The Yellow Fever Virus Vaccine Induces a Broad and
Polyfunctional Human Memory CD8?T Cell Response1
Rama S. Akondy,* Nathan D. Monson,* Joseph D. Miller,* Srilatha Edupuganti,*
Dirk Teuwen,¶Hong Wu,* Farah Quyyumi,* Seema Garg,* John D. Altman,* Carlos Del Rio,*
Harry L. Keyserling,‡Alexander Ploss,§Charles M. Rice,§Walter A. Orenstein,*
Mark J. Mulligan,* and Rafi Ahmed2*†
The live yellow fever vaccine (YF-17D) offers a unique opportunity to study memory CD8?T cell differentiation in humans
following an acute viral infection. We have performed a comprehensive analysis of the virus-specific CD8?T cell response using
overlapping peptides spanning the entire viral genome. Our results showed that the YF-17D vaccine induces a broad CD8?T cell
response targeting several epitopes within each viral protein. We identified a dominant HLA-A2-restricted epitope in the NS4B
protein and used tetramers specific for this epitope to track the CD8?T cell response over a 2 year period. This longitudinal
analysis showed the following. 1) Memory CD8?T cells appear to pass through an effector phase and then gradually down-
regulate expression of activation markers and effector molecules. 2) This effector phase was characterized by down-regulation of
CD127, Bcl-2, CCR7, and CD45RA and was followed by a substantial contraction resulting in a pool of memory T cells that
re-expressed CD127, Bcl-2, and CD45RA. 3) These memory cells were polyfunctional in terms of degranulation and production
of the cytokines IFN-?, TNF-?, IL-2, and MIP-1?. 4) The YF-17D-specific memory CD8?T cells had a phenotype
(CCR7?CD45RA?) that is typically associated with terminally differentiated cells with limited proliferative capacity (TEMRA).
However, these cells exhibited robust proliferative potential showing that expression of CD45RA may not always associate with
terminal differentiation and, in fact, may be an indicator of highly functional memory CD8?T cells generated after acute viral
infections. The Journal of Immunology, 2009, 183: 7919–7930.
T cell memory has advanced a great deal through exhaustive stud-
ies in acute viral infections that cause protective, long-lasting
memory (2–5). We know from these studies that Ag-driven clonal
expansion results in a dynamic antiviral CD8?T cell response
initially manifested as pathogen clearance via cytotoxic molecules
and effector cytokines and later by the presence of a small popu-
lation of memory cells that can be rapidly recruited to blunt sub-
sequent infections. We have also begun to understand the differ-
ences between the properties of poor and high quality memory
CD8?T cells based on their cytotoxicity, cytokine production,
homeostatic turnover, and proliferative potential (6–17).
mmune memory forms the basis of vaccine-induced protec-
tion, and memory CD8?T cells form an important cellular
component of this immunity (1). Our understanding of CD8?
The yellow fever vaccine strain 17D (YF-17D),3a live attenuated
form of the wild-type virus, has not only been extremely effective in
adults (18, 19). A single immunization confers protection, and virus-
neutralizing Abs can be detected for up to 30 years post-vaccination
(20). The live viral nature of the vaccine combined with its efficacy is
useful for studying how humans generate functional immunity in the
context of an acute viral infection and also as a potential expression
vector for recombinant vaccines (21, 22). Some of the studies that
address the complex interactions between the virus and the immune
system suggest that the ability of YF-17D to infect dendritic cells and
activate multiple TLRs may be crucial for generating the robust
adaptive immune response seen after vaccination (23–25). The im-
mune memory developed as a consequence comprises the ability to
rapidly produce neutralizing Abs as well as the CD8?T cell ef-
fectors (26–28) that are likely required for killing cells infected
with the virus that escape the humoral response. This cellular
memory has been of interest to our group because it presents a rare
opportunity to study primary CD8?T cell responses in humans.
CD8?T cells that recognize HLA-B35-restricted epitopes in E,
NS1, NS2B, and NS3 proteins have been reported in vaccinees
(26). In addition, we demonstrated recently that the magnitude of
the total effector CD8?T cells response against YF-17D (or the
smallpox live viral vaccine) could be measured using the transient
Ki67?Bcl-2lowHLA-DR?CD38?phenotype of effector T cells
(28, 29). However, there are gaps in our knowledge regarding the
breadth of the response, the differentiation of virus-specific effector
*Emory Vaccine Center and the Hope Clinic,†Department of Microbiology and Im-
munology, and‡Department of Pediatrics, Emory University School of Medicine,
Atlanta, GA 30022;§Center for the Study of Hepatitis C, Laboratory of Virology and
Infectious Disease, Rockefeller University, New York, NY 10065; and¶Sanofi Pas-
teur, Lyon, France
Received for publication December 1, 2008. Accepted for publication October
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health (NIH) U19 Grant
AI057266 (to R.A.) and in part by Sanofi-Pasteur, Lyon, France. C.M.R. receives
support from the Greenberg Medical Research Institute, the Starr Foundation, the
Foundation for the National Institutes of Health through the Grand Challenges in
Global Health initiative (grant identification nos. 334 and 574), General Clinical Re-
search Center Grant M01-RR00102 (to Rockefeller University Hospital), and Center
for Translational Science Award Grant 1UL1 RR024143-01 (to Rockefeller Univer-
sity Hospital) from the NIH National Center for Research Resources.
2Address correspondence and reprint requests to Dr. Rafi Ahmed, 1510 Clifton
Road., G211, Atlanta, GA 30322. E-mail address: rahmed@.emory.edu
3Abbreviations used in this paper: YF-17D, yellow fever virus strain 17D; ICC, intra-
cellular cytokine; PD-1, programmed death-1; TEMRA, effector memory RA T cell.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
and memory CD8?T cells, and the functional qualities of these
To address the above questions, we used a peptide library en-
compassing the entire YF-17D genome for estimating the magni-
tude of the T cells targeting each viral protein and the range of
epitopes recognized. Using a strategy involving direct ex vivo
stimulation of cells with overlapping peptides and detection of
cytokine production (30), we identified an immunodominant class
I-restricted epitope that permitted longitudinal tracking of YF-
17D-specific CD8?T cells in individual vaccinees using MHC
class I tetramers. This analysis offers novel perspectives for two
reasons. First, our study has the advantage that the subjects were
primary vaccinees from a geographical area that is not endemic for
yellow fever, i.e., the United States (19). Thus, an accurate deter-
mination of the breadth and magnitude of the primary YF-17D-
specific response could be made while avoiding the possible cross-
reactivity introduced by prior exposure to either the yellow fever
virus or other closely related flaviviruses. Second, we performed a
longitudinal tetramer-based analysis and followed YF-17D-spe-
cific CD8?T cells from the time they first appeared in circulation
until 2 years later, in the same group of vaccinees. Hence, we were
able to study various stages of memory CD8?T cell differentiation
longitudinally without the confounding effects introduced by a
cross-sectional analysis. This analysis will be valuable in under-
standing the basic tenets of human memory T cell differentiation
after acute viral infections.
Materials and Methods
Study subjects and blood samples
Healthy volunteers (18–40 years of age) were recruited in the study after
informed consent. Approval for all procedures was obtained from the
Emory University Institutional Review Board (Atlanta, GA). A single dose
(0.5 ml containing at least 105PFU) of 17D live-attenuated yellow fever
vaccine strain was administered subcutaneously. The recommendations es-
tablished by the Advisory Committee on Immunization Practices (Depart-
ment of Health and Services, Centers for Disease Control and Prevention,
Atlanta, GA) were followed for selection and vaccination of individuals in
the study. In addition, individuals with a previous history of vaccination
with YF-17D or exposure to flaviviruses as evidenced by serology or a
history of travel to endemic areas were excluded from the study. Blood
samples were analyzed before and at various times postvaccination as in-
dicated in the text. Seroconversion after vaccination was confirmed by
assaying the neutralizing Ab titers for YF-17D (data not shown). PBMC
were purified from cell preparation tubes (BD Biosciences), and EDTA
blood samples were used to quantify viral RNA by real-time PCR as de-
scribed elsewhere (28).
YF-17D peptide library
To map T cell epitopes, a library of overlapping peptides spanning the entire
YF-17D polyprotein was made. All 851 peptides comprising the library were
15-aa long (Synpep) and nonamidylated, with neighboring peptides overlap-
ping by 11 aa. The peptides were organized into 60 pools based on a matrix
such that each pool had 24–30 peptides, each at a concentration of 10 ?g/ml,
and any two pools had no more than one peptide in common (30, 31).
Cell preparation, stimulation, and intracellular cytokine (ICC)
PBMC were purified from cell preparation tubes (BD Biosciences) accord-
ing to standard protocols and cryopreserved in 90% FCS plus 10% DMSO.
For mapping T cell epitopes, freshly isolated PBMC were stimulated with
each of 60 peptide pools. Assays for confirming epitopes in the single
peptides identified using the pools were done using cryopreserved PBMC
that were revived, rested overnight at 37°C, and stimulated with 10 ?g/ml
peptides. Fresh or revived PBMC were stimulated for 6 h in 96-well round-
bottom plates in the presence of brefeldin A (1 ?l/ml) and anti-CD28/
CD49d (10 ?l/ml) and then stained for relevant T cell markers and cyto-
kines. Culture medium for all cellular assays was RPMI 1640 containing
10% FCS, 2 mM glutamine, 100 IU/ml penicillin, and 100 ?g/ml strepto-
mycin (RPMI 1640 plus 10% FCS).
Staining and flow cytometry analysis
All mAbs except anti-granzyme B (Caltag), programmed death (PD)-1
(provided by Dr. G. J. Freeman, Dana Farber Cancer Institute, Boston,
MA), and CCR7 (R&D Systems) were obtained from BD Biosciences.
A2-NS4B 214–222 tetramers were made in-house. For ICC after in vitro
stimulation, cells were stained for T cell markers by incubation with the
relevant Abs at room temperature for 30 min, washed with PBS, and then
stained for cytokines using anti-IFN-?, TNF-?, IL-2 and Mip-1? Abs after
cell permeabilization with the Cytofix/Cytoperm kit (BD Biosciences). De-
granulation in stimulated cells was measured by including anti-CD107a-PE
(10 ?l/well) in the initial culture medium. For phenotypic analysis of A2-
NS4B?CD8?T cells, 100–200 ?l of unprocessed whole blood was in-
cubated at room temperature first with tetramer for 10 min and then for a
further 30 min with Abs for surface markers. This was followed by a
10-min lysis of RBC using FACS lysing solution (BD Biosciences), wash-
ing with PBS, and fixing in 1% p-formaldehyde. For staining intracellular
proteins like Bcl-2, Ki-67, granzyme B, and perforin, cells were permeabilized
and stained using the Cytofix/Cytoperm kit (BD Biosciences) according to the
manufacturer’s instructions. Data were acquired on a FACSCalibur (BD Bio-
Jo (Tree Star) software. In addition, the programs SPICE (version 4.1.5) and
Vaccine Research Center, Bethesda, MD) were used to quantify CD8?T cells
positive for the various combinations of functions assayed. Statistical analysis
and graphical representation of data was performed using GraphPad Prism
CFSE labeling and in vitro expansion of CD8?T cells
PBMC from vaccinated subjects were labeled for 5 min with 1 ?M CFSE
(Molecular Probes) in PBS at room temperature; cold FCS was then added and
cells were washed extensively with RPMI 1640 plus 10% FCS. CFSE-labeled
cells were incubated with or without the NS4B 214–222 peptide (10 ?g/ml)
for 6 days, at the end of which flow cytometry and analysis were performed as
described above. Responding CD8?T cells were identified either by tetramer
staining or by ICC staining after a 6-h recall with peptide.
YF-17D elicits a broad diversity of memory CD8?T cells
To define the breadth of the primary antiviral CD8?T cell re-
sponse, we created a library of overlapping peptides encompassing
the entire viral polyprotein. This library was organized into 60
pools of multiple peptides based on a 24 ? 36 matrix such that
each peptide was present in precisely two pools (30). Responses to
all of these pools were studied in nine healthy subjects vaccinated
2 mo previously with a single s.c. injection of YF-17D. PBMC
isolated from vaccinees were stimulated with individual pools fol-
lowed by ICC staining assays, and responding virus-specific CD8?
T cells were identified by their ability to produce IFN-?. Of 60
peptide pools tested with this assay, between eight and 25 pools
were capable of eliciting IFN-? production in different vaccinees.
Many pools contained peptides from adjacent sequences in the
viral polyprotein and thus had epitopes primarily from one protein.
IFN-? production stimulated by these pools was used to estimate
the contribution of individual viral proteins toward eliciting a
CD8?T cell response (Fig. 1). Overall, each vaccinee had CD8?
T cells targeting multiple proteins and several epitopes within each
protein, resulting in broad cellular immunity. Despite variation in
their magnitude, CD8?T cells specific for each of the 10 YF-17D
proteins were detected, indicating that every viral protein was im-
munogenic. IFN-??CD8?T cells specific for E, NS3, and NS5
were elicited in all vaccinees with an average of 10.7% (SD 8.0),
16.7% (SD 7.0), and 27.7% (SD 14.0) of the total responding
CD8?T cells specific for E, NS3, and NS5 respectively. NS1 also
elicited frequent (in 8/9 vaccinees) but lower numbers (mean
7.2%; SD 3.7) of CD8?T cells, whereas the magnitude of C-, M-,
NS2A-, and NS2B-reactive CD8?T cells was low (?3%) when
detected. Strikingly, in five vaccinees the NS4B component was
dominant and accounted for ?42.1% (SD 13.5) of the total
IFN-??CD8?T cells (Fig. 1).
7920YFV-17D INDUCES POLYFUNCTIONAL HUMAN CD8?T CELL MEMORY
YF-17D harbors a dominant HLA-A2-restricted epitope in the
Although vaccinees had CD8?T cells recognizing multiple
epitopes, IFN-? production in some pools containing NS4B-de-
rived peptides consistently dominated the CD8?T cell response in
five vaccinees. These pools had either the peptide NS4B 209–223
or NS4B 213–227 in common. Both peptides were individually
capable of stimulating CD8?T cells from the same five vaccinees,
confirming that these peptides contained MHC class I-restricted
epitopes (Fig. 2, A and B). Notably, all the above vaccinees were
derived from YF-17D proteins and IFN-? production was assayed by ICC. The percentage of IFN-??CD8?T cells for individual vaccinees is shown.
YF-17D elicits a broad diversity of memory CD8?T cells. PBMC from YF-17D-vaccinated individuals were stimulated with peptide pools
7921 The Journal of Immunology
positive for the HLA-A2 serotype (data not shown), suggesting
that the response was HLA-A2 restricted. An epitope prediction
algorithm (32) was used to identify putative HLA-A0201-re-
stricted epitopes in the 15-mer NS4B 209–223 and NS4B 213–227
peptides. Of the several nonamer epitopes predicted, three epitopes
with the highest scores (and hence the best binding motifs), NS4B
213–221, NS4B 214–222, and NS4B 215–223 were tested by ICC
staining for their ability to stimulate IFN-? production. Only one
peptide, NS4B 214–222 (amino acid sequence LLWNGPMAV)
generated IFN-?-producing CD8?T cells in HLA-A2?vaccinees,
identifying it as the HLA-A2-restricted CD8?T cell epitope (Fig.
2, A and B). In addition, control peptides that had a single amino
acid substitution (either L214 to V214 or L215 to E215) did not
stimulate IFN-? production, thus validating the specificity of the
NS4B 214–222 epitope. MHC-peptide tetramers containing HLA-
A0201 and the NS4B 214–222 peptide were prepared and tested in
individuals. Tetramer-stained CD8?cells were seen in vaccinated
HLA-A2?but not HLA-A2?individuals, confirming the specific-
ity of the epitope (Fig. 2C). This tool enabled us to perform a
longitudinal analysis of YF-17D-specific CD8?T cells.
Kinetics of the YF-17D-specific primary CD8?T cell response
tracked by MHC class I tetramers
We analyzed the dynamics of viral replication after the vaccination
of HLA-A2?individuals who did not have a history of exposure
to flaviviruses (Fig. 3). Most vaccinees did not have detectable
viral RNA in the first 2 days following vaccination; on day 3,
several vaccinees tested positive for viral RNA (mean 114 copies/
ml; SD 50), and the number of positives increased further such that
all vaccinees had YF-17D genomes in the plasma by day 5. The
highest level of viral RNA in circulation was seen on day 5 for
most vaccinees (mean 3598 copies/ml; SD 1813) and on day 7 for
others (mean 1443 copies/ml; SD 613), after which it declined
quickly. By day 11 all except two vaccinees had no viral RNA in
the plasma, and by day 14 viral genomes were absent.
Ag dictates the size of the effector CD8?T cell response and the
kinetics of initial expansion (33). We used A2-NS4B 214–222
tetramers to quantify the size and examine the kinetics of the YF-
17D-driven primary CD8?T cell response in vaccinees. Strik-
ingly, A2-NS4B tetramer?CD8?T cells were seen in nearly all
HLA-A2?vaccinees (19 of 21). The A2-NS4B?CD8?cells were
often detectable as early as 11 days after vaccination and definitely
after 14 days in all responding individuals, coincident with the
decline in plasma viral RNA. Tetramer?cells continued to expand
till 30 days postvaccination, when the peak tetramer frequency
ranging from 0.5 to 17% of CD8?T cells was seen in the vacci-
nees; it diminished by ?3.6-fold by day 90. The size of the A2-
NS4B?pool at this time was roughly proportional to that seen on
day 30; vaccinees with high peak frequencies retained high num-
bers of memory cells. We were able to track these cells for an
additional time in five vaccinees from the same cohort and found
that tetramer?cells were readily detectable even 1 year after vac-
cination (Fig. 3B). In conclusion, HLA-A2?vaccinees have a ro-
bust and long-lived response to the NS4B 214–222 epitope, show-
ing expansion, contraction, and memory kinetics typical of CD8?
T cells elicited in a primary acute viral infection.
Effector and memory differentiation of YF-17D-specific CD8?
To understand the differentiation of effector and memory CD8?T
cells and to gain an insight into the generation of YF-17D-specific
CD8?memory, we performed a detailed longitudinal analysis of
tetramer-stained CD8?T cells in 15 HLA-2?vaccinees (Fig. 4).
Because rapid clonal expansion is the hallmark of a virus-driven
CD8?T response, we assessed proliferation of YF-17D-specific
CD8?T cells using Ki-67, a marker that is tightly associated with
cycling CD8?T cells (34). Compared with naive CD8?T cells
that were Ki-67 negative, ?95% of the A2-NS4B?cells detected
early on (days 11 to 14) were Ki-67 positive. The Ki-67 staining
declined significantly (present in ?5% of the cells) by day 30 and
the NS4B protein. A, Sequences of the peptides used to confirm and iden-
tify the immunogenic nonamer epitope in NS4B. B, PBMC from HLA-
A2?vaccinees were cultured in the presence or absence of 15-mer peptides
(NS4B 209–223 or NS4B213–227) and ICC staining was performed (up-
per panels). To confirm the nonamer epitope in these peptides, PBMC were
stimulated with either the NS4B 214–222 peptide or with control peptides
that differed by one amino acid (lower panels). Plots gated on CD8?T cells
for one representative donor are shown. C, Identification of YF-17D-spe-
cific CD8?T cells using MHC class I tetramers. Peripheral blood from
HLA-A2?or HLA-A2?individuals vaccinated with YF-17D 2 wk previ-
ously was stained with the A2-NS4B tetramer. Plots are gated on all lym-
phocytes and numbers indicate the percentage of tetramer?cells from total
YF-17D harbors a dominant HLA-A2 restricted epitope in
7922YFV-17D INDUCES POLYFUNCTIONAL HUMAN CD8?T CELL MEMORY
decreased to baseline by day 90. The proliferation seen early on
was coupled with decreased expression of the antiapoptotic protein
Bcl-2, suggesting that a majority of these cells were destined to
undergo apoptosis. Progressive reexpression of Bcl-2 occurred
concomitantly with loss of Ki-67 and differentiation to a memory
phenotype. Expression of the activation markers HLA-DR and
CD38 mirrored the kinetics seen with Ki-67. Activated but not
naive CD8?T cells also expressed the chemokine receptor CCR5.
Although we observed that the CCR5 expression kinetics was sim-
ilar to those of the proliferation and activation markers, unlike the
other markers CCR5 was retained in 40–50% of A2-NS4B?mem-
ory cells. Potent effector properties were evidenced by high levels
of granzyme B (Fig. 4), granzyme A, and perforin (data not shown)
expression until as late as 30 days after YF-17D vaccination, and
tetramer?cells continued to express granzyme B later, although at
We analyzed the expression of markers associated with other
relevant functions of CD8?T cells (Fig. 4). CD127 (IL-7R?)
has been shown earlier to be an important marker that is down-
regulated on effectors and selectively reexpressed on a subset
destined to form precursors of the memory pool (10, 35). We
observed substantial but not complete down-regulation of
CD127 on A2-NS4B?CD8?T cells over the 30 days following
vaccination, and a gradual reexpression thereafter. Coexpres-
sion of CCR7, a homing marker, and CD45RA, a transmem-
brane tyrosine phosphatase, is characteristic of naive cells (36).
Both markers were down-regulated in the YF-17D-specific
CD8?T cells during effector differentiation. However in con-
trast to the lack of CCR7 on a majority of memory cells,
CD45RA was reexpressed over time. We next examined the
profiles of signaling molecules that regulate T cell activation.
PD-1 is a regulatory receptor of the CD28 family that is tran-
siently expressed on activated T cells during acute viral infec-
tions (37). Its up-regulation by CD8?T cells (transient in acute
and long-term in chronic viral infections) inhibits the activa-
tion, expansion, and acquisition of effector CD8?T cell func-
tions (38). A2-NS4B?cells transiently up-regulated PD-1 be-
tween 11 and 14 days after YF-17D vaccination, similar to
reports with other acute viral infections in mice (38). To assess
the ability to respond to costimulatory signals at the different
stages of differentiation, we evaluated the expression of two
critical signaling proteins, CD27 and CD28. CD27 was uni-
formly expressed at high levels during all stages of the differ-
entiating virus specific CD8?T cells, whereas CD28 expression
was uniform in effectors (day 11–14) and heterogeneous on
memory cells. CD11a, a component of the adhesion molecule
LFA-1 (39), was expressed by YF-17D-experienced but not na-
ive CD8?T cells as described in studies with other infections
representative vaccinee is shown. Plots show all lymphocytes but the numbers indicate the percentage of A2-NS4B?cells from total CD8?T cells.
B, YF-17D genomes in the plasma (red line; mean ? SD) and the percentage of A2-NS4B?(black lines; each for one vaccinee) in YF-17D vaccinees
over 1 year is shown.
Kinetics of the YF-17D-specific primary CD8?T cell response tracked by MHC class I tetramers. A, Tetramer kinetics in one
7923The Journal of Immunology
To summarize, the primary antiviral response was characterized
by a pronounced activation and expansion phase that led to in-
creasing frequencies of A2-NS4B?cells by day 30. At this stage,
YF-17D-specific cells were at the end of the expansion phase and
showed a phenotype intermediate to that of effector and memory
CD8?T cells. Substantial contraction resulted in a pool of mem-
ory cells that had lost activation and proliferation markers, uni-
formly expressed CD45RA and CD27, and had heterogeneous ex-
pression with respect to proteins such as CCR5, CD28, and
The YF-17D-specific CD8?T cell memory is fine tuned over time
Memory differentiation is a gradual and continuous process result-
ing in subtle changes in the memory pool that may not be obvious
differentiation of YF-17D-specific
CD8?T cells. A, Results from the lon-
gitudinal phenotypic analysis of A2-
NS4B?cells present in blood of 10 to
15 vaccinees are summarized. The day
11, 14, 30, and 90 data are from the
same group of vaccinees. The day 0
data are from a separate group and rep-
resent expression of the relevant
marker on naive (CD45RA?CCR7?)
CD8?T cells. The percentage of A2-
NS4B?CD8?T cells expressing the
relevant marker for each individual
(open circles) and the group mean
(horizontal line) are shown. MFI,
Mean fluorescence intensity. B, Flow
plots representing the phenotype of
YF-17D-specific CD8?T cells. Plots
are gated on total CD8?T cells (black
background) or YF-17D-specific (A2-
NS4B?) CD8?T cells (red dots).
Numbers show the percentage of A2-
NS4B?CD8?T cells in the gate.
Effector and memory
7924 YFV-17D INDUCES POLYFUNCTIONAL HUMAN CD8?T CELL MEMORY
7925 The Journal of Immunology
over a short period of time. We followed A2-NS4B?memory cells
at 3 mo and 2 years after vaccination in the same three individuals
to determine any such gradual changes (Fig. 5). After 2 years, the
frequency of A2-NS4B?memory cells in the blood of these indi-
viduals had decreased from 1.3, 0.43, and 0.36% at 3 mo to 0.13,
0.12, and 0.06%, respectively (data not shown). Phenotypic anal-
ysis revealed that strikingly, ?95% of the A2-NS4B?cells con-
tinued to express CD45RA even 2 years later, verifying that reex-
pression of this molecule was not a transient phase of memory
differentiation (Fig. 5A). In addition, uniform CD27 expression
and heterogeneous CD28 expression were observed at both early
(3 mo) and late (2 years) memory stages. However, we did observe
a subtle increase over time with respect to CCR7 and CD127, two
markers associated with high-quality immune memory (Fig. 5A).
The expression of CD45RA has been associated with senescence
in several studies (41), whereas CD127 confers the ability to re-
spond to IL-7 mediated survival signals (4). To further dissect the
issue of whether these memory cells were still capable of differ-
entiation, we examined two markers, CD56 and CD57, associated
with terminal effectors (Fig. 6). A2-NS4B?CD8?T cells did not
express either CD56, which is thought to identify CD8?T cells
with direct cytolytic activity (42), or CD57, which is associated
with replicative senescence (43), suggesting that YF-17D-specific
memory cells were not terminally differentiated even though they
expressed CD45RA and were mostly CCR7 negative, a phenotype
often associated with terminal effectors.
YF-17D elicits polyfunctional, long-lived memory CD8?T cells
CD8?T cells have a spectrum of functions to achieve viral
control. We evaluated five functions (degranulation and secre-
tion of IFN-?, TNF-?, Mip1?, and IL-2) of CD8?T cells rec-
ognizing the A2-NS4B epitope by stimulation of PBMC in vitro
with the NS4B 214–222 peptide followed by ICC staining and
multicolor flow cytometry. The kinetics of IFN-?-producing
CD8?T cells (Fig. 6A) matched that seen using tetramer-based
analysis (Fig. 3), with highest frequencies seen 30 days post-
vaccination. Upon assessing the composition of the cytokine-
producing population, we found that at every time point ana-
lyzed the cells that simultaneously produced IFN-? and TNF-?
dominated the response, and a prominent fraction of these cells
additionally produced IL-2 (Fig. 6A). Surface mobilization of
CD107a (lysosome-associated membrane protein (LAMP)-1)
after peptide stimulation indicates the ability to release cyto-
lytic granules (44). Independent and simultaneous measurement
of four functions showed that the majority of virus-specific
CD8?T cells from days 11 through 90 were CD107a?as well
as capable of IFN-?, TNF-?, and Mip-1? production at the
same time (Fig. 6B). Notably, this polyfunctional CD8?T cell
memory was retained even 2 years after vaccination (Fig. 6C).
Successful pathogen clearance depends on both quantity and
quality of CD8?T cells, and our findings show that highly
polyfunctional memory CD8?T cells are elicited and main-
tained after YF-17D vaccination.
A cardinal property of memory CD8?T cells is their ability to
undergo rapid proliferation upon reencountering Ag (5). This is an
important component of protective immunity; a higher prolifera-
tive potential implies a larger pool of secondary effectors. To ex-
amine the proliferative potential of A2-NS4B-specific CD8?T
cells, CFSE-labeled PBMC from vaccinees were stimulated in
vitro with the relevant peptide and the dilution of CFSE was as-
sayed as a measure of cell division (Fig. 7A). Expansion of A2-
NS4B?cells at day 90 was much higher (7.2-fold) compared with
day 14 (2.3-fold). This proliferative potential was preserved even
when ?95% of tetramer?cells were CD45RA?(supplemental
over 2 years. A, Comparison of the phenotype YF-17D-specific memory
CD8?T cells 3 mo and 2 years after vaccination. Representative flow plots
(gated on total CD8?T cells) from one of three vaccinees are shown. B,
Representative flow plots (gated on total CD8?T cells) show expression of
the late differentiation markers CD56 and CD57 on YF-17D-specific mem-
ory CD8?T cells in an individual vaccinated 2 years earlier.
The YF-17D-specific CD8?T cell memory is fine-tuned
7926YFV-17D INDUCES POLYFUNCTIONAL HUMAN CD8?T CELL MEMORY
with YF-17D. PBMC were isolated from vaccinees and responses to the NS4B 214–222 peptide were measured by ICC staining. Representative flow plots
(gated on total CD8?T cells) from one vaccinee are shown. B, Functional profile of the YF-17D-specific CD8?T cell response. All possible combinations
of four functions (degranulation by CD107a staining and secretion of IFN-?, TNF-?, and Mip-1?) are shown on the x-axis. Bars indicate the percentage
of the total response contributed by CD8?T cells with the combination of responses on the x-axis. Responses are grouped according to the number of
functions and the data summarized by the pie charts. Each slice of the pie represents the fraction of the total response that consists of CD8?T cells positive
for a given number of functions. Mean data from four YF-17D-immunized individuals are shown. C, Functional profile of the YF-17D-specific memory
CD8?T cells 2 years after vaccination. Plots gated on CD8?T cells show the cytokines produced in response to the stimulation of PBMC with the NS4B
214–222 peptide. Data from one representative vaccinee are shown.
YF-17D elicits polyfunctional, long-lived memory CD8?T cells. A, Kinetics of cytokine-producing CD8?T cells in individuals immunized
7927 The Journal of Immunology
Fig. 1).4Thus, YF-17D-specific CD8?T cells progressively ac-
quire proliferative potential over the course of time. The quality of
memory CD8?T cells generated in chronic viral infections is in-
ferior to that in response to acute viral infections (9). Ag-driven
proliferation in particular is reported to be lower (45, 46). We were
able to make a direct comparison in this regard between YF-17D,
an acute virus, and CMV, a chronic virus, in an individual vacci-
nated 1 year previously with YF-17D. This vaccinee had CD8?T
cells specific for A2-NS4B as well as for a CMV pp65-derived
epitope (A2-NLV). A2-NS4B?CD8?T cells expanded to higher
levels (8.6-fold) than A2-NLV-specific CD8?T cells (1.45-fold)
upon stimulation with the corresponding peptides, indicative of the
superior proliferation capability in YF-17D memory CD8?T cells
(Fig. 7B). Strikingly, A2-NS4B?cells that diluted CFSE could be
detected in an individual immunized 10 years previously (Fig. 7C).
This vaccinee had no history of recent travel to a yellow fever
endemic area, strongly suggesting that long-term persistence of
YF-17D-specific CD8?T cell memory does not require antigenic
To summarize, YF-17D generates a CD8?T cell response
that is high in magnitude, broadly diverse, and polyfunctional.
This results in memory CD8?T cells that progressively acquire
potent proliferative potential and retain it long after the virus is
Live viral vaccines like YF-17D offer a rare opportunity to study
the progressive differentiation of human Ag-specific CD8?T cells
that are generated as part of a protective antiviral immune re-
sponse. We have studied the magnitude, breadth, and dynamics of
the YF-17D-specific CD8?T cell response as a model to study the
generation and maintenance of CD8?T cell memory in the context
of acute viral infections. We used a set of overlapping peptides
spanning the entire virus to analyze the total antiviral CD8?T cells
and report several interesting findings. Firstly, each vaccinee re-
sponded to multiple proteins, and although some proteins such as
E, NS3, and NS5 were targeted more commonly, most viral pro-
teins contained CD8?T cell epitopes. Secondly, the magnitude of
the NS4B-specific response was particularly high in HLA-A2?
and not HLA-A2?vaccinees. We identified an immunodominant,
HLA-A2-restricted epitope (NS4B 214–222) responsible for this.
MHC class I tetramers specific for this epitope were used for
longitudinal analysis of A2-NS4B?CD8?T cells in a cohort of
HLA-A2?vaccinees. A2-NS4B?CD8?T cells had an activated
Ki67?Bcl-2lowHLA-DR?CD38?phenotype on day 14 that was
progressively lost by day 90. This differentiation was also accom-
panied by the acquisition of proliferation potential. Notably,
although the earliest detectable A2-NS4B?cells were heteroge-
neous for many markers, nearly all had the activated Ki67?Bcl-
2lowHLA-DR?CD38?phenotype, indicating a prominent phase
of activation and expansion. Lastly, A2-NS4B?memory CD8?T
cells were detected in circulation 2 years after vaccination. Inter-
estingly, a majority of these cells were CD45RA?CCR7?, a phe-
notype that has been associated with terminal differentiation and
lack of proliferative potential in many studies (6, 41, 47). How-
ever, they retained high proliferation potential 5–10 years after
vaccination and produced multiple cytokines upon antigenic re-
call in vitro. Overall, these characteristics may be used to un-
derstand the qualities of antiviral CD8?T cell memory in
Using an immunodominant epitope identified in YF-17D to
quantify virus-specific CD8?T cells over time, we observed ex-
pansion, contraction, and long-term maintenance typical of a pri-
mary CD8?T cell response. Tetramer?CD8?T cells were first
detected 11 days after vaccination, reached highest frequency at 30
days, diminished to ?30% of the peak by day 90, and only 5–10%
of the peak cell numbers were seen 1 year later. However, it is
evident that YF-17D-specific memory persists even longer, be-
cause A2-NS4B?cells were detectable in subjects vaccinated
5–10 years previously. Significantly, these individuals had no his-
tory of recent exposure to the virus, strongly suggesting that the
persistence of YF-17D CD8?memory is Ag independent.
Based on an estimated 1–3 ? 1011total CD8?T cells in humans
(48) and the percentage of tetramer?cells observed in the current
study (0.5–17% at the peak), the approximate number of A2-
NS4B?CD8?T cells is between 5 ? 108and 2 ? 109, compa-
rable to the number of total YF-17D-specific CD8?T cells (1.5–
9 ? 109) approximated on the basis of IFN-? production in our
earlier study (28). We found that at day 14 A2-NS4B?cells
formed a subset of total responding CD8?T cells; both popula-
tions had the Ki67?Bcl-2lowHLA-DR?CD38?activated pheno-
type (28). However, we saw discordance between the times at
which these two populations peaked. The highest frequency of
total activated cells was seen 2 wk after vaccination, whereas the
frequency of A2-NS4B?cells continued to increase for an addi-
tional 2 wk. This suggested that the YF-17D-specific CD8?T cells
continued to expand beyond day 14. In keeping with this, we could
detect Ki-67?CD8?T cells at day 21 with a frequency that was
70–75% lower than that seen at day 14 (data not shown). Because
naive CD8?T cell expansion is Ag driven, this indicated that
YF-17D could persist for slightly longer (possibly in tissues other
4The online version of this article contains supplemental material.
erative potential. A, Proliferative potential of YF-17D-specific effector (day
14) and memory (day 90) CD8?T cells. PBMC from vaccinated individ-
uals were labeled with CFSE and stimulated (Stim) or not (Unstim) with
the A2-NS4B peptide for 6 days. CFSE dilution and staining with the
A2-NS4B tetramer was used to identify divided cells. The percentage of
CD8?T cells that are A2-NS4B?cells is shown as the fold increase above
the percentage in blood. B, Expansion of YF-17D-specific memory CD8?
T cells compared with the expansion of CMV-specific CD8?T cells from
the same individual. CFSE-labeled PBMC were stimulated with either A2-
NS4B or the CMV-NLV peptide for 6 days. The percentage of CD8?T
cells that are tetramer?is shown as the fold increase above the percentage
in blood. C, Retention of proliferation potential by YF-17D-specific mem-
ory CD8?T cells. PBMC from an individual vaccinated with YF-17D 10
years previously were labeled and stimulated as described above and A2-
NS4B specific responses were measured. Plots are gated on CD8?T cells.
YF-17D elicits memory CD8?T cells with a robust prolif-
7928YFV-17D INDUCES POLYFUNCTIONAL HUMAN CD8?T CELL MEMORY
than blood) than indicated by the viral genomes in circulation that
are undetectable by day 14.
Elaboration of the breadth of YF-17D epitopes recognized
showed that vaccinees had CD8?T cells specific for multiple pro-
teins with variation among donors in the size of the response to
each protein. However, the hierarchy of CD8?T cell reactivity
established early on was retained through the memory stage as
evidenced in stimulations with recombinant vesicular stomatitis
virus expressing YF-17D proteins (data not shown). Interestingly,
specificity of CD8?T cells in HLA-A2?vaccinees was highly
skewed toward NS4B, where we later identified an epitope. In
some vaccinees up to 86.6% of the proliferating (Ki-67?) CD8?T
cells recognized the NS4B 214–222 epitope and the frequency was
as high as 17% of total CD8?T cells at the peak of expansion,
suggesting that the NS4B 214–222 epitope was highly immuno-
genic. Binding affinity of the peptide for MHC class I is an im-
portant factor contributing to the immunogenicity of CD8?T cell
epitopes, with most known peptide epitopes having high (IC50?
50 nM) or intermediate (IC50of 50–500 nM) binding affinities. We
speculate that the high binding affinity of the NSB 214–222 pep-
tide predicted for HLA-A2 alleles (IC50? 10 nM; Ref. 49) is
instrumental for the immunogenicity of the NS4B 214–222 pep-
tide. Also, the response to NS4B was much more vigorous com-
pared with that directed toward the more frequently recognized E,
NS3, and NS5 proteins. A similar observation has been reported
recently in a murine vaccinia infection where Oseroff and col-
leagues find that the most frequently recognized epitopes differ
from the immunodominant ones (50).
Studies of total CD8?T cell populations in healthy individ-
uals have provided useful T cell differentiation models by cat-
egorizing CD8?T cells into subsets based on their distinct phe-
notypes and finding functions unique to each subset. In one
widely accepted model, Ag-experienced CD8?T cells are clas-
sified into central memory cells (CD45RA?CCR7?), effector
memory cells (CD45RA?CCR7?), or CD45RA?effector mem-
ory cells (TEMRA; CD45RA?CCR7?) (13, 36). Unexpectedly,
YF-17D-specific memory cells shared the CD45RA?CCR7?phe-
notype of TEMRAcells. Cells with this phenotype are reported to
have very little proliferative capacity and to be sensitive to apo-
ptosis and associated with senescence. However, in contrast to the
“bulk” TEMRApopulation, A2-NS4B?cells had a high prolifera-
tive potential and did not express other markers of terminal dif-
ferentiation (CD56 and CD57). Recent reports showed that mem-
ory CD8?T cells for two acute viruses, vaccinia (7, 28) and B19
(51), were CD45RA?. Together with our findings, this strongly
suggests that CD45RA can be reexpressed in the absence of anti-
genic stimulation. Thus, the presence of CD45RA in Ag-experi-
enced cells may not always be associated with the lack of prolif-
erative potential and in fact may be an indicator of highly
functional memory CD8?T cells generated after acute viral
The smallpox vaccine (Dryvax) is another highly efficacious live
viral vaccine that offers some interesting comparisons with the
yellow fever vaccine. Possibly due to factors like the route of
vaccination used and the complexity and tropism of each virus,
there are some differences between the CD8?T cell responses
elicited by them. Firstly, Dryvax elicits a CD8?T cell response 4-
to 10-fold larger than that seen for YF-17D (28). Secondly, the
contraction of YF-17D-specific CD8?T cells seems more pro-
tracted (4-fold contraction by day 90) than that of Dryvax-specific
CD8?T cells (10- to 20-fold contraction by day 90) (28). Lastly,
in Dryvax vaccinees the peak of tetramer?cells occurs at the same
time as the effector (Ki-67?Bcl-2low) peak, unlike the discordance
seen for YF-17D vaccinees. Overall however, both vaccines elicit
a brisk, broadly targeting, polyfunctional CD8?T cell response (7,
28, 52) with similarities in the phenotype of virus specific-CD8?
T cells, such as the reexpression of CD45RA on memory cells.
Given the remarkable safety and efficacy of YF-17D, it has re-
cently been exploited to develop vaccine candidates against other
flaviviral diseases by substitution of the YF-17D M and E genes
with those of the target virus. YF-17D-based West Nile, Dengue,
and Japanese encephalitis chimeric vaccines have already been
evaluated in several phase I–III human trials (53–55). This strategy
is also being used to test vaccines against some pathogens unre-
lated to YF-17D, such as the malaria parasites of the plasmodium
genus (56–58). The current study may provide a useful benchmark
for evaluation of the responses to these vaccines that use YFV-17D
as a vector. In addition, the YF-17D epitope identified may be
useful in a simultaneous comparison of CD8?responses to vector
and recombinant proteins in chimeric vaccines, which may be use-
ful for ruling out vector-dominant responses as reported in a study
with an modified vaccinia Ankara recombinant vaccine (59).
We define the attributes of a human CD8?T cell response that
generates high-quality immune memory by performing a compre-
hensive analysis of the CD8?T cells elicited after vaccination with
the efficacious yellow fever live virus vaccine. The YF-17D-spe-
cific CD8?T cells displayed broad specificity, high magnitude,
multiple functions, robust proliferative potential, and long-term
persistence, all characteristics of protective cellular immunity. We
saw an interesting discordance between the “terminal effector-like”
phenotype of yellow fever memory CD8?T cells and their robust
recall potential. This observation restates the importance of mul-
tiparameter analysis for evaluating an immune response. Recently
available systems biology approaches to analyzing changes in
global gene expression profiles induced by the vaccine have
proved useful for examining the YF-17D-specific innate immune
response in depth (29). The ability to track YF-17D-specific CD8?
T cells using tetramers raises the possibility of defining the signa-
ture of high quality CD8?T cell memory in humans.
We thank K. Araki and R. Aubert for input and critical reading of the
manuscript and A. Popkowski for technical assistance. We also thank
Mario Roederer, National Institutes of Health, Vaccine Research Center,
for providing the PESTLE and SPICE software.
The authors have no financial conflict of interest.
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7930YFV-17D INDUCES POLYFUNCTIONAL HUMAN CD8?T CELL MEMORY