![Overall cytokine profile of peripheral blood leukocytes from healthy adults at day 0, day 7, day 15 and day 30 following YF-17DD primo-vaccination. Cytokine studies were performed after in vitro short-term incubation in the absence (non-stimulated culture) or in the presence of specific antigen stimuli (YF-17DD stimulated culture), as previously reported by Vitelli-Avelar et al. [22], consisting of a five step platform: (i) establishment of the global median of cytokine + cells; (ii) classification low ( for all cytokines) and high cytokine-producers ( – inflammatory and – regulatory), using the global median values as the cut-off edge (doted lines), as illustrated for monocytes in the top panels; (iii) creation of gray-scale diagrams (bottom panels) for each leukocyte subset, representing for each volunteer (V1–V10) the low cytokine-producers ( ), high cytokine-producers ( for predominant inflammatory profile; for predominant regulatory profile) and mixed cytokine-producers ( for simultaneous high inflammatory and regulatory profile); (iv) classification of the overall leukocyte cytokine profile predominant for each volunteer (column chart) and (v) definition of the final frequency of each cytokine pattern at day 0, day 7, day 15 and day 30 (pie chart).](https://www.researchgate.net/profile/Jose-Geraldo-Ribeiro-2/publication/45820590/figure/fig4/AS:352543289954311@1461064279160/Overall-cytokine-profile-of-peripheral-blood-leukocytes-from-healthy-adults-at-day-0-day_Q320.jpg)
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Vaccine 29 (2011) 583–592
Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Characterization of main cytokine sources from the innate and adaptive immune
responses following primary 17DD yellow fever vaccination in adults
Maria Luiza Silvaa,b, Marina Angela Martinsa,d, Luc¸ andra Ramos Espírito-Santo a,e,
Ana Carolina Campi-Azevedoa, Denise Silveira-Lemosa, José Geraldo Leite Ribeirof,
Akira Hommag, Erna Geessien Kroonh, Andréa Teixeira-Carvalhoa,
Silvana Maria Elói-Santosa,c, Olindo Assis Martins-Filhoa,∗
aLaboratório de Biomarcadores de Diagnóstico e Monitorac¸ ão, Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz, Brazil
bCentro de Pós-graduac¸ ão, Curso Patologia – Faculdade de Medicina – Universidade Federal de Minas Gerais, Brazil
cDepartamento de Propedêutica Complementar – Faculdade de Medicina – Universidade Federal de Minas Gerais, Brazil
dDepartamento de Bioquímica e Imunologia – ICB – Universidade Federal de Minas Gerais, Brazil
eUniversidade Estadual de Montes Claros, Campus Universitário Professor Darcy Ribeiro – Vila Mauricéia, Montes Claros, MG, Brazil
fSecretaria Estadual de Saúde do Estado de Minas Gerais, Brazil
gInstituto Biomanguinhos – Fundac¸ ão Oswaldo Cruz, Brazil
hDepartamento de Microbiologia – ICB – Universidade Federal de Minas Gerais, Brazil
article info
Article history:
Received 23 March 2010
Received in revised form 29 July 2010
Accepted 7 August 2010
Available online 21 August 2010
Keywords:
17DD vaccine
Yellow fever
Cytokines
Innate and adaptive immunity
abstract
The mechanisms of immune response following yellow fever (YF-17DD) vaccination are still poorly
understood. In this study, we have performed a longitudinal investigation (days 0, 7, 15 and 30) to char-
acterize the cytokine profile of innate and adaptive immunity following YF-17DD first-time vaccination.
Data from non-stimulated cultures demonstrated a prominent participation of the innate immunity with
increased frequency of TNF-␣+neutrophils and IFN-␥+NK-cells at day 7 besides TNF-␣+monocytes at day
7, day 15 and day 30. Increased frequency of IL-10+monocytes was observed at day 15 and day 30, and
decreased percentage of IL-4+NK-cells were detected at day 7, day 15 and day 30. Time-dependent and
oscillating cytokine pattern was observed in CD4+T-cells, with low percentage of IL-12+, IL-4+and IL-10+
cells at day 7 and increased frequency of TNF-␣+cells at day 15 besides IFN-␥+and IL-5+cells at day 15
and day 30. Later changes with increased percentage of IL-12+and IL-5+CD8+T-cells were observed at day
30. Increased frequency of IL-10+B-cells was observed at day 15, when seroconversion was detected in all
vaccinees. The overall cytokine analysis of non-stimulated leukocytes showed a transient shift towards
a pro-inflammatory profile at day 7, mainly due to changes in the innate immunity, which draws back
toward a mixed/regulatory pattern at day 15 and day 30. The changes induced by the in vitro YF-17DD
vaccine-stimulation were mainly observed at day 0 and day 7 (before seroconversion) with minor changes
at day 15 and day 30 (after seroconversion). These data support the hypothesis that a complex network
with mixed pro/anti-inflammatory cytokine profile is associated with the establishment of the protective
immunity following YF-17DD primo-vaccination, free of adverse events.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Yellow fever (YF) is a severe viral disease of humans caused
by YF virus, a mosquito-transmitted member of the Flaviviridae
family with clinical spectrum ranging from subclinical infection to
overwhelming pansystemic disease [1,2]. The disease is endemic
∗Corresponding author at: Laboratório de Biomarcadores de Diagnóstico e
Monitorac¸ ão – Centro de Pesquisas René Rachou, Fundac¸ ão Oswaldo Cruz, Avenida
Augusto de Lima, 1715 – Barro Preto – Belo Horizonte – Minas Gerais – 30 190 002,
Brazil. Tel.: +55 31 3349 7764; fax: +55 31 3295 3115.
E-mail address: oamfilho@cpqrr.fiocruz.br (O.A. Martins-Filho).
in sub-Saharan Africa and tropical regions of South America and
also in several Caribbean islands. In the past 20 years, the global
epidemic arboviral activity has dramatically increased with more
than 200,000 cases and 30,000 deaths registered a year [3,4]. The
disease can be prevented by a live attenuated vaccine, originally
prepared from the 17D strain, and developed following successive
passages of wild-type YF strain Asibi in mouse and chicken tissue.
Two substrains are currently used worldwide, 17D-204 and 17DD,
which are at passages 235–240 and 287–289, respectively, from
wild-type Asibi virus [5,6].
A single immunization induces seroconversion in more than 95%
of recipients with long-lasting neutralizing antibody levels besides
YF-specific T-cell responses with minimal incident of severe side
0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2010.08.046
584 M.L. Silva et al. / Vaccine 29 (2011) 583–592
effects [5,7–9]. Since 1937, this vaccine has protected more than
400 million humans from YF and it is considered as one of the most
successful live attenuated vaccines available [10–12]. Despite this
outstanding efficacy, the mechanisms underlying the robust immu-
nity triggered by the 17D-204 and 17DD YF vaccines are still poorly
understood.
Although, the humoral immune response induced by the YF vac-
cine is generally considered as the main mediator of protection
against infection with wild-type YF virus, the cellular immunity,
both innate and adaptive, has been pointed out as an important
event playing pivotal role to generate an effective protection fol-
lowing YF vaccination with YF-17DD (FIOCRUZ), YF-VAX (Aventis
Pasteur) and YF-VAX (Connaught Laboratories) [9,13–18].
Recently, a novel proposal has been raised by the scientific
community, suggesting that the induction of complex immune
response, including activation/modulation events as well as a
mixed cytokine profile, is in fact triggered by the YF-17D vaccina-
tion, which seems to play a pivotal role in inducing a protective
immunity and in avoiding adverse reactions in primo-vaccinees
[13–17,19,20].
In the current report, we have performed a detailed investi-
gation of intracytoplasmic cytokine pattern of peripheral blood
innate and adaptive leukocytes aiming to characterize the kinetics
and the major sources of cytokines following first-time vaccina-
tion with YF-17DD. Our findings confirmed the existence of mixed
pattern of cytokines following YF vaccination, highlighting in the
innate immunity compartment the relevance of neutrophils and
monocytes as an early source of TNF-␣and NK-cells supplying IFN-
␥, both counterbalanced by enhanced levels of IL-10+monocytes.
In the adaptive immunity environment the early and transient
downregulation of cytokine synthesis by CD4+T-cells were prompt
changed toward a mixed profile characterized by enhanced fre-
quency of IFN-␥+, TNF-␣+and IL-5+CD4+T-cells with CD8+T-cells
pointed as a late source of IL-12 and IL-5. B-cells were identi-
fied as the source of IL-10 at day 15, when seroconversion was
observed in all vaccinees. The overall cytokine signature showed
that the transient pro-inflammatory profile observed at day 7,
mainly due to the innate immunity cells, draws back toward a
mixed or modulated pattern at day 15 and day 30 in most vac-
cinees.
2. Patients, material and methods
2.1. Study population
This longitudinal investigation consisted of four consecutive
analysis of 10 healthy volunteers, aged 21–51 years with no his-
tory of previous YF vaccination or infection with the wild-type
YF virus, confirmed by negative plaque reduction neutralization
test (PRNT), carried out at Laboratório de Virologia, Departamento
de Microbiologia, Instituto de Ciências Biológicas, Universidade
Federal de Minas Gerais under supervision of Dra. Erna Geessien
Kroon.
Each volunteer was vaccinated subcutaneously with a single
0.5 mL dose of YF-17DD vaccine (Batch# 007VFA 010Z) as rec-
ommended by the manufacturer (Bio-Manguinhos, Oswaldo Cruz
Foundation, Brazil). Following vaccination, all volunteers were
questioned about clinical signs and symptoms, and none adverse
events were reported.
Peripheral blood samples (10 mL) were collected into
VacutainerTM tubes containing sodium heparin (Becton Dick-
inson, San Jose, CA, USA) at four consecutive periods including:
before vaccination (day 0) and seven (day 7), fifteen (day 15) and
thirty (day 30) days after YF-17DD primo-vaccination.
All subjects signed an informed consent, approved by the
Ethical Committee of the Centro de Pesquisas René Rachou
(CPqRR/FIOCRUZ – protocol # 03/2002), Belo Horizonte, Minas
Gerais, Brazil.
2.2. In vitro short-term whole blood culture
The short-term whole blood cultures were performed as
described by Peruhype-Magalhães et al. [21], modified as fol-
lows: 500 L aliquots of heparinized peripheral blood were
dispensed into triplicates of 14 mL polypropylene tubes (Falcon®,
B.D. Pharmingen, San Diego, CA) and incubated for 6 h at 37 ◦C
ina5%CO
2humidified atmosphere in the presence of 500 Lof
RPMI 1640 (GIBCO – Grand Island, NY) plus 50 L vaccine dilu-
ent (non-stimulated culture) or in the presence of 500 L of RPMI
1640 (GIBCO – Grand Island, NY) plus 50 L of live attenuated
YF-17DD vaccine (lot# 02UVFB005Z – BioManguinhos – FIOCRUZ)
at final concentration of 106viral particles/mL (YF-17DD stimu-
lated culture). After priming, 10 g/mL of Brefeldin A-BFA (Sigma
– Chemical Company – St Louis, MO) were added and the sam-
ples were re-incubated for an additional 4 h at 37 ◦Cina5%
CO2humidified atmosphere. Following incubation, the cultures
were treated with 2 mM ethylenediamine tetraacetic acid—EDTA,
(Sigma – Chemical Company – St Louis, MO) and maintained at
room temperature for 15 min. Following EDTA treatment, the trip-
licates were pooled together into one 14 mL polypropylene tubes
and then washed once with 6 mL of FACS buffer (0.015 M phos-
phate buffered saline—PBS, supplemented with 0.5% bovine serum
albumin—BSA and 0.1% sodium azide) by centrifugation at 600 ×g
for 7 min at room temperature. After centrifugation, the super-
natant was discarded and the cell pellet was resuspended in 1 mL
of FACS buffer. The cell suspension was immediately submitted
to immunophenotyping and intracytoplasmic cytokine analysis by
flow cytometry.
Positive control cultures were performed in order to evaluate
the sample viability. For this purpose, 500 L aliquots of whole
blood were incubated in the presence of 500 L of RPMI 1640
plus Phorbol 12-Myristate 13-Acetate-PMA, ionomycin and BFA at
final concentration of 25 ng/mL, 1 g/mL and 10 g/mL, respec-
tively. Positive control cultures were incubated for 4 h at 37 ◦C
ina5%CO
2humidified incubator. Positive control (PMA stimu-
lated culture) characterized by high levels of IFN-␥+and TNF-␣+
cells were used in all immunophenotyping and intracytoplasmic
cytokine analysis to confirm cell viability of all blood samples (data
not shown).
2.3. Immunophenotyping and intracytoplasmic cytokines
analysis by flow cytometry
The analysis of intracytoplasmic cytokines in peripheral blood
leukocyte subsets was performed as described by Peruhype-
Magalhães et al. [21]. Briefly, 200 L aliquots of EDTA-treated
cultures were dispensed into five 12 ×75 mm polystyrene tubes
and individually stained with anti-human cell surface mark-
ers monoclonal antibodies (mAbs), including anti-CD4 (clone
RPA-T4), CD8 (clones RPA-T8), CD14 (clone TüK4), CD16 (clone
3G8) and CD19 (clones 4G7 or SJ25-C1) labeled with fluores-
cein isothiocyanate (FITC) or TRI-COLOR (TC), purchased from
BD Pharmingen (San Diego, CA, USA) or Caltag (Burlingame, CA,
USA). Following incubation for 30 min at room temperature in
the dark, stained samples were gently treated while vortexing
with 2 mL of FACS erythrocyte lysing solution (Becton Dickin-
son Biosciences) and re-incubating for an additional 10 min at
room temperature in the dark. After erythrocyte lysis, the sam-
ples were centrifuged at 600 ×gfor 7 min at room temperature,
the supernatant discarded and the cell pellet resuspended and
M.L. Silva et al. / Vaccine 29 (2011) 583–592 585
incubated with 2 mL of FACS permeabilizing solution (FACS buffer
supplemented with 0.5% of saponin) for 10 min at room tem-
perature in the dark. Following incubation the samples were
centrifuged at 600 ×gfor 7 min at room temperature, the super-
natant decanted gently and the cell pellet was washed with 3 mL
of FACS buffer. After centrifugation, the cells were resuspended
in 200 L of FACS buffer and distributed in 30 L aliquots over
96-well U-bottomed microplates. Cells were then stained with
20 L of phycoerythrin (PE)-labeled anti-cytokine mAbs, includ-
ing anti-IFN-␥(clone B27), TNF-␣(clone Mab11), IL-12 (clone
C11.5.14), IL-10 (clone JES3-9D7), IL-4 (clone MP4-25D2) and IL-
5 (clone TRFK5), all purchased from BD Pharmingen (San Diego,
CA, USA), previously diluted 1:50 in sterile FACS permeabiliz-
ing solution. After incubation for 30 min at room temperature
in the dark, the cells were washed twice with 200 L of FACS
permeabilizing solution and FACS buffer, respectively, fixed with
200 L of FACS fixing solution (10 g/L paraformaldehyde, 10.2 g/L
sodium cacodylate and 6.63 g/L sodium chloride, pH 7.2) and
stored at 4 ◦C in the dark prior flow cytometry acquisition within
24 h.
2.4. Flow cytometry acquisition and analysis
Flow cytometry acquisition and analysis were performed in a
FACScaliburTM flow cytometer equipped with four colors detec-
tion system (Becton Dickinson, San Jose, CA, USA), using the
CELLQUEST software (Franklin Lakes, NJ, USA). After acquir-
ing 30,000 events/tube, distinct gating strategies were used
to analyze the different cytokine-expressing leukocytes sub-
sets, including innate (neutrophils, monocytes and NK cells)
and adaptive immunity cells (CD4+, CD8+T-cell subsets and B-
cell).
Selective analysis of neutrophils was performed by establishing
a specific scatter gate using the dot plot distribution of anti-CD16-
FITC and laser side scatter (SSC) to discriminate the neutrophils
as SSChighCD16high+ . Analysis of monocytes was performed using
the dot plot distribution of anti-CD14-TC and SSC to discriminate
the monocytes as SSCintCD14high+ cells. The selection of NK cells,
T-cell subsets and B-cells was performed by initially gating the
lymphocytes on forward scatter (FSC) versus SSC dot plot distri-
bution, followed by analysis on anti-CD16-FITC, anti-CD19-FITC,
anti-CD8-FITC or anti-CD4-TC. Following the selection of leuco-
cytes subset, the frequency of cytokine+cells was determined
using quadrant statistics over FL-1/anti-cell surface marker-FITC
or FL-3/anti-cell surface marker-TC versus FL-2/anti-cytokine-PE
dot plot distribution. The results were expressed as percentages of
cytokine+cells for different gated leucocytes subpopulations ana-
lyzed.
2.5. Analysis of overall cytokine profile
The analysis of overall cytokine profile was assessed to charac-
terize the general cytokine pattern of each vaccinee as previously
suggested by Vitelli-Avelar et al. [22], modified as follows: the
percentages of cytokine+cells were compiled using a five step plat-
form, as follows: (i) establishment of the global median of cytokine+
cell for each leukocyte subset, taking all values from day 0, day
7, day 15 and day 30 as illustrated for monocytes in Fig. 5 – top
graphs; (ii) classification of each leukocyte subsets as low (for
all cytokines) and high cytokine-producers (䊉for inflammatory
cytokines: IFN-␥, TNF-␣and IL-12; for regulatory cytokines: IL-10,
IL-4 and IL-5), using the global median values as the cut-off edge,
as illustrated for monocytes in Fig. 5 – top graphs; (iii) creation
of gray-scale diagrams for each leukocyte subset, representing for
each volunteer (V1–V10) the low cytokine-producers ( ), high
cytokine-producers ( for predominant inflammatory profile;
for predominant regulatory profile) and mixed cytokine-producers
(for simultaneous high inflammatory and regulatory profile)
(Fig. 5); (iv) classification of the overall leukocyte cytokine pro-
file predominant for each volunteer (Fig. 5 – column chart); and (v)
definition of the final frequency of each cytokine pattern at day 0,
day 7, day 15 and day 30 (Fig. 5 – pie chart).
2.6. Statistical analysis
Statistical analysis was performed by paired Wilcoxon’s Ttest
for comparisons between day 0, day 7, day 15 and day 30, using the
GraphPad Prism 4.03 software package (USA). Significant differ-
ences are identified in the figures by the letters ‘a’, ‘b’, ‘c’ and ‘d’ for
comparisons between day 0, day 7, day 15 and day 30, respectively.
Significant differences between non-stimulated and YF-17DD stim-
ulated cultures are highlighted by asterisks. In all cases, significance
was considered at p≤0.05.
3. Results
3.1. Prominent participation of innate immunity is observed in
healthy adults after YF-17DD primo-vaccination
Aiming to characterize the pro- and anti-inflammatory cytokine
profiles of innate immunity cells following YF-17DD vaccination,
we have analyzed the ex vivo intracytoplasmic cytokine pattern
of peripheral blood leukocytes after short-term culture of whole
blood samples, particularly in the absence of exogenous stimuli,
here referred as non-stimulated culture (Fig. 1). Our data showed a
prominent involvement of innate immunity cells, as demonstrated
by increased frequency of TNF-␣+neutrophils at day 7 as com-
pared to day 0. Moreover, analysis of monocytes demonstrated
increased percentage of TNF-␣+cell at day 7, day 15 and day 30
as compared to day 0 and increase of IL-10+cells at day 15 as com-
pared to day 0 and day 7, and at day 30 as compared to day 0.
Furthermore, our data showed increased frequency of IFN-␥+NK-
cells at day 7 as compared to day 0, and decreased percentage of
IL-4+NK-cells at day 7, day 15 and day 30 as compared to day
0.
The analysis of the cytokine patterns of innate immunity
leukocytes in the YF-17DD stimulated culture also demonstrated
significant differences on the cytokine profile of neutrophils,
monocytes and NK-cells (Fig. 1). Significant decrease of IL-10+neu-
trophils was observed at day 30 as compared to day 7. Moreover,
significant increase of IL-10+monocytes was found at day 30 as
compared to day 7. Analysis of NK-cells showed decreased levels
of IL-4+cells at day 7 as compared to day 0.
Comparative analysis of non-stimulated and YF-17DD stimu-
lated cultures pointed out a prominent and selective increase of
TNF-␣+monocytes at all times evaluated and an increase of IFN-␥+
NK-cell at day 0 (Fig. 1 – asterisks).
Taken together, these findings emphasize that the YF-17DD
vaccination comprises a complex cytokine network at the
innate immunity compartment, involving both pro- and anti-
inflammatory cytokines produced by neutrophils, monocytes and
NK cells. The major changes in the innate immunity compartment
are summarized in Fig. 4.
3.2. Time-dependent and oscillating changes in the cytokine
profile of CD4+T-cells with late involvement of CD8+T-cells is the
hallmark of adaptive cellular immune response following
YF-17DD primo-vaccination
In order to evaluate the dynamic of events in the cytokines
network concerning the adaptive cellular immune response, we
have characterized the cytokine profile of circulating CD4+and
586 M.L. Silva et al. / Vaccine 29 (2011) 583–592
Fig. 1. Intracellular cytokine profile of innate immunity leukocytes in peripheral blood of healthy adults at day 0, day 7, day 15 and day 30 following YF-17DD primo-
vaccination. Phenotypic and intracytoplasmic cytokine studies were performed after in vitro short-term incubation in the absence (non-stimulated culture – left axis for
TNF-␣+monocytes) or in the presence of specific antigen stimuli (YF-17DD stimulated culture – right axis for TNF-␣+monocytes), using anti-CD16-FITC mAbs to identify
neutrophils (SSChighCD16high+ ) and NK-cells (CD16+lymphocytes) or anti-CD14-FITC for monocytes (SSCintCD14high+) together with anti-cytokines-PE mAbs to determine
inflammatory (top panels) and regulatory (bottom panels) cytokines+cells. The results are expressed in box plot format, with boxes stretching from the 25th percentile to
the 75th percentile and the line across the box representing the median values. Significant differences at p< 0.05 are identified by the letters “a” and “b” for comparison with
day 0 and day 7, respectively. Differences between non-stimulated and YF-17DD stimulated cultures at p< 0.05 are identified by *.
CD8+T-cells after short-term incubation in vitro either in the
absence or presence of YF-17DD stimuli (Fig. 2). Data analysis of
non-stimulated culture demonstrated a time-related change in the
cytokine profile of CD4+T-cells, with decreased frequency of IL-
12+and IL-4+cells at day 7 as compared to day 0 and day 30 and
decreased percentage of IL-10+cells as compared to day 0 and day
15. On the other hand, increased frequency of TNF-␣+CD4+T-cells
was observed at day 15 as compared to day 7. Moreover, increased
frequency of IFN-␥+and IL-5+CD4+T-cells was observed at day 15
and day 30 as compared to day 0 and day 7.
Differently, the CD8+T-cells exhibited later changes in the
cytokine pattern, as demonstrated by enhanced frequency of IL-
12+and IL-5+CD8+T-cells at day 30 as compared to day 0, day 7,
and day 15, respectively.
The analyses of YF-17DD stimulated culture demonstrated
similar time-dependent and oscillating changes in the cytokine
profile of CD4+and CD8+T-cells. Data analysis pointed towards
decreased frequency of IL-12+, IL-4+and IL-10+CD4+T-cells at
day 7. On the other hand, increased percentage of IL-5+CD4+
T-cells was observed at day 7, day 15 and day 30 and IFN-␥+
CD4+T-cells at day 30. Similarly, increased frequency of IL-12+
CD8+T-cells was observed late at day 30 as compared to day
7.
The comparison between non-stimulated and YF-17DD stimu-
lated culture also demonstrated a time-dependent impact on CD4+
T-cells with decreased frequency of IL-10+cells observed at day 0
and increased percentage of IL-4+, IL-10+and IFN-␥+cells observed
at day 7, day 15 and day 30, respectively (Fig. 2 – asterisks).
Together these data also highlights that the YF-17DD vaccina-
tion comprises a complex and time-dependent cytokine network at
the adaptive cellular immunity compartment involving both pro-
and anti-inflammatory cytokines produced by CD4+and CD8+T-
cells. The major changes in the adaptive immunity compartment
are summarized in Fig. 4.
3.3. The increased frequency of IL-10+B-cell is observed at day 15
when seroconversion was already observed in all YF-17DD adult
primo-vaccinees
Aiming to characterize the phenotypic features of the humoral
immune response, we have analyzed the ex vivo levels of seric neu-
tralizing antibodies and also characterized the in vitro cytokine
profile of circulating B-cells after short-term incubation in the
absence or in the presence of YF-17DD stimuli (Fig. 3). Data analysis
demonstrated that despite three out of ten vaccinees seroconverted
at day 7, at day 15 all vaccinees already presented seroconver-
sion pattern as demonstrated by PRNT titer above the cut-off
line (200 mUI/mL). Analysis of intracytoplasmic profile in the non-
stimulated cultures demonstrated increased frequency of IL-10+
B-cells at day 15 concomitant with the seroconversion of all vac-
cinees. Despite no changes observed in the cytokine profile of
B-cells upon YF-17DD stimuli, the comparative analysis of non-
stimulated and YF-17DD stimulated cultures revealed an increase
in the percentage of TNF-␣+B-cells at day 30 (Fig. 3 – aster-
isk). The change in the B-cell compartment is summarized in
Fig. 4.
3.4. The analysis of the overall cytokine profile of circulating
leukocytes further emphasize the complex cytokine network
triggered by the YF-17DD adult primo-vaccination
Aiming to further characterize the cytokine profile of periph-
eral blood leukocytes following the YF-17DD primo-vaccination,
we have used a novel strategy to access the overall cytokine profile
M.L. Silva et al. / Vaccine 29 (2011) 583–592 587
CD 4+T-cells
days aer vaccinaon
% of cytokine+cells
InflammatoryRegulatory
% of cytokine+cells
InflammatoryRegulatory
CD 8+T-cells
IFN-
γ
+
a,b,c
a,b
TNF-
α
+
IL-4+
IL-12+
b
b
IFN-
γ
+TNF-
α
+
IL-4+
a,b
0 7 15 30 0 7 15 30
IL-5+
a
IL-5+
a
a,b
b
a,d
IL-12+
a,d
d
a,b,c
0
0.5
1
0
0.5
1
*
0
0.5
1
0
0.5
1
*
0
0.5
1
a,b
0
0.5
1
0
0.5
1
0
0.5
1
0
0.5
1
0
0.5
1
a
d
IL-10+
IL-10+
a,c
*
*
0
1.3
2.6
0
1.3
2.6
071530
c
Fig. 2. Intracellular cytokine profile of adaptive cellular immunity lymphocytes in peripheral blood of healthy adults at day 0, day 7, day 15 and day 30 following YF-17DD
primo-vaccination. Phenotypic and intracytoplasmic cytokine studies were performed after in vitro short-term incubation in the absence (non-stimulated culture )orinthe
presence of specific antigen stimuli (YF-17DD stimulated culture ) using anti-CD4-FITC mAbs to identify T-helper cells (CD4+lymphocytes) and anti-CD8-TC mAbs to label
cytotoxic T-cells (CD8+lymphocytes) together with anti-cytokines-PE mAbs to determine inflammatory (top panels) and regulatory (bottom panels) cytokines+lymphocytes.
The results are expressed in box plot format, with boxes stretching from the 25th percentile to the 75th percentile and the line across the box representing the median
values. Significant differences at p< 0.05 are identified by letters “a”, “b”, “c” and “d” for comparison with day 0, day 7, day 15 and day 30, respectively. Differences between
non-stimulated and YF-17DD stimulated cultures at p< 0.05 are identified by *.
proposed by Vitelli-Avelar et al. [22] to determine the kinetic fre-
quency of cytokine producers ( for low, for high-inflammatory,
for high-regulatory and for mixed cytokines producers) at day
0, day 7, day 15 and day 30 following YF-17DD primo-vaccination.
(Fig. 5 – gray-scale diagrams, columns and pie charts).
The overall cytokine analysis of non-stimulated leukocytes
showed a transient shift from a predominant mixed pattern at day
0 (observed in 60% of the subjects) towards a pro-inflammatory
profile at day 7 (observed in 60% of primo-vaccinees), mainly due
to changes in the innate immunity compartment. This inflam-
matory profile draws back toward a mixed/regulatory pattern at
day 15 and day 30 with most vaccinees (80%) tending to restore
the mixed or regulatory overall cytokine profile observed at day
0.
Upon YF-17DD stimuli, the major changes were observed at day
0 and day 7 (before seroconversion) with minor changes at day 15
and day 30 (after seroconversion). At day 0 the antigen stimula-
tion shifted the cytokine profile toward an inflammatory pattern
in 2 out of 10 volunteers. On the other hand, at day 7 the antigen
stimulation was able to completely abolish the inflammatory pro-
file previously observed in the non-stimulated culture. At day 15,
when seroconversion is already observed in all vaccinees, no evi-
dent change in the overall cytokine profile was observed between
non-stimulated and YF-17DD stimulated cultures, highlighting the
588 M.L. Silva et al. / Vaccine 29 (2011) 583–592
B-cells
days aer vaccinaon
Inflammatory
Regulatory
% of cytokine+cells
TNF-
α
+
*
0
0.5
1
IL-10+
071530
b
0
0.5
1
PRNT
days aer vaccinaon
071530
ter (mIU/mL)
10
100
1,000
10,000
100,000
Fig. 3. Phenotypic features of adaptive humoral immunity in peripheral blood of healthy adults at day 0, day 7, day 15 and day 30 following YF-17DD primo-vaccination.
YF-17DD specific seric antibody levels (left panel) were quantified by plaque reducing neutralizing test (PRNT ) and the results expressed as mIU/mL. The dashed line
represents the cut-off line used to segregate negative and positive results (200 mIU/mL). Phenotypic and intracytoplasmic cytokine data (right panels) were obtained after
in vitro short-term incubation in the absence (non-stimulated culture ) or in the presence of specific antigen stimuli (YF-17DD stimulated culture ), using anti-CD19-FITC
mAbs to identify B-cells (CD19+lymphocytes) together with anti-cytokines-PE mAbs to determine inflammatory (top panels) and regulatory (bottom panels) cytokines+
lymphocytes. All results are expressed in box plot format, with boxes stretching from the 25th percentile to the 75th percentile and the line across the box representing
the median values. Significant difference at p< 0.05 is identified by the letter “b” for comparison with day 7. Difference between non-stimulated and YF-17DD stimulated
cultures at p< 0.05 is identified by *.
day 7 day 15 day 30
↑
↑
Neu TNF-
α
+a
↑
CD8+IL-12+b
↓
CD4+IL-10+a,c
↓
CD4+IL-4+a,d
↑
CD4+IL-5+a
↑
CD4+IL-5+a,b
↑
CD4+IFN-
γ
+a,b
↑
CD4+IFN-
γ
+a,b
↑
CD4+TNF-
α+
b
↑
CD8+IL-5+a,b,c
↑
CD19+IL-10+b
Non-smulated culture
↓
CD4+IL-12+a,d
↑
Mon IL-10+a
↑
Mon IL-10+a
↑
Mon TNF-
α
+a
↑
Mon TNF-
α
+a
↑
Mon TNF-
α
+a
↓
NK IL-4+a
↓
NK IL-4+a
↓
NK IL-4+a
Innate immunity
Adapve immunity
↑
NK IFN-
γ
+a
Fig. 4. Major changes in the cytokine profile of innate and adaptive immunity leuko-
cytes in peripheral blood samples from healthy vaccinees at day 7, day 15 and day
30 following YF-17DD primo-vaccination. Increased (↑) and decreased (↓) levels of
inflammatory (in black) and regulatory (in gray) cytokines highlight the complex
cytokine network triggered by the YF-17DD primo-vaccination. Letters “a”, “b”, “c”
and “d” represent significant differences at p< 0.05 for comparisons with day 0, day 7,
day 15 and day 30, respectively. Rectangles emphasize persistent changes observed
during the kinetic follow-up study.
ability of PRNT antibodies to minimize the impact of YF antigen on
the immune system. At day 30, the YF-17DD antigenic stimulation
was accompanied by similar frequency of vaccinees with mixed
cytokine profiles and induced a slight shift towards an inflamma-
tory pattern (20–40%) reflecting the increased levels of TNF-␣+
monocytes and B-cells and IFN-␥+CD4+T-cells observed at day 30
following in vitro stimulation of YF-17DD antigens (Figs. 1–3).
4. Discussion
The major goal of this study was to identify the major sources of
pro- and anti-inflammatory cytokines, aiming to add new elements
to the complex cytokine network triggered by the YF-17DD primo-
vaccination. These findings may help not only for the understanding
of the immunological mechanism underlying the establishment of
protective immunity but also supply new insights to support the
investigation of severe adverse diseases following YF-17D vaccina-
tion. Moreover, considering the outstanding performance of the YF
vaccine, the detailed identification of each cell source of cytokine
may also help the rational development of new vaccines.
For several decades, it has been postulated that a potent pro-
inflammatory immune response, mediated by Type-1-cytokines,
such as IFN-␥and TNF-␣, was crucial to the development of anti-YF
protective immunity [23–26]. The pioneer investigation of Whee-
lock and Sibley have demonstrated that YF 17D-204 vaccinees
displayed increased plasmatic levels of IFN-␥(measured by a bioas-
say), peaked at 24 h after viremia, which declined rapidly thereafter
[23]. In addition, elevated levels of the IFN-dependent enzyme 2,5-
oligoadenylate synthase have been demonstrated in T and B-cells
M.L. Silva et al. / Vaccine 29 (2011) 583–592 589
Fig. 5. Overall cytokine profile of peripheral blood leukocytes from healthy adults at day 0, day 7, day 15 and day 30 following YF-17DD primo-vaccination. Cytokine studies
were performed after in vitro short-term incubation in the absence (non-stimulated culture) or in the presence of specific antigen stimuli (YF-17DD stimulated culture), as
previously reported by Vitelli-Avelar et al. [22], consisting of a five step platform: (i) establishment of the global median of cytokine+cells; (ii) classification low (for all
cytokines) and high cytokine-producers (䊉– inflammatory and – regulatory), using the global median values as the cut-off edge (doted lines), as illustrated for monocytes
in the top panels; (iii) creation of gray-scale diagrams (bottom panels) for each leukocyte subset, representing for each volunteer (V1–V10) the low cytokine-producers
(), high cytokine-producers ( for predominant inflammatory profile; for predominant regulatory profile) and mixed cytokine-producers ( for simultaneous high
inflammatory and regulatory profile); (iv) classification of the overall leukocyte cytokine profile predominant for each volunteer (column chart) and (v) definition of the final
frequency of each cytokine pattern at day 0, day 7, day 15 and day 30 (pie chart).
590 M.L. Silva et al. / Vaccine 29 (2011) 583–592
early after the immunization with the YF 17D-204 vaccine [24].
Reinhardt et al. have observed an increased level of neopterin, a
protein induced by INF-␥, following primary YF-17D-204 immu-
nization [25]. Moreover, increased plasmatic levels of TNF-␣have
also been reported at day 2 and day 7 after primo-vaccination with
YF-17D-204 [26].
Martins et al. have hypothesized that a controlled microenviron-
ment including mechanism of activation/modulation of both innate
and adaptive immunity seems to be the key to the development
of protective immunity triggered by the YF-17DD vaccine [14,15].
Moreover, Gauche et al. have suggested that a mixed Th1/Th2
cytokine response that appears early and is persistent after primo-
vaccination, characterized by increased levels of IL-2, IFN-␥and
TNF-␣besides IL-4 and IL-10 profiles. These findings are observed
following in vitro stimulation of peripheral blood mononuclear cells
(PBMC) isolated from YF-17D-204 vaccinees [16]. Santos et al. have
recently reported that a mixed cytokine profile is observed in YF-
17-DD primo-vaccinees [17]. Despite these new insights toward the
novel concept of mixed cytokine signature following the YF vacci-
nation, the cell sources of specific cytokines triggered after 17DD
vaccination are still unknown.
The current study have added new elements to the complex
cytokine network triggered by the YF-17DD vaccination, demon-
strating that the transient inflammatory profile observed at day
7 was mainly due to increased frequency of TNF-␣+neutrophils
and monocytes and IFN-␥+NK-cells triggered after vaccination
suggesting an effective role of innate immune cells to the estab-
lishment of this microenvironment. TNF-␣is the major component
of the immune system involved in the control of virus infec-
tion through direct antiviral activity, usually in association with
interferons and the induction of apoptosis [27]. Hacker et al.
have previously reported an increase in the seric levels of TNF-
␣early after the YF-17D-204 vaccination [26]. Our findings add
new elements to this observation, suggesting that monocyte and
neutrophils might represent the major sources of TNF-␣at day
7 after YF-17D vaccination. The increased frequency of TNF-␣+
cells neutrophils and monocytes as well as the increased percent-
age of IFN-␥+NK-cells may reflect the establishment of anti-viral
immune response triggered by the viremia typically reported
from days 3 to 7 post-vaccination following 17D vaccination
[25,28]. Corroborating this data, Martins et al. have demonstrated
that increased frequency of macrophage-like (CD14+CD16+), acti-
vated monocytes (CD14+CD16High+), pro-inflammatory monocytes
(CD14+CD16+HLA-DR++) as well as increased frequency of activated
neutrophils (CD28+, CD16High+ and CXCR4+) can be observed at
day 7 after YF-17DD vaccination [15]. Moreover, Neves et al. have
described an increased expression of activation markers in circu-
lating NK-cells [18].
Our data demonstrated that the increased frequency of TNF-
␣+monocytes can be observed throughout the post-vaccination
period up to day 30. We believe that this phenomenon may be
important to control the occurrence of the severe adverse dis-
ease after YF-17D vaccination (YEL-AVD), since we have previously
reported that YEL-AVD is associated with impaired TNF-␣synthesis
by monocytes at day 15 after vaccination [20].
It was interesting to notice that the early and persistent increase
in the percentage of TNF-␣+monocytes observed throughout the
post-vaccination period was counterbalanced after day 15 by an
increased frequency of monocytes IL-10+cells, electing the mono-
cytes as an important cell-type involved in modulatory event
triggered by the YF-17DD vaccination. These data are consistent
with those previously reported by Martins et al., demonstrating
a significant increase of IL-10R+on monocytes at day 15 after
YF-17DD vaccination [15]. It has been demonstrated that endoge-
nous IL-10 produced by human monocytes/macrophages is able
to inhibit the production of pro-inflammatory cytokines by mono-
cytes/macrophages, such as IL-1, IL-6, IL-8 and TNF-␣, and also
inhibit the synthesis of IL-1, IL-8 and TNF-␣by neutrophils. This
may represent an important mechanism controlling the transient
pro-inflammatory response observed at day 7 after YF vaccination.
Moreover, it has been demonstrated that IL-10 also regulates the
growth and/or differentiation of NK-cells, T-cell subsets and B-cells
[29,30].
In the context of adaptive immune system, our results show an
early and transient downregulation of cytokine synthesis by CD4+
T-cells (IL-12, IL-4 and IL-10). We believe that the impairment of
cytokine synthesis by CD4+T-cells observed at day 7 after YF-17DD
vaccination may be due in part to the direct impact of peak of
viremia temporally observed at this time after primo-vaccination.
This may also represent a modulatory mechanism to counter bal-
ance the intense activation of the innate immunity observed not
only in neutrophils, monocytes and natural killer cells but probably
also in DCs, representing an important cross-talk between innate
and adaptive immunity that re-enforces the hypothesis of simulta-
neous activation/modulation following YF-17DD vaccination.
In agreement with the hypothesis that a mixed cytokine
signature is triggered by the YF-17D vaccine [13–17], our
data demonstrated a simultaneous increase in pro- and anti-
inflammatory cytokines in T-cell subsets at day 15 and day 30.
Specifically, increased levels of IFN-␥+, TNF-␣+and IL-5+CD4+T-
cells observed at day 15 followed by persistent increase in the
percentage of IFN-␥+and IL-5+CD4+T-cells concomitant with late
increase IL-12+and IL-5+CD8+T-cells at day 30. The late recruit-
ment of CD8+T-cells following YF-17DD vaccination has been
already reported by Martins et al., as demonstrated by increased
frequency of CD8+HLA-DR+at day 30 after 17DD vaccination [14].
Santos et al. have also presented data that support the existence
of mixed cytokine signature at day 15 after vaccination, as demon-
strated by increased levels of IFN-␥and IL-4 following in vitro
stimulation of PBMC from YF-17DD vaccinees [17]. In our study, we
did not detect any major source of IL-4+cells throughout the post-
vaccination periods analyzed. In fact, we have observed a decrease
in the frequency of IL-4+cells, mainly in NK-cells (day 7, day 15
and day 30) and CD4+T-cells (day 7). We believe that method-
ological particularities, such as the use of long-term incubation of
isolated PBMCs in the absence of autologous plasma [17] instead
the use of short-term incubation of whole blood samples may be
related to these apparently distinct data. In our model, the presence
of autologous plasma, rich on YF-neutralizing antibodies probably
had a strong impact in the microenvironment that does not turn out
in expressive IL-4 synthesis. Further analyses are currently under
evaluation to investigate this hypothesis.
The increased frequency of IL-5+T-cells at day 15 and day 30 may
represent an important mechanism linked to the B-cell activation
process. Some studies had demonstrated levels of interleukin IL-
5 in culture supernatant following 17D-YF vaccination [16,31].In
mouse, IL-5 has been showed to be essential for the differentiation
of antibody-secreted B-cells [32,33], whereas in human this still
remain controversial [34–36]. Our results demonstrated a signifi-
cant increased frequency of IL-10+B-cells at day 15 concomitant
with the seroconversion of all vaccinees. Interestingly, Martins et
al. have already reported that at day 15 post-vaccination a signifi-
cant increase in the expression of IL-10R can be observed in parallel
with enhanced frequency of activated B-cells [14]. In humans, IL-
10 potentiates DNA replication on B-cells and is also a very potent
factor to induce B-cell differentiation and secretion of immunoglob-
ulins [37–40]. The IL-10/IL-10R interaction has been pointed out as
an important event to prevent apoptosis and enhances the prolif-
eration and differentiation of B-cells towards plasma cells [41,42].
In attempt to characterize the overall cytokine profile of periph-
eral blood leukocytes following the YF-17DD primo-vaccination,
we have used a novel strategy to access the overall cytokine profile
M.L. Silva et al. / Vaccine 29 (2011) 583–592 591
proposed by Vitelli-Avelar et al. [22]. Our results demonstrated that
the mixed cytokine pattern of whole blood cells before YF-17DD
vaccination (day 0) was transitorily shifted toward an inflamma-
tory profile at day 7, mainly due to changes in the innate immunity
cells, with the most relevant finding being the enhanced frequency
of TNF-␣+monocytes. This transient inflammatory cytokine pattern
shifted toward a mixed/regulatory profile in 80% of the vacci-
nees remaining with this feature at day 30 in most vaccinees.
This mixed/regulatory pattern could be in part supported by the
increase of TNF-␣+/IL-10+monocytes as well as IFN-␥+/TNF-␣+/IL-
5+CD4+T-cells, IL-12+/IL-5+CD8+T-cells and IL-10+B-cells.
Additional analysis revealed that the 17DD stimulation in vitro
was able to induce even before vaccination (day 0) an inflammatory
profile in 20% of the volunteers, probably due to the direct action
of the viral antigen on the innate immunity cells, since these sub-
jects were not previously primed by YF antigens. Moreover, at day
7 the overall inflammatory profile observed in the non-stimulated
culture was overturned toward a mixed/regulatory pattern proba-
bly due to an over stimulation of the innate immunity cells by the
antigenic booster in vitro. The massive antigenic stimulation, rep-
resented by the in vivo viremia plus the exogenous antigen stimuli
in vitro led to changes in the cytokine profile of leukocyte subsets
of both innate and adaptive compartments with a general impact
in the overall frequency of cytokine profile. Interestingly, at day 15
and day 30, when all primo-vaccinees already presented YF neu-
tralizing antibodies, the in vitro 17DD antigen stimulation induced
minor changes in the overall cytokine profile.
In conclusion, our findings confirmed previous reports, suggest-
ing that a parallel activation/modulation microenvironment with a
mixed cytokine signature is the key to the establishment of protec-
tive immunity mechanisms triggered by the YF-17D vaccine. The
novel contribution of this work was the possibility to identify the
major sources of pro- and anti-inflammatory cytokines that con-
tribute to this complex microenvironment, highlighting the role of
the innate immunity (TNF-␣+neutrophils, TNF-␣+monocytes and
IFN-␥+NK-cells, counterbalanced by IL-10+monocytes) as well as
the adaptive immunity (IFN-␥+and TNF-␣+CD4+T-cells and IL-12+
CD8+T-cells modulated by IL-5+CD4+T-cells, IL-5+CD8+T-cells
and IL-10+B-cells).
Acknowledgments
This work was supported by Centro de Pesquisas René Rachou
– FIOCRUZ-MG and Instituto Bio-manguinhos – FIOCRUZ-RJ by the
grant # carta compromisso 05/05 05/08, CNPq (Grant #422782/09)
and FAPEMIG (APQ 01183-08). The authors thank the program for
technological development in tools for health – PDTIS – FIOCRUZ
for the use of its facilities. EGK, ATC and OAMF thank CNPq for fel-
lowships (PQ). ACCA acknowledge the FAPEMIG for the fellowship
(PDJ 00265/09).
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