45 isoforms.  However, unlike canonical 
CD45RO+ CD45RA- memory cells the bulk of the antigen specific CD8 T cells transition 
to a CCR7- CD45RA+ phenotype similar to the “TEMRA” population that accumulates in 
some elderly individuals.  Unlike the TEMRA cells, these antigen specific cells retain 
proliferative capacity have the characteristics of effector memory cells.  To date, little is 
known of the regulation of CD45 isoform expression nor its role in phenotype of effector 
and memory CD8 T cells.  Better understanding of the role and regulation of this 
abundant phosphatase could allow for improved understanding of the requirement to 
induce effective long term CD8 memory in response to vaccine change. 
3.
Plan to determine the output of naïve T cells from thymus and determine the telomere 
length in T cells.
4.
Plan to study the antibody secreting cells and memory B cell responses post-
vaccination.
5.
Plan to study the epigenetics, specifically, the methylation and demethylation patterns of 
yellow fever specific CD8 T cells as they move from naïve to effector to memory cells 
after vaccination. To accomplish this goal, we plan to perform leukapheresis of 
participants who are positive for the HLA-A202 allele.
6.
Plan to study the draining axillary lymph node cells for germinal center reactivities after 
vaccination, in particular CD4+ T follicular cell, and their functions. 
7.
Assays proposed in Dr. Crotty’s lab in La Jolla, CA:  
-
Yellow fever-specific T-B cell co-culture assay in vitro to measure the B cell helper ability 
of the YFV-specific CD4 T cells
-
YFV-specific T cell proliferation in vitro
-
YFV specific cytokine/chemokine production in vitro
-
Phenotypical characterization of YFV-specific CD4 T cells by Flow Cytometry. This 
analysis also requires HLA typing of the donors.
1.4
Potential Risks and Benefits
1.4.1
Potential Risks
YF-VAX® is an FDA approved vaccine that is routinely recommended for persons 9 months of 
age or older who are traveling or living in a country where yellow fever is endemic. However, 
there are some risks associated with YF-VAX®. Mild reactions include soreness, redness, and 
swelling at the injection site, mild headache, muscle aches and mild fever and in up to 25% of 
the vaccines 5 to 10 days after vaccination. Severe reactions to the vaccine have been reported 
but are extremely rare. (Please refer to the Vaccine Information Sheet and Section 1.2).

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1.4.2
Known Potential Benefits
Currently, CDC and WHO recommend vaccination for persons 9 months of age or older who are 
traveling to or living in a yellow fever endemic area13.  YF-VAX® is highly efficacious and is 
protective from yellow fever virus infection, which is potentially life-threatening.
2
OBJECTIVES
2.1
Primary:
1.
Isolation and characterization of yellow fever vaccine-specific adaptive immune 
responses: characterize the magnitude and quality of yellow fever virus-specific T 
cell responses, antibody secreting cells and memory B cells.
2.
Determine the signatures of innate immune responses: cytokines, chemokines, 
dendritic cells and microarray analyses on peripheral blood mononuclear cells.
2.2
Secondary:
1.
Compare the magnitude and kinetics of T and B cell responses, antibody secreting 
cells and memory B cells at various timepoints. 
2.
Isolation and characterization, and phenotypic analysis of Epstein-Barr virus, 
cytomegalovirus and yellow fever virus-specific CD8 T cells.
3
STUDY DESIGN
This is an observational study of immune responses to YF-17D vaccination in healthy adults.  
The study will be conducted at the Hope Clinic of Emory Vaccine Center. 
A total of 250 healthy participants (18-45 years of age) will be enrolled into four study arms: A, 
B, C & D. Arm enrollment is determined by Human Leukocyte Antigen (HLA) type, current 
needs of the lab and/or willingness to participate in sampling procedures. 
All participants will receive YF-17D on Day 0 at the FDA-approved dose and route of 
administration. Post-vaccination procedures are determined by arm assignment. 
The study arms are as follows:

Arm A (n = 200): HLA-A202+– post-vaccination blood draws 

Arm B (n = 10): HLA-A202+ – post-vaccination blood draws and leukapheresis 

Arm C (n = 20): HLA-A202+ – post-vaccination blood draws and fine needle aspirate 

Arm D (n = 20): HLA-A202– – post-vaccination blood draws 
In general, follow up visits will occur on Days 3, 7, 14, 21, 28, 90, 180, and 360 post-
vaccination. 
Please see Study Schema in Appendix A for detailed flow chart of study Arms A-D.

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4
STUDY ENROLLMENT 
4.1
Subject Inclusion Criteria
1.
Able to understand and give informed consent
2.
Age 18-45 years
3.
Participant agrees not to take any live vaccines 30 days before or after (14 days for 
inactivated, including COVID-19 vaccination) yellow fever vaccination
4.
Women of childbearing potential must agree to use effective birth control throughout the 
duration of the study.  A negative urine pregnancy test must be documented prior to 
vaccination.   Participants who have a history of surgical sterilization or post-menopausal 
status >1 year, are not required to have a pregnancy test.
4.2
Subject Exclusion Criteria
1.
Prior receipt of a yellow fever vaccine 
2.
Lived in a country/area which is endemic for yellow fever
3.
Travel to country/area which is endemic for yellow fever. Subject to investigator 
discretion
4.
History of previous yellow fever, West Nile, Dengue, St. Louis encephalitis, Japanese 
encephalitis vaccination or infection
5.
Any history of allergy to eggs, chicken or gelatin or to any previous vaccine
6.
A history of a medical condition resulting in impaired immunity (such as HIV infection, 
cancer, particularly leukemia, lymphoma, use of immunosuppressive or antineoplastic 
drugs or X-ray treatment). Persons with previous skin cancers or cured non-lymphatic 
tumors are not excluded from the study
7.
History of HIV infection
8.
Active Hepatitis B or Hepatitis C infection
9.
COVID-19 infection in the last 30 days. Symptoms of COVID-19 must be completely 
resolved before yellow fever vaccine receipt.
10. History of any chronic medical conditions that are considered progressive (ex, diabetes, 
heart disease, lung disease, liver disease, kidney disease, gastrointestinal diseases 
and uncontrolled hypertension). Use of systemic immunosuppressive medications (ex, 
prednisone) for 2 weeks or more in the past 3 months
11. History of excessive alcohol consumption, drug abuse, psychiatric conditions, social 
conditions or occupational conditions that in the opinion of the investigator would 
preclude compliance with the trial
12. Thymus gland problems (such as myasthenia gravis, DiGeorge syndrome, thymoma) or 
removal of thymus gland or history of autoimmune disorder

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13. Recipient of a blood products or immune globulin product within 42 days of the 
vaccination visit.  Participants who received COVID mAbs for treatment are not 
excluded
14. Pregnant women and nursing mothers or women who are planning to become pregnant 
for the duration of the study
15. Any condition in the opinion of the investigator that would interfere with the proper 
conduct of the trial
4.3
Randomization Procedures
There is no randomization in this study. The arm to which a subject is assigned to is determined 
by their HLA type, current lab needs and/or willingness to participate in sampling procedures.  
5
STUDY INTERVENTION
5.1
Study Product Description
YF-VAX is manufactured by Sanofi Pasteur as one-dose vials will be purchased from the 
manufacturer. Vaccine will be stored at the Emory Investigational Drug service between 2 to 8oC 
per the manufacturer’s instructions. 
5.2
Dosage, Preparation and Administration of Study Product
YF-VAX will be stored, prepared and dispensed from the Hope Clinic Investigational Drug 
Services (IDS) – Hope Clinic Satellite Location. 
The vaccine will be administered by a nurse at the Hope Clinic with a 5/8th to ¾ inch long needle 
via subcutaneous injection.  
5.3
Accountability Procedures for the Study Vaccine
The IDS pharmacist will maintain accurate drug accountability logs which will be kept at the IDS 
pharmacy until the completion of the study. Upon completion of the study, a final drug 
accountability will be done.  
6
STUDY PROCEDURES
6.1
     Screening Visit
When a potential participant presents for the screening visit, the informed consent will be 
reviewed, and all questions answered. Once the subject signs the informed consent, a study 
participant number will be assigned.  Eligibility will then be determined for each participant 
based primarily on information gathered during the screening visit. Procedures conducted 
during the screening visit may include:

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
Collection of medical history

Collection of current medications  

Physical exam

Vital signs

Blood sample collection

HLA typing (if not previously known)
At this visit, if the participant reports having received a vaccination against COVID-19 in the past 
14 days or had a COVID-19 infection in the past 30 days, the enrollment visit will be delayed. All 
symptoms following a COVID-19 infection must be resolved before the enrollment visit.
6.2
Enrollment/Vaccination Visit (Day 0)
This visit will serve as an enrollment and vaccination visit for all participants. This visit may 
occur on the same day as the Screening Visit if the participant’s HLA type is known prior to 
Screening. At this visit, the following activities may be performed:

Physical exam

Vital signs

Collection of current medications

Blood sample collection

Urine pregnancy test (for women of childbearing potential)

AE collection (any AE present prior to vaccination will be considered medical history)

Vaccine administration

Reactogenicity symptom diary distribution 
Prior to vaccine administration, participants will be asked to read the Vaccine Information Sheet 
for the Yellow Fever Vaccine per CDC guidelines. After vaccination, participants will be 
observed for a minimum of 20 minutes for any immediate hypersensitivity reactions and will be 
given the International Certificate of Vaccination. 
6.3
Follow-up Visits
The follow-up schedule that a participant follows will be dictated their Arm assignment and 
current needs of the lab (designated as optional visits). Please see Tables 1 and 2 for a full 
Schedule of Activities (SOA). The following activities may take place at follow-up visits.

Review of current medications

Blood Sample Collection

Collection of Solicited AEs

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6.4
Leukapheresis Procedure
Participants in Arm B will undergo this procedure on either Day 14 or 28. Collection of CD8 T 
Cells via leukapheresis will be performed in accordance with the standard practices of the 
Emory University Hospital Hemapheresis Center. Typically, in this procedure, approximately 12 
liters of blood will be processed over about 3 to 4 hours using peripheral veins for venous 
access. Samples will be sent from the Hemapheresis Center to the Emory Vaccine Center Labs.
6.5
Fine Needle Aspirate Procedure
Participants in Arm C will undergo this procedure on either Day 14 or 28. Tissue sampling of an 
axillary lymph node will be carried out percutaneously by fine needle aspiration (FNA). This 
procedure will be performed in accordance with the standard practices of the Winship Cancer 
Center by Emory Radiology Department providers. The procedure involves tissue retrieval with 
a needle under sonographic guidance. Approximately 2-4 passes will be made to retrieve 
cytologic material. Samples will be sent from Winship to the Emory Vaccine Center Labs.

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6.6
Schedule of Activities
Table 1. Schedule of Activities – Arms A (Standard), B (Leukapheresis), C (FNA) & D (HLA-A202–)
Visit
Screen
Day 0/ 
Enrollment
Day 31
Day 71
Pre-
Leuka2 
Day 14
Day 211
Day 28
Day 90
Day 180
Day 360
Window (Days)
–
–
 –1  
± 1
–2 prior 
to leuka
± 1
± 5
± 5
± 28
± 30
± 90
Informed Consent
X
Medical History
X
X3
Current Medications
X
X3
X
X
X
X
X
X
X
X
Physical Exam
X
Urine Pregnancy Test
X
X
X3
X3
Vitals
X
X3
Confirm Eligibility
X
Vaccination
X
Symptom Diary
Distribute
Collect
Leukapheresis2,4
X
X
FNA4, 5
X
X
AE Collection
X
X
X
X
X
Blood Sample (Max Volume)6
20 ml
20 ml
24 ml
24 ml
5 ml
74 ml7
74 ml
74 ml7
106 ml
106 ml
106 ml
1Visit may be optional based on current needs of the lab. 
2Only applicable for Arm B participants. 
3If needed. 
4Only if undergoing FNA procedure that day.  
5Participants have procedure on either Day 14 OR 28 – not both. Specific day depends on the current needs of the lab. Day 14 window: -2/+4 days; Day 28 window: ± 4 days.
6Only applicable for Arm C participants. 
7Blood volume will be adjusted as per the laboratory needs at that point in time, no more than the maximum allowable.
8Blood sample not collected on the same day a participant undergoes leukapheresis. 

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7
SAFETY ASSESSMENT
7.1
Adverse Events and Serious Adverse Events
An adverse event (AE) is any untoward medical occurrence in a clinical investigation subject 
administered a pharmaceutical product regardless of its causal relationship to the study 
treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom or disease temporally associated with the use of a medicinal 
(investigational) product. 
Any medical condition that is present at the time that the subject is screened will be considered 
as baseline and not reported as an AE. However, if the severity of any pre-existing medical 
condition increases, it will be recorded as an AE. The start and stop date of each reported AE 
will be recorded on the appropriate CRF. AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
AEs occurring during the trial-collection and reporting period will be documented appropriately 
regardless of relationship to study product. At each study visit, the investigator will inquire about 
the occurrence of AE/SAEs since the last visit.  AEs will be followed through resolution.
A summary report of all AEs will be reviewed at regularly scheduled quarterly meetings by the 
study team. 
Serious Adverse Event (SAE): An AE or suspected adverse reaction is considered “serious” if, 
in the view of either the Principal Investigator (PI) or appropriate sub-investigator, it results in 
any of the following outcomes:

Death,

A life-threatening AE*,

Inpatient hospitalization or prolongation of existing hospitalization,

A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or

A congenital anomaly/birth defect.

Important medical events that may not result in death, be life-threatening, or require 
hospitalizations may be considered serious when, based upon appropriate medical 
judgment they may jeopardize the patient or subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an 
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.
* Life-threatening AE. An AE is considered “life-threatening” if, in the view of either the PI, sub-
investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It 
does not include an AE that, had it occurred in a more severe form, might have caused death.

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7.2
Collection of Adverse Events
AEs and SAEs will be collected via a participant-completed reactogenicity symptom diary,  
participant interviews and/or direct evaluation.  Participants may also reach out to study staff to 
report AEs. If needed, the subject may be asked to return to the Hope Clinic for additional safety 
procedures to further evaluate an AE or SAE. 

Solicited injection site and systemic reactogenicity AEs will be collected from the time of 
study vaccination through 7 days after study vaccination.

Unsolicited non-serious AEs will be collected from the time of study vaccination through 
approximately 30 days after study vaccination.

SAEs (regardless of relation to study product) will be collected for 12 months post-
vaccination (or last study visit, whichever occurs first).  
7.3
Assessment of Severity of Adverse Events
AEs must be assessed for severity and relationship to study product (see definitions below). 
AEs will be assessed by a licensed study PI or sub-investigator using the FDA CBER Toxicity 
Grading Scale for Healthy Adult and Adolescent Volunteers enrolled in Preventive Vaccine 
Clinical Trials (Sept 2007) (see Appendix B) except:  

Unintentional weight loss is required to be reported as an AE only if it is considered to be 
potentially deleterious to the participant’s health

Injection Site Erythema or Redness and Injection Site Induration or Swelling will not 
consider interference with usual social and functional activities such that:

Grade 1 is: 2.5 to < 5 cm in diameter OR 6.25 to < 25 cm2 surface area;

Grade 2 is: ≥ 5 to < 10 cm in diameter OR ≥ 25 to < 100 cm2 surface area;

Grade 3 is: ≥ 10 cm in diameter OR ≥ 100 cm2 surface area OR Ulceration OR 
Secondary infection OR Phlebitis OR Sterile abscess OR Drainage;

Grade 4 is: Potentially life-threatening consequences (e.g., abscess, exfoliative 
dermatitis, necrosis involving dermis or deeper tissue)
Additionally, chills and arthralgia will be graded according to the following criteria:

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Mild 
(Grade 1)
Moderate
(Grade 2)
Severe 
(Grade 3)
Potentially Life 
Threatening 
(Grade 4)
Chills
Symptoms causing no 
or minimal 
interference with 
usual social & 
functional activities
Symptoms causing 
greater than minimal 
interference with 
usual social & 
functional activities 
Symptoms causing 
inability to perform 
usual social & 
functional activities
N/A
Arthralgia
Joint pain causing 
no or minimal
Interference with 
usual social & 
functional activities
Joint pain causing 
greater than minimal 
interference with 
usual social & 
functional activities
Joint pain causing 
inability to perform 
usual and & functional 
activities
Disabling joint pain 
causing inability to 
perform basic self-
care functions
For events not included in the protocol-defined grading system, the following guidelines will be 
used to quantify severity:

Mild (Grade 1): Events require minimal or no treatment and do not interfere with the 
subject’s daily activities.

Moderate (Grade 2): Events result in a low level of inconvenience or concern with 
therapeutic measures. Moderate events may cause some interference with functioning 
and daily activities.

Severe (Grade 3): Events interrupt the subject’s daily activities and may require systemic 
drug therapy or other treatment. Severe events are usually incapacitating.

Potentially life threatening (Grade 4): ER visit or hospitalization
7.4
Relationship to Study Intervention
The study products that must be considered in determining relationships of AEs are:
•
YF-VAX 
The licensed study physician’s assessment of an AE’s relationship to study product is part of the 
documentation process, but it is not a factor in determining what is or is not reported in this trial. 
If there is any doubt as to whether a clinical observation is an AE, the event should be reported. 
Study investigators will assess all AEs as being related or unrelated to the vaccination. 
7.4.1
Assigning AE Relationship to Study Product
An inability to exclude or “rule out” a relationship to the study product is not, in and of
itself, sufficient to deem an AE “related.”
Use the following definitions when reporting attribution to study product:

Choose “Related” if there is a reasonable possibility* that the AE may be related to the 
study product. This choice suggests that the AE is more likely to be related than not 
related to the study product(s). 

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
Choose “Unrelated” if there is not a reasonable possibility* that the AE is related to the 
study agent. This choice suggests that the AE is more likely to be related to another 
cause rather than the study product(s).
*The U.S. Federal Drug Administration has published a final rule amending the IND
safety reporting requirements under 21 CFR part 312.

The final rule set a higher standard for reasonable possibility of relationship of study 
product to an adverse event.

It defined that, for the purposes of IND safety reporting by the study sponsor, 
“reasonable possibility” means there is evidence to suggest a causal relationship 
between the study product and the adverse event.
7.5
Reporting Adverse Events 
Because YF-VAX® is an FDA-approved vaccine, AEs that meet the following criteria will be 
reported to the Vaccine Adverse Events Reporting System (VAERS):

Local and Systemic Reactogenicity AEs ≥ Grade 3

Related AEs ≥ Grade 3

Related SAEs
AEs and SAEs will be reported to the Emory IRB per their guidelines. 
8
LABORATORY
8.1
Laboratory Assays or Procedures
The blood will be processed to isolate peripheral blood mononuclear cells (PBMCs) and serum. 
Based on laboratory supply availability, either Cell Preparation Tube (CPT) or Acid Citrate 
Dextrose (ACD) will be used. 
Plasma from all time points will be separated from whole blood and will be frozen at -80oC. 
Peripheral blood mononuclear cells will be isolated on all study days. The 17D vaccine-induced 
viremia will be determined by TaqMan real-time PCR assay of the plasma (Applied Biosystems, 
Foster City, CA). The lower limit of detection for this assay is 25copies/ml and will be performed 
in the Molecular Virology Core of the Emory Vaccine Center.
The following assays will be performed for innate immunity:
1.
Serum analyses of cytokines and chemokines that regulate adaptive immunity. 
2.
Phenotype, activation status and frequencies of dendritic cell (DC) subsets and DC 
precursors in the blood
3.
Microarray analyses. 
The following assays will be performed for the adaptive immunity:
1.
Magnitude of the YFV-specific effector T cell response

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2.
Quality of the YFV-specific T cell response by determining the expression of various 
markers of cytolysis (granzymeB and perforin), homing (CD62L, CCR7, a4b7, CLA), 
cytokine receptors (CD127, CD122), costimulation (CD27, CD28), inhibition (PD-1), 
adhesion (CD11a) and survival (Bcl-2) will be analyzed using 11-parameter flow 
cytometry.
3.
Output of naïve T cells from the thymus using the TREC assay
4.
YFV-specific serological responses
5.
Analysis of the YFV-specific ASC variable gene repertoire
6.
Gene profiling of the YFV vaccine-induced antibody secreting cells (ASC)
7.
Yellow Fever-specific T-B co-culture assay in vitro to measure the B cell helper ability 
of the YFV-specific CD4 T cells 
8.
YFV-specific T cell proliferation in vitro
9.
YFV-specific cytokine/chemokine production in vitro
10.
Phenotypical characterization of YFV-specific CD4 T cells by Flow Cytometry. This 
analysis requires also an HLA typing assay of the donors.
11.
YFV-specific CD4 and CD8 T cells in axillary lymph node aspirates
The PBMCs will be stained using a combination of surface markers to identify activated or YFV 
tetramer specific CD8 T cells.  The CD8 T cell populations of interest will be isolated using flow 
cytometry-based sorting. The sorted populations will be processed for RNA isolation and 
microarray analysis performed using affymetrix gene arrays. Gene expression values will be 
obtained using robust multiarray analysis (RMA).
A small part of the PBMCs will be stored for validation of expression of certain signature genes 
by RT-PCR and for in vitro functional assays.  Plasma will be stored and later tested for 
antibodies against yellow fever virus. At all visits PBMCs are harvested and the following tests 
will be done to analyze the T cell responses:

FACS analysis for activation markers (such as CD69, CD38, CD45, CD62L, etc.) on 
CD8 and CD4 T cells and B cells.

CFSE labeling of PBMCs, stimulation with antigenic peptides and evaluation of 
proliferative responses as seen by CFSE dilution in flow cytometric analysis.
8.2
Specimen Preparation, Handling, and Shipping
The blood tubes will be labeled at the Hope Clinic with a unique identifier and will be transported 
to the Emory Vaccine Center via courier service in appropriate transport containers.
9
STATISTICAL CONSIDERATIONS
This is an exploratory non-placebo-controlled analysis of variation in the cellular and antibody 
specific immune responses generated against YFV. This will provide us with descriptive data to 
analyze the immune responses. The microarray data generated will be analyzed using 
Benjamin’s Fisher test.

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SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS
All source documents will be kept at the Hope Clinic in a locked cabinet in a locked office. The 
building is secured by a monitored alarm system.
11
QUALITY CONTROL AND QUALITY ASSURANCE
Per Hope Clinic Standard Operating Procedures (SOPs), a portion of the study charts 
(approximately 25%) will be audited by the Hope Clinic Quality Management (QM) personnel  
and all charts will be audited for informed consent compliance.
12
ETHICS/PROTECTION OF HUMAN SUBJECTS
This study will be conducted in compliance with all applicable federal regulations and 
institutional policies. The study team will get IRB approval of all applicable study documents 
prior to use. Informed consent will be sought from all participants prior to any study activities 
occurring.  
13
DATA HANDLING AND RECORD KEEPING
Participant data will be maintained at the Hope Clinic in a locked file cabinet. Biological samples 
and immunological assays data will be labeled with only a unique study code number and 
maintained in the laboratory of Dr. Rafi Ahmed and Dr. Bali Pulendran. The links to the unique 
codes will be stored at the Hope Clinic and will not be available to the laboratory personnel. 
The study records will be maintained up to ten years after the completion of the study.
14
PUBLICATION POLICY
The PI and/or sub-investigators will participate in manuscript preparation and submission to 
journals.
15
LITERATURE REFERENCES
1.
Lindenbach B, Rice C. Flaviviridae: the viruses and their replication. . In: Knipe D, 
Howley P (eds). Fields Virology. Volume 1. Fourth Edition ed: Lippincott Williams & 
Wilkins; 2001, pp 991-1041.
2.
Lindsey NP, Schroeder BA, Miller ER, et al. Adverse event reports following yellow fever 
vaccination. Vaccine 2008; 26: 6077-6082.
3.
Burke D, Monath T. Flaviviruses. In: Knipe D, Howley P (eds). Fields Virology. Volume 1. 
Fouth Edition ed: Lippincott Williams & Wilkins; 2001, pp 1043-1125.

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4.
Monath T. Yellow fever.  Vaccines. Third Edition ed: WB Saunders Co; 1999, pp 815-
879.
5.
Adverse events associated with 17D-derived yellow fever vaccination--United States, 
2001-2002. MMWR Morbidity and mortality weekly report 2002; 51: 989-993.
6.
Martin M, Weld LH, Tsai TF, et al. Advanced age a risk factor for illness temporally 
associated with yellow fever vaccination. Emerging infectious diseases 2001; 7: 945-
951.
7.
Miller JD, van der Most RG, Akondy RS, et al. Human effector and memory CD8+ T cell 
responses to smallpox and yellow fever vaccines. Immunity 2008; 28: 710-722.
8.
Akondy RS, Monson ND, Miller JD, et al. The yellow fever virus vaccine induces a broad 
and polyfunctional human memory CD8+ T cell response. Journal of immunology 
(Baltimore, Md : 1950) 2009; 183: 7919-7930.
9.
Dutton RW, Bradley LM, Swain SL. T cell memory. Annual review of immunology 1998; 
16: 201-223.
10.
Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation: implications 
for vaccine development. Nature reviews Immunology 2002; 2: 251-262.
11.
Murali-Krishna K, Altman JD, Suresh M, et al. Counting antigen-specific CD8 T cells: a 
reevaluation of bystander activation during viral infection. Immunity 1998; 8: 177-187.
12.
Jameson SC. Maintaining the norm: T-cell homeostasis. Nature reviews Immunology 
2002; 2: 547-556.
13.
Staples JE, Gershman M, Fischer M. Yellow fever vaccine: recommendations of the 
Advisory Committee on Immunization Practices (ACIP). MMWR Recommendations and 
reports : Morbidity and mortality weekly report Recommendations and reports 2010; 59: 
1-27.

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APPENDICES 
APPENDIX A. Flow chart for Yellow Fever Immune Response Study 

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APPENDIX B. Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventative Vaccine Clinical Trials (FDA, September 2007) 
Tables for Clinical Abnormalities
Local Reaction to 
Injectable Product
Mild (Grade 1)
Moderate(Grade 2)
Severe (Grade 3)
Potentially Life 
Threatening (Grade 
4)
Pain
Does not 
interfere with 
activity
Repeated use of non- 
narcotic pain reliever
> 24 hours or 
interferes with activity
Any use of narcotic 
pain reliever or 
prevents daily 
activity
Emergency room (ER) 
visit or hospitalization
Tenderness
Mild discomfort 
to touch
Discomfort with 
movement
Significant 
discomfort at rest
ER visit or 
hospitalization
Erythema/Redness *
2.5 – 5 cm
5.1 – 10 cm
> 10 cm
Necrosis or exfoliative 
dermatitis
Induration/Swelling **
2.5 – 5 cm and 
does not 
interfere with 
activity
5.1 – 10 cm or 
interferes with activity
> 10 cm or prevents 
daily activity
Necrosis
*In addition to grading the measured local reaction at the greatest single diameter, the measurement should be 
recorded as a continuous variable.
**Induration/Swelling should be evaluated and graded using the functional scale
Vital Signs*
Mild 
(Grade 1)
Moderate 
(Grade 2)
Severe 
(Grade 3)
Potentially Life 
Threatening 
(Grade 4)
Fever (°C) **
(°F) **
38.0 – 38.4
100.4 – 101.1
38.5 – 38.9
101.2 – 102.0
39.0 – 40
102.1 – 104
> 40
> 104
Tachycardia - beats per 
minute
101 – 115
116 – 130
> 130
ER visit or hospitalization 
for arrhythmia
Bradycardia - beats per 
minute***
50 – 54
45 – 49
< 45
ER visit or hospitalization 
for arrhythmia
Hypertension (systolic) - 
mm Hg
141 – 150
151 – 155
> 155
ER visit or hospitalization 
for malignant 
hypertension
Hypertension (diastolic) 
- mm Hg
91 – 95
96 – 100
> 100
ER visit or hospitalization 
for malignant 
hypertension
Hypotension (systolic) – 
mm Hg
85 – 89
80 – 84
< 80
ER visit or hospitalization 
for hypotensive shock
Respiratory Rate – 
breaths per minute
17 – 20
21 – 25
> 25
Intubation
*Subject should be at rest for all vital sign measurements.
**Oral temperature; no recent hot or cold beverages or smoking.
*** When resting heart rate is between 60 – 100 beats per minute. Use clinical judgement when characterizing 
bradycardia among some healthy subject populations, for example, conditioned athletes.

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Systemic (General)
Mild 
(Grade 1)
Moderate
(Grade 2)
Severe 
(Grade 3)
Potentially Life 
Threatening (Grade 
4)
Nausea/vomiting
No interference with 
activity or 1 – 2 
episodes/24 hours
Some interference 
with activity or > 2 
episodes/24 hours
Prevents daily 
activity, requires 
outpatient IV 
hydration
ER visit or 
hospitalization for 
hypotensive shock
Diarrhea
2 – 3 loose stools or
< 400 gms/24 hours
4 – 5 stools or
400 – 800 gms/24 
hours
6 or more watery 
stools or
> 800gms/24 hours 
or requires 
outpatient IV 
hydration
ER visit or 
hospitalization
Headache
No interference with 
activity
Repeated use of non- 
narcotic pain
reliever > 24 hours or 
some interference 
with activity
Significant; any 
use of narcotic 
pain reliever or 
prevents daily 
activity
ER visit or 
hospitalization
Fatigue
No interference with 
activity
Some interference 
with activity
Significant; 
prevents daily 
activity
ER visit or 
hospitalization
Myalgia
No interference with 
activity
Some interference 
with activity
Significant; 
prevents daily 
activity
ER visit or 
hospitalization
Systemic Illness
Mild 
(Grade 1)
Moderate
(Grade 2)
Severe 
(Grade 3)
Potentially Life 
Threatening (Grade 
4)
Illness or clinical adverse 
event (as defined 
according to applicable 
regulations)
No interference with 
activity
Some 
interference with 
activity not 
requiring medical 
intervention
Prevents daily 
activity and 
requires 
medical 
intervention
ER visit or 
hospitalization