Trypanosoma cruzi Adjuvants Potentiate T Cell-Mediated
Immunity Induced by a NY-ESO-1 Based Antitumor
Caroline Junqueira1,2, Ana Tereza Guerrero3, Bruno Galva ˜o-Filho1,2, Warrison A. Andrade1,2,4, Ana
Paula C. Salgado1, Thiago M. Cunha5, Catherine Ropert1, Marco Anto ˆnio Campos1, Marcus L. O. Penido2,
Lu ´cia Mendonc ¸a-Previato6, Jose ´ Oswaldo Previato6, Gerd Ritter7, Fernando Q. Cunha5,
Ricardo T. Gazzinelli1,2,4*
1Laborato ´rio de Imunopatologia, Centro de Pesquisas Rene ´ Rachou, Fundac ¸a ˜o Oswaldo Cruz, Belo Horizonte, Brazil, 2Departamento de Bioquı ´mica e Imunologia,
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 3Instituto Cerrado Pantanal, Fundac ¸a ˜o Oswaldo Cruz, Campo Grande, Brazil, 4Division of Infectious Diseases
and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 5Departamento de Farmacologia, Faculdade de
Medicina de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Ribeira ˜o Preto, Brazil, 6Instituto de Biofı ´sica Carlos Chagas Filho, Centro de Cie ˆncias da Sau ´de, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 7Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan–Kettering Cancer Center, New York, New York,
United States of America
Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the
development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived
from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like
Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as
immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the
bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were
comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4+T and
CD8+T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi
elicited interferon-gamma (IFN-c) production by CD4+T cells. On the other hand, the parasite derived CpG ODNs, but not
GIPLs, elicited a potent IFN-c response by CD8+T lymphocytes. The side effects were also evaluated by local pain
(hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an
analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1
expressing melanoma was associated with the magnitude of both CD4+T and CD8+T cell responses elicited by a specific
Citation: Junqueira C, Guerrero AT, Galva ˜o-Filho B, Andrade WA, Salgado APC, et al. (2012) Trypanosoma cruzi Adjuvants Potentiate T Cell-Mediated Immunity
Induced by a NY-ESO-1 Based Antitumor Vaccine. PLoS ONE 7(5): e36245. doi:10.1371/journal.pone.0036245
Editor: Mauricio Martins Rodrigues, Federal University of Sa ˜o Paulo, Brazil
Received November 28, 2011; Accepted March 29, 2012; Published May 2, 2012
Copyright: ? 2012 Junqueira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from The Atlantic Philanthropies/Ludwig Institute for Cancer Research (LICR) – Clinical Discovery Program; Conselho
Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico, Brazil (CNPq) – Instituto Nacional de Cie ˆncia e Tecnologia de Vacinas; and Fundac ¸a ˜o de Amparo a ` Pesquisa
do Estado de Minas Gerais, Brazil (FAPEMIG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
NY-ESO-1 is a human cancer/testis antigen that is frequently
expressed in a variety of cancer cells, but not in normal adult
tissues apart from testis [1,2]. Both humoral and T cell-mediated
immunity (TCMI) specific for NY-ESO-1 develop in patients with
NY-ESO-1-positive tumors; and several major histocompatibility
complex (MHC) class II and I restricted peptides have been
defined as the epitopes recognized by CD4+T as well as CD8+T
lymphocytes, respectively [3,4,5]. The immunogenicity and tissue
distribution indicate that NY-ESO-1 is an excellent candidate
antigen for prophylactic and therapeutic anticancer vaccines.
Hence, different vaccine formulations employing NY-ESO-1 have
been developed aiming at efficient antitumor activity. Most
formulations combine heterologous prime-boost protocols to
achieve satisfactory immunogenicity and tumor regression in
experimental models [6,7]. Importantly, different clinical trials
have shown the ability of NY-ESO-1 vaccines to induce specific
cytolytic T lymphocytes as well as CD4+T cell-mediated immune
responses in humans [8,9].
However, the quality of the T cell response and protection
against tumors still remains a major challenge for vaccine
development. One of the main difficulties is the limited availability
of licenced immunological adjuvants that induce strong and long-
lasting TCMI with the least undesirable effect. The discovery that
activation of Toll-Like Receptors (TLRs) promote the initiation
and development of both T cell and B cell responses has intensified
the search for new immunological adjuvants . Indeed, various
PLoS ONE | www.plosone.org1 May 2012 | Volume 7 | Issue 5 | e36245
microbial components as well as synthetic components previously
shown to work as immunological adjuvants were proven to be
TLR agonists . When exposed to microbial components, cells
from the innate immune system, synthesize high levels of pro-
inflammatory cytokines and express co-receptors, in order to
initiate the activation process of naı ¨ve T cells, bridging the innate
and acquired immunity . Importantly, dendritic cells (DCs)
activated with TLR agonists will produce interleukin (IL)-12 and
influence the differentiation of CD4+T cells into the T helper type
1 (Th1) phenotype, which orchestrate the establishment of cell-
mediated immunity as well as the production of interferon-gamma
(IFN-c)-inducible Ig isotypes that are often involved in host
resistance to tumors [13,14,15]. Furthermore, activation of antigen
presenting cells favors cross-presentation, allowing presentation of
exogenous antigens via MHC class I [16,17]. Currently, several
vaccines based on association of tumor antigens with defined TLR
agonists (e.g., Poly I:C, Monophosphoryl Lipid A, Flagellin, CpG
oligodeoxynucleotides, Imiquimods) are being tested in pre-clinical
and clinical trials [15,18,19].
We have previously shown that glycosylphosphatidylinositol
(GPI) anchors linked to mucin-like glycoproteins, and the
ceramide-containing GPI anchors, also termed glycoinositolpho-
spholipids (GIPL), present in outer plasmatic membrane of the
Trypanosoma cruzi are immunostimulatory molecules for TLR2 and
TLR4, respectively [20,21]. It was also demonstrated that CpG
oligodeoxynucleotide (CpG ODN) motifs derived from the T. cruzi
genome activate TLR9 . We believe that this is the molecular
basis of the highly polarized Th1 response and strong TCMI
elicited during infection with T. cruzi parasites.
In this study, we evaluated the T. cruzi derived TLR agonists as
immunological adjuvants in vaccine formulations employing
ovalbumin (OVA) or NY-ESO-1 as antigens. Our results show
that formulations containing either CpG ODNs or GIPL induced
immune responses mediated by CD4+Th1 lymphocytes. In
particular, parasite derived CpG ODNs, but not GIPL, elicited a
potent IFN-c response by CD8+T lymphocytes. We also
evaluated adjuvant-induced hypernociception and showed that
there was no correlation with the quality of the immune response,
and alum was the main cause of ‘‘pain’’ in the vaccine
formulations. Immune-mediated protection against melanoma
development directly correlated with the magnitude of IFN-c
responses by both NY-ESO-1-specific CD4+T as well as CD8+T
cells. Finally, the use of the analgesic Paracetamol (PCM) did not
alter the immunogenicity and protective immunity elicited by
these novel vaccine formulations employing parasite adjuvants.
Materials and Methods
Mice experiments were approved by and conducted according
to animal welfare guidelines of the Ethics Committee of Animal
Experimentation from Universidade Federal de Minas Gerais
under the title ‘‘Parasite derived adjuvants for cancer vaccines’’ and
approved protocol number 19/2008.
Mice and cell lines
C57BL/6 mice, originally obtained from Jackson Laboratory,
were kept under standard pathogen-free conditions. Six- to eight-
week-old females, weight-matched, were used in the different
experimental groups. CHO transfected cells  and B16F10 wild
type (WT) and NY-ESO-1-transfected  cell lines were
maintained in culture in RPMI 1640 supplemented with
100 IU/ml penicillin, 10 mg/ml streptomycin, 10% heat-inacti-
vated fetal bovine serum (FBS), 1 mg/ml tylosin and maintained at
37uC in a 5% CO2incubator. B16F10 cell line expressing NY-
ESO-1 were supplemented with 300 mg/ml geneticin.
Removal of lipopolysaccharides from ovalbumin
Chicken OVA (Sigma-Aldrich, St. Louis, MO) was diluted in
pyrogen-free saline at 10 mg/ml and depleted of the endotoxin
activity using five cycles of Triton X-114 extractions . OVA
concentration was determined by Bradford assay, and adjusted to
1 mg/ml. The endotoxin levels in purified OVA were measured
by Gel-Clot Limulus amoebocyte lysate reagent (Charles River
Laboratories, Wilmington, MA) and found to be below the limit
of detection (0.03 EU/ml).
NY-ESO-1: recombinant protein and T cell epitope
The recombinant NY-ESO-1 protein was produced under
Good Manufacturing Practice (GMP) at the Ludwig Institute for
Cancer Research/Cornell University Partnership Production
Facility in Ithaca, New York.
In silico prediction of MHC class I (H2-Kb, H2-Db and H2-Lb)
and class II (H2-IAb) ligands from NY-ESO-1 were determined by
the software Bimas  and SYFPEITHI , respectively. The
MHC restricted peptides from Ovalbumin (OVA CD4+T cell
epitope – ISQAVAAHAEINEAGR and OVA CD8+T cell
epitope – SIINFEKL) and the restricted peptides from NY-ESO-1
(NY-ESO-1 CD4+T cell epitopes - CD4-1 QAEGRGTGGSTG-
NAN, CD4-2 AGGPGEAGATGGRGP, and CD4-3 FYLAMP-
FATPMEAEL; as well as NY-ESO-1 CD8+T cell epitopes - CD8-
1 TVSGNILTI, CD8-2 SCLQQLSLL, and CD8-3 LLEFYLAM)
were synthesized by standard N-9-fluorenylmethyloxycarbonyl on
a PSSM8 multispecific peptide synthesizer (Shimadzu, Kyoto,
Japan) by solid- phase synthesis with a scale of 30 mM and a purity
.85%, as determined by reverse-phase HPLC. Their identities
were confirmed by Autoflex III Maldi-TOF/TOF Mass Spec-
trometer (Bruker Daltonics, Billerica, MA). For in vitro lympho-
cytes restimulation, each peptide was used at 10 mM final
T. cruzi GIPLs: purification and in vitro
The isolation and purification of GIPL has been previously
described in detail [23,28]. Briefly, epimastigotes of T. cruzi (Y and
Tulahuen strains) were grown in BHI-hemin medium supple-
mented with 5% FBS. T. cruzi in stationary growth phase were
extracted three times with cold water and the remaining cell pellet
extracted with 45% aqueous phenol. The aqueous layer from the
phenol extraction was dialyzed and applied to a column of Bio-Gel
P-100 (Bio-Rad, Hercules, CA). The excluded material was
lyophilized and the free GPIs extracted by chloroform/methanol/
water (10:10:3). The virtual absence of contaminating peptidic
material was confirmed by the absence of peptide-derived signals
in nuclear magnetic resonance spectroscopy and mass spectrom-
etry analyses of the purified material. The GIPL preparation tested
negative for Lipopolysaccharides (LPS) content using a Limulus
amebocyte lysate test, with a limit of detection of 0.03 EU/ml
(Charles River Laboratories, Wilmington, MA).
The CHO reporter cell lines (CHO/CD14, expressing
endogenous functional TLR4; 7.19/CD14/TLR2, expressing
TLR2; and the 7.19 clone, expressing neither TLR2 nor
functional TLR4) were generated as described . These cell
lines contain a human CD25 gene reporter under the control of E-
selectin promoter and CD25 expression is completely dependent
upon NF-kB translocation. Macrophage-activating lipopeptide
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2 kDa (MALP-2; Alexis Biochemicals, San Diego, CA) and LPS
(Sigma-Aldrich, St. Louis, MO) were used as controls at 10 gg/ml
and 200 gg/ml, respectively. TLR4+cells that showed activation
were additionally treated 15 min with 1 mg/ml of polymyxin B
(Sigma-Aldrich, St. Louis, MO), an inhibitor of LPS, prior to
GIPLs exposure. Cells were exposed to the different molecules,
and analyzed 18 hours after stimulation, through staining with PE
CD25 (Caltag Laboratories, Burlingame, CA) and flow cytometry
analyzes by BD CellQuest Pro Software (Becton, Dickinson and
Company, Franklin Lakes, NJ). The results were obtained by
subtracting the percentage of cells expressing the reporter gene in
stimulated versus not stimulated cell populations. Ten thousand
cells were analyzed in each sample.
CpG ODN: sequences and in vitro immunostimulation
Table 1 shows the sequences of mouse B-class-like CpG ODNs,
mouse-human hybrid B class-like CpG ODNs and human B-class-
like CpG ODNs derived from T. cruzi genome . T. cruzi
derived CpG ODNs, as well as positive and negative controls for
GpG ODNs were synthesized by Alpha DNA (Montreal, Quebec,
Canada) as phosphorothioate ODNs and purified by oligonucle-
otide purification cartridge.
For IL-12 production assays, inflammatory macrophages from
mice injected with 1,5 ml of 3% thioglycolate were plated at
56106cells/ml, and incubated at 37uC and 5% CO2for 72 h in
the presence or absence of LPS or CpG ODNs at different
concentrations. IL-12 concentrations were determined in cell
culture supernatants with DuoSet ELISA (R&D Systems,
Minneapolis, MN). Two hundred thousand peripheral blood
mononuclear cells (PBMCs) were cultured in 96-well plates in the
presence of CpG ODNs at different concentrations associated with
DOTAP (Roche, Indianapolis, IN) for 24 h, and interferon-alpha
(IFN-a) was measured in the cell culture supernatant with DuoSet
ELISA (R&D Systems, Minneapolis, MN).
Vaccine formulations and immunization protocols
Vaccine formulations were prepared with 10 mg/ml OVA or
5 mg/ml NY-ESO-1 and TLR agonists co-adsorbed in 30% (v/v)
of alum Rehydragel L.V. solution (Reheis, Berkeley Heights, NJ)
for 1 hour at room temperature in a tube rotator. After
incubation, saline solution was added to each sample to the final
concentration of 100 mg/ml of the antigen. The final concentra-
tion of each agonist was used as follows: 10 mg/ml Synthetic
MPLA (InvivoGen, San Diego, CA); 10 mg/ml Pam3Cys (Alexis
Biochemicals, San Diego, CA); 180 mg/ml CpG [positive control
(+), negative control (2), or T. cruzi derived B344, B297, B128];
500 mg/ml GIPL (TcGY or TcGTH). All the procedures were
developed using endotoxin free supplies in a sterile environment.
Four to six mice per group were immunized with alum, alum
plus OVA or alum plus OVA plus TLR agonists. Each mouse
received three subcutaneous (s.c.) doses of vaccine formulations 14
days apart. Sera were collected 9 days after the last immunization
and spleens were collected 12 days later for analysis of immune
responses. Mice that were immunized with NY-ESO-1 received
only two immunizations 21 days apart. In parallel, a group of mice
were treated with 10 mg/kg of PCM by the oral route 30 minutes
prior to each immunization dose.
Measurement of antibody and T cell responses
Vaccinated mice were bled from the retro-orbital plexus under
ether anesthesia. Antigen-specific antibodies were measured in
sera from immunized mice by enzyme-linked immunosorbent
assay (ELISA). Secondary Ab, peroxidase-conjugated goat anti-
mouse total Immunoglobulin G (IgG), IgG1 or IgG2c (South-
ernBiotech, Birmingham, AL) were used and the reactions were
detected with 3,39,5,59-tetramethylbenzidine reagent (Sigma-
Aldrich, St. Louis, MO).
For IFN-c production assays, splenocytes from vaccinated mice
were prepared in complete RPMI supplemented with 100 U/ml
rIL-2 (R&D Systems, Minneapolis, MN), plated at 56106cells/ml
and incubated at 37uC and 5% CO2for 72 h in the presence or
absence of epitopes derived from OVA or NY-ESO-1 proteins.
IFN-c concentrations were determined in cell culture supernatants
with DuoSet ELISA (R&D Systems, Minneapolis, MN). To
confirm peptides specificity, CD8+T and CD4+T cells were
isolated from total splenocytes using Dynabeads (Invitrogen Dynal,
Oslo, Norway), and plated at 56106cells/ml for peptides addition.
Total splenocyte from vaccinated mice were stained ex vivo with
FITC CD3 (BD Pharmingen, San Jose, CA) and PE-Cy5 CD8
(BD Pharmingen, San Jose, CA) antibodies as well as PE H2-Db
CD8 NY-ESO-1127–135tetramer and analyzed in a FACSCalibur
(Becton, Dickinson and Company, Franklin Lakes, NJ).
Evaluation of mechanical hypernociception
The term hypernociception rather than hyperalgesia or
allodynia is used to define the decrease in nociceptive withdrawal
threshold . Mechanical hypernociception was tested in mice as
previously reported . In a quiet room, mice were placed in
acrylic cages (12610617 cm) with wire grid floors, 15–30 min
before the start of testing. The test consisted of evoking a hindpaw
flexion reflex with a handheld force transducer (electronic
anesthesiometer; IITC Life Science, Woodland Hills, CA) adapted
Table 1. Sequences of synthesized CpG oligonucleotide.
CpG 7909 [CpG(+)]*TCGTCGTTTTGTCGTTTTGTCGTT
GpG [CpG (2)] TCCAGGACTTCTCTCAGGTT
CpG 2007TCGTCGT TGTCGTTTTGTCGTT
*CpG ODNs used for immunization protocolsz.
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with a 0.5-mm2polypropylene tip. The investigator was trained to
apply the tip perpendicularly to the central area of the plantar
hindpaw with a gradual increase in pressure. The gradual increase
in pressure was manually performed in blinded experiments. The
upper limit pressure was 15 g. The end-point was characterized by
the removal of the paw followed by clear flinching movements.
After paw withdrawal, the intensity of the pressure was
automatically recorded, and the final value for the response was
obtained by averaging three measurements. The animals were
tested before and after treatments. The results are expressed by the
delta (n) withdrawal threshold (in g) calculated by subtracting the
zero time mean measurements from the mean measurements at
the indicated times after drug or solvent (controls) injections.
Withdrawal threshold was 9.060.2 g (mean6S.E.M.) before
injection of solvent or hypernociceptive agents.
B16F10 or B16 NY-ESO-1 melanoma challenge
Mice vaccinated with recombinant NY-ESO-1 were challenged
s.c. at day 21 after boost with 56104B16F10 melanoma cells
expressing or not the cancer antigen NY-ESO-1. Tumor growth
was monitored during 40 days.
All the statistic analysis were performed by GraphPad Prism
Software Version 5.0 b (GraphPad Software, Inc., La Jolla, CA).
The non-parametric group comparison was developed by Mann-
Whitney test and the parametric data by T test. The tumor
development data was analyzed by two-way ANOVA with
additional Bonferroni post-test analysis. Survival curves were
analyzed by Log-rank test. Differences were considered significant
T. cruzi derived GIPLs activate TLR4 and promote
antigen-specific IgG2c and CD4+T cell responses
The ability of GIPLs to activate TLR2 and TLR4 was
investigated in CHO cells functional for TLR4 (TLR4+) or not
functional for TLR4 and stably transfected with TLR2 (TLR2+)
(Figure 1). As positive controls, we used LPS and MALP-2 as
TLR4 and TLR2 agonists, respectively. CHO cells transfected
only with CD25 reporter gene were used as negative controls
(TLR22TLR42). In this system, expression of CD25 is completely
dependent upon NF-kB translocation. In Figure 1A it is shown
that GIPLs derived from the Y strain of T. cruzi (GY) leads to 15%
enhancement of CD25 expression in TLR4+cells. To certify that
activation of TLR4+cells were not due to LPS contamination, the
same experiments were performed in the presence of polymyxin B
(PB), a compound that is known to bind LPS and prevent TLR4
activation. The experiment shown in Figure 1B demonstrates
activation of TLR4+cells by T. cruzi GIPL even after treatment
with PB. In contrast activation with LPS was completely blocked
by pre-treatment with PB. Thus, we consider that the activation of
TLR4+CHO cells by GIPLs from the Y strain of T. cruzi was not
due to a LPS contamination.
The GIPLs were also compared with well-established TLR2
and TLR4 agonists, Pam3Cys and MPL, respectively, for their
capacity to promote immunological responses in an immunization
protocol using OVA as antigen. All the evaluated TLR agonists
associated with alum plus OVA induced antigen specific total IgG,
as well as IgG1 and IgG2c. In contrast, mice that received only
alum plus OVA induced antigen specific total IgG and IgG1, but
not IgG2c isotype (Figure 1C). Splenocytes from vaccinated mice
were restimulated with OVA-specific peptides for CD4+T and
CD8+T cells. The levels of IFN-c in the supernatant of splenocyte
cultures was measure by ELISA (Figure 1D). Our data shows that
MPL and Pam3Cys promoted IFN-c production by OVA-specific
CD4+T and CD8+T cells. In contrast, the GIPL from T. cruzi
induces IFN-c production only by CD4+T cells. Since the GIPL
derived from the Y strain of T. cruzi (GY) was the best parasite
adjuvant to induce CD4+T cell responses, it was chosen to be used
in the vaccine formulations employing NY-ESO-1 as antigen.
CpG ODNs derived from the T. cruzi genome activate
TLR9 and promote antigen specific IgG2c as well as CD4+
T and CD8+T cell responses
As previously reported , CpG ODNs derived from T. cruzi
genome activated human and mouse cells through TLR9 to
produce IFN-a and IL-12 respectively (Fig. 2A and 2B). The T.
cruzi derived CpG motifs B344, B297 and B128 were compared to
the bacterial CpG motif (CpG 7909), as a positive control, for their
capacity to induce immune response in a vaccine formulation
containing OVA as antigen (Fig. 2C and 2D). Similar levels of
anti-OVA antibodies were detected in sera from mice immunized
with distinct TLR9 agonists, including the IgG2c isotype (Fig. 2C).
The IFN-c production by splenocytes restimulated with OVA-
specific peptides demonstrates that all the CpG ODNs were able
to induce IFN-c production by both CD4+T and CD8+T cells
(Fig. 2D). The best results were obtained in mice immunized with
CpG ODNs 7909 and B344, indicating a potential for the T. cruzi
derived CpG ODN B344 to be used in vaccine formulations
combined with NY-ESO-1.
Mapping of CD4+T and CD8+T cell epitopes from the
cancer/testis antigen NY-ESO-1
The ORF of the NY-ESO-1 gene was analyzed for regions that
bind to MHC Class I or II, in order to identify NY-ESO-1 specific
T cell epitopes. To select the best CD4+T cell epitope and CD8+
T cell epitope, three different epitopes for MHC class I and three
for MHC class II were tested in an ex vivo assay (Figure 3). C57BL/
6 mice were immunized with alum alone, alum plus NY-ESO-1,
and alum plus NY-ESO-1 plus CpG ODN 7909. The IFN-c
production by NY-ESO-1-specific T cells was detected after
restimulation of splenocytes with the different peptides (Fig. 3A).
The CD8-1 and CD4-3 epitopes induced higher levels of IFN-c
production and were selected to be used in the next experiments.
A schematic illustration shows the localization and sequence of
NY-ESO-1 epitopes selected for this study (Fig. 3B). The
specificity of selected peptides was confirmed employing highly
purified CD4+T or CD8+T cell subsets. Our results show that
CD4+T and CD8+T cells produced IFN-c only when stimulated
with peptides CD4-3 or CD8-1, respectively (Fig. 3C).
A T. cruzi derived CpG motif promotes both CD4+T and
CD8+T cell responses to NY-ESO-1 and delays
development of the B16F10 melanoma cell line
To test the ability of the T. cruzi derived TLR agonists to induce
protective immunity to tumor development, we immunized mice
with alum plus NY-ESO-1 associated with GIPL GY, CpG ODN
B344 or positive and negative controls. The capacity of the vaccine
formulations to induce immunological responses anti-NY-ESO-1
and to protect mice against a challenge with B16F10 melanoma
cell line expressing NY-ESO-1 was measured (Figure 4). The
immunized mice were evaluated for the serum levels of anti-NY-
ESO-1 total IgG, IgG1 and IgG2c isotypes. The levels of total
IgG, IgG1 and IgG2c were similar when comparing the mice that
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received different TLR agonists. In contrast, mice that received
alum plus NY-ESO-1 without a TLR agonist produced high levels
of antigen-specific total IgG and IgG1, but not IgG2c (Fig. 4A).
The cellular immune responses were evaluated by measuring the
levels of IFN-c produced by splenocytes from immunized mice
after restimulation with NY-ESO-1 derived CD4+T or CD8+T
cell epitopes. Our results show that CpG ODN B344 and 7909
(positive control) induced similar levels of IFN-c (Fig. 4B).
Importantly, in mice challenged with B16F10 melanoma express-
ing NY-ESO-1, the CpG ODN B344 was the most effective TLR
agonist in delaying tumor growth. No protection was observed in
mice that received the wild type (non-transfected) B16F10
melanoma, indicating that the delay of tumor growth was
mediated by NY-ESO-1-specific immune responses (Fig. 4C).
Treatment with paracetamol alleviates adjuvant-induced
hypernociception, but does not affect the protective
immunity induced by the NY-ESO-1 vaccine
One important aspect of immunological adjuvants is the side
effect including the development of inflammation and pain in the
site of vaccine injection. In this context, we evaluated whether the
immunostimulatory effect of parasite adjuvants and complete
vaccine formulation was associated with local hypernociception
(decrease in nociceptive threshold). As positive controls we used
MPL, Pam3Cys and bacterial CpG motifs. As expected, all the
TLR agonists, positive controls or the enquired ones, induced
significant increase in hypernociception (Fig. 5A). In a second set
of experiments, we used the complete vaccine formulation,
including alum and NY-ESO-1 as adjuvant (Fig. 5B). Our results
Figure 1. T. cruzi derived GIPLs are TLR4 agonists and promote high levels of antigen-specific IgG2c antibodies as well as IFN-c
production by CD4+T cells. (A) CHO cells control (TLR22/TLR42) or expressing TLR2 (TLR2+) or TLR4 (TLR4+) were either left untreated (solid gray)
or exposed to 100 mg/ml of GIPLs from Trypanosoma cruzi Tulahuen (GTH), Y strain (GY) (black line). MALP-2 (10 gg/ml) and LPS (200 gg/ml) were
used as positive controls for activation of TLR2+or TLR4+, respectively. (B) TLR4+cells were activated with different preparations of GIPLs in the
presence of polymyxin B (PB). LPS was used as control. (C) OVA specific immune responses induced by immunization with TLR2 or TLR4 agonists
associated with OVA absorbed in alum. Mice were immunized with three doses on days 0, 14 and 28. The production of total IgG, IgG1 and IgG2c
were assessed by ELISA using the sera from immunized mice, at day 9 after the second boost. (D) To assess the levels of IFN-c production by T
lymphocytes from vaccinated mice, splenocytes were collected 21 days after the third immunization dose and stimulated with either CD4+T or CD8+
T cell epitopes from OVA. The results are representative of two independent experiments yielding similar results. Asterisks indicate that differences
were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.
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show that alum on its own induced an augmentation of
hypernociception, which was not further augmented by the
association with specific TLR agonists. Thus, we did not find a
correlation with the quality of the immune response (i.e., TCMI
and protective immunity) and ‘‘pain’’. In fact, the presence of alum
was the determinant factor for hypernociception in our vaccine
formulations. Importantly, PCM partially blocked hypernocicep-
tion in mice inoculated with our vaccine formulations. Parallel
experiments were performed by immunizing mice with the
previously described formulations with NY-ESO-1 antigen in
association or not with the PCM treatment (Fig. 6). Neither
humoral (Fig. 6A) nor cellular (Fig. 6B) immune responses were
Figure 2. Immunostimulatory and adjuvant activity of TLR9 agonists derived from T. cruzi genome. (A) PBMCs derived from healthy
donors were stimulated with human B-class-like CpG ODNs derived from the T. cruzi genome with four different concentrations (3.0, 1.0, 0.3, and
0.1 mM) and the levels of IFN-a measured in the cell culture supernatants 24 h later. The CpG ODN 2007 was used as positive controls for human B-
class ODNs. PBMC experiments were performed in three different donors, yielding similar results. (B) Proinflammatory activity of mouse B class-like
CpG motifs was evaluated in inflammatory macrophages from WT (C57BL/6), TLR42/2and TLR92/2mice. ODNs were tested at different
concentrations (1.5, 0.3 and 0.06 mM) and LPS, as well as CpG ODN 7909 were used as positive controls for TLR4 and TLR9 activation, respectively. IL-
12 (p40) was measured in the macrophage culture supernatants 24 h after cellular stimulation. (C) C57BL/6 mice received three immunization doses
with alum alone, OVA plus alum or OVA plus alum associated with either CpG ODNs B344, B287, B128 or 7909 (positive control). The levels of OVA-
specific total IgG, IgG1 and IgG2c were assessed by ELISA. (D) Amount of IFN-c secreted by splenocytes after stimulation with OVA derived CD4+T or
CD8+T cell epitopes was evaluated in culture supernatants 72 hours post-stimulation. Asterisks indicate that differences were statistically significant,
when comparing T cell response from mice receiving different vaccine formulations.
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Figure 3. Mapping of immunostimulatory CD4+T and CD8+T cell epitopes present in NY-ESO-1. (A) C57BL/6 mice were immunized with
alum alone, alum plus rNY-ESO-1 associated or not with CpG ODN 7909 to evaluate the immunostimulatory activity of peptides encoding the
putative CD4+T and CD8+T cell epitopes from NY-ESO-1. Mice received three immunization doses at day 0, 14 and 28. Splenocytes were harvested 21
days after the last immunization dose, restimulated in vitro with different NY-ESO-1-specific peptides, and the levels of IFN-c production measured in
the cell culture supernatants by ELISA. Asterisks indicate that differences in IFN-c responses to a specific CD4+T or CD8+T cell peptides (CD8-1, CD8-3
and CD4-3) were statistically significant (p,0.001), when comparing splenocytes from mice receiving the same vaccine formulation, stimulated with
CD8-2, CD4-1, CD4-2, or left unstimulated. (B) A schematic illustration shows the sequence and position of immunostimulatory CD4+T and CD8+T
cell epitopes selected from NY-ESO-1 to be used in this study. (C) CD4+T and CD8+T lymphocytes were enriched from total spleen cells of
immunized mice by magnetic beads. Each subpopulation was restimulated with CD4-3 and CD8-1 peptides and IFN-c production evaluated by ELISA
after 72 hours incubation.
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impaired with the analgesic administration. Importantly, the
vaccine-induced delay in tumor development was not affected by
treatment with PCM (Fig. 6C). The frequency of antigen-specific
CD8+T cells was evaluated by staining splenocytes with anti-CD3,
anti-CD8 and NY-ESO-1 tetramer. The frequency of NY-ESO-1-
T cells in spleen of vaccinated mice was
proportional to the IFN-c production (Fig. 6D).
Most of the commercially available vaccines that are considered
highly effective in eliciting strong and long-lasting protective
immunity are thought to be mediated by neutralizing antibodies,
as is the case of Tetanus Toxoid, Polyomielitis, Small Pox and
Measles vaccines [31,32]. In contrast, development of efficient
vaccines that elicit TCMI, largely responsible for mediating
protective immunity to infections such as tuberculosis and
leishmaniasis, as well as cancer, is a difficult task [18,31,33]. A
major challenge for the development of vaccines that induce
TCMI, is the establishment of ideal formulations to induce strong
and long-lasting protective T cell immunity, mediated by CD8+T
lymphocytes [18,31]. In particular, immunological adjuvants
capable of eliciting a strong and long-lived TCMI with the least
side effect is a main Achille’s heel for development of human
The finding that activation of innate immune receptors
promotes the development of Th1 lymphocytes and TCMI had
stimulated the search for new TLR agonists, as potential
immunological adjuvants [11,12]. Our previous studies demon-
strated that GPI anchors and GIPLs from T. cruzi are able to
induce the synthesis of pro-inflammatory cytokines via TLR2 and
TLR4, respectively [20,21]. Furthermore, various CpG ODN
sequences derived from T. cruzi genome are also able to stimulate
cytokine synthesis, including IL-12, both by macrophages and
dendritic cells via TLR9 . In the present study, we tested in vivo
the T. cruzi derived Pathogen Associated Molecular Patterns
(PAMPs) as immunological adjuvants. We showed that GIPLs
from T. cruzi are potent inducers of antigen-specific immune
responses, as measured by IFN-c production by CD4+lympho-
cytes as well as serum levels of IgG2c. Furthermore, CpG ODNs
derived from T. cruzi, in especial the B344 induced an antigen-
specific immune response by CD8+T lymphocytes, leading to a
delay in tumor development in an antigen-specific manner.
Figure 4. Evaluation of antibody and T cell responses as well as protective immunity elicited by immunization with different
formulations containing the tumor-associated NY-ESO-1 antigen. C57BL/6 mice were subjected to three immunization doses on days 0, 14
and 28. (A) Serum levels of NY-ESO-1-specific total IgG, IgG1 and IgG2c; and (B) IFN-c production by splenocytes stimulated with NY-ESO-1 CD4+T
and CD8+T peptides cells were evaluated by ELISA. (C) Control and immunized mice were challenged with 56104B16F10 melanoma cell expressing
or not NY-ESO-1. The tumor growth was evaluated every 4 days for 40 days after challenge. Asterisks indicate that differences in IFN-c responses to
NY-ESO-1 CD4+T and CD8+T cell peptide and tumor growth are statistically significant, when comparing mice receiving different vaccine
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Figure 5. Hypernociception induced by TLR agonists and the NY-ESO-1 vaccine formulations. (A) Different TLR agonists were injected in
the footpad of mice and hypernociception evaluated at the indicated time points. (B) Vaccine formulations containing alum; alum plus NY-ESO-1; or
alum plus NY-ESO-1 associated with TLR agonists were given to mice that were left untreated or treated with PCM orally, 30 minutes prior injection
with different vaccine formulations. Asterisks mean significant difference when comparing PBS group with TLR agonists experimental groups
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Figure 6. Evaluation of antigen-specific immune response after PCM administration. C57BL/6 mice were subjected to three immunization
doses on days 0, 14 and 28. Thirty minutes prior each immunization dose, a group of mice received 10 mg/Kg of PCM by the oral rout. (A) Serum
levels of NY-ESO-1-specific total IgG, IgG1 and IgG2c; and (B) IFN-c responses by splenocytes stimulated with NY-ESO-1 CD4+T and CD8+T cell
peptides were evaluated by ELISA. (C) Control and immunized mice were challenged with 56104B16F10 melanoma cell expressing or not NY-ESO-1.
The tumor growth was evaluated every 4 days for 40 days after challenge. Asterisks indicate that differences in IFN-c responses to NY-ESO-1 CD4+T
and CD8+T cell peptide and curve of tumor growth are statistically significant, when comparing mice receiving different vaccine formulations. (D)
The frequency of CD8+T cells NY-ESO-1-specific were evaluated by flow cytometry using as marker FITC CD3, PECy5 CD8 and PE NY-ESO-1 tetramer.
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Acquired immunity to tumors involves both humoral and
cellular compartments. While antigen-specific antibodies have
been demonstrated to mediate protection , CD8+
lymphocytes are thought to be the main effector cells, which
mediate cytotoxic activity against tumor cells . In addition,
both CD8+T as well as CD4+T lymphocytes are important
sources of IFN-c that has various roles in inducing effector
mechanisms thought to mediate antitumor activities, which
includes activation of effector functions by macrophages .
Thus, a critical step for developing effective vaccines is to learn
how to induce the appropriate immune response necessary to
control tumor growth. Different strategies have been employed to
induce strong T cell-mediated immunity and in particular CD8+T
cells specific for tumor antigens. These strategies include the use of
plasmids (naked DNA)  as well as live attenuated viral and
bacterial vectors, such as adenovirus , MVA , Salmonella sp.
 and Listeria monocytogenis , respectively. In addition, the
discovery that Toll-like receptors are activated by viral and
bacterial products, also named PAMPs (e.g., LPS, DNA, RNA,
lipopeptides, flagelin, Poli-IC) as well as synthetic components (e.g.
imiquimod, CpG ODN, Pam3Cys)  with proinflammatory
and immunologic activity has boosted the field of vaccine
Briefly, cells from the innate immune system exposed to
microbial/synthetic products, synthesize high levels of pro-
inflammatory cytokines, such as IL-12 and TNF-a, that are
responsible for initiation of IFN-c synthesis by natural killer cells
[41,42]. In addition, when DCs are exposed to certain PAMPs
they express co-receptors, in order to initiate the activation process
of naı ¨ve T cells, making a bridge between the innate and acquired
immunity [11,14,18,41,42,43]. Furthermore, DCs activated with
TLR agonists will produce IL-12, which will influence the
differentiation of CD4+T cells into the Th1 lymphocytes, a main
source of IFN-c, and orchestrate the establishment of TCMI. IFN-
c is critical for class switch of Ig isotypes  and activation of
effector mechanisms displayed by macrophages that are often
involved in host resistance to tumors [35,36]. Moreover, activation
of antigen presenting cells favors cross-presentation, allowing
presentation of exogenous antigens via MHC class I and favoring
the development of antigen-specific CD8+T cells . Of note,
vaccine formulations containing TLR agonists associated with self-
antigens, often result in break of tolerance and development of
strong immune response to the employed self-antigen . The
vaccine formulation employing CT antigens, including NY-ESO-1
and TLR agonists as immunological adjuvants are being widely
tested in pre-clinical and clinical trials, both in prophylactic and
therapeutic vaccines to fight cancer cells [15,18]. Some of these
formulations also employ combinations of different adjuvants
aiming at the induction of a more robust immune response against
the tumor antigens [6,46,47].
Intracellular protozoan parasites, such as Toxoplasma gondii, T.
cruzi as well as Leishmania major are known to induce a strong and
long lasting TCMI, which is characterized by highly polarized
Th1 lymphocytes and strong CD8+T cell responses [48,49]. The
induction of Th1 lymphocytes and TCMI during protozoan
infection is highly dependent of IL-12  as well as Myeloid
differentiation primary response gene (88) (MyD88) and TLRs
[49,51]. Studies performed in our laboratory have dedicated to
indentify parasite PAMPs that are critical for activating TLRs and
initiating the IL-12 production and TCMI during protozoan
infections . We identified and characterized the structure of
GPI anchors derived from T. cruzi as agonists of TLR2 and TLR4
[20,21]. The lipid moiety of GPI anchors and GIPLs from T. cruzi
was shown to be an essential moiety that determines the pro-
inflammatory activity of these glycolipids, both in rodent and
human cells [20,21,52,53]. In more recent studies, we determined
that both DNA and RNA activate, respectively, TLR9 and TLR7
and are critical parasite components for induction of IL-12 and
host resistance to a primary infection with T. cruzi [22,54,55].
Furthermore, we identified multiple CpG motifs that activate
either mouse or human TLR9 . While the CpG motifs with
optimal immunostimulatory activity in mouse and human cells are
distinct, the results presented here, employing a CpG motif for
mouse, indicate the potential use of the CpG ODNs for human
One of the main impediments for approval of new immuno-
logical adjuvants for vaccine formulations is their side effect. Due
to their intrinsicpro-inflammatory
adjuvants often induce undesirable effects, for instance, local
edema, rubor and pain, and even systemic effects, such as
headache and fever [56,57,58]. Here, we addressed this question
by testing hypernociception, a measure of ‘‘pain’’, as a
consequence of inflammation elicited by our vaccine formulations.
Whereas, each of the TLR agonists used in our formulations
induced hypernociception on its own, the alum, an immunological
adjuvant approved for use in human vaccines, was sufficient to
induce the maximal score of ‘‘pain’’. Furthemore, addition of
specific TLR agonists did not augment the hypernociception
scores induced by alum. Importantly, treatment with PCM, which
acts at least in part through inhibition of prostaglandin release
, alleviated the intensity of hypernociception without affecting
the quality of the immune responses elicited by the vaccine
formulations containing the different parasite adjuvants.
In conclusion, our experiments employing vaccine formulations
containing NY-ESO-1 demonstrate that T. cruzi derived TLR
agonists, e.g. GIPLs and CpG ODNs, are efficient immunological
adjuvants. Importantly, the use of T. cruzi derived CpG ODNs as
immunological adjuvant in our vaccine formulation resulted in a
significant delay in the growth of the B16F10 melanoma cell line
expressing NY-ESO-1. The protective immunity correlated with
the magnitude of CD8+T cell response induced by a specific TLR
agonist. Finally, our results show that in our vaccine formulations
alum was the main component inducing hypernociception, which
was alleviated by the administration of PCM. Thus, parasite
adjuvants should be further explored in the development of
vaccine formulations, aiming to induce both humoral and cellular-
mediated immune responses.
We are thankful to Andrew Simpson, Jonathan Skipper and Lloyd Old
from the LICR, New York, and Denise Golgher for incentive, scientific
discussions and suggestions during the development of this work. We must
also acknowledge the LICR - Cornel University for the recombinant NY-
ESO-1 protein; and Dr. Jonathan Cebon from LICR-Melbourne for the
B16F10 and B16-NY-ESO-1 cell lines.
Conceived and designed the experiments: CJ ATG TMC GR FC RTG.
Performed the experiments: CJ ATG BGF WA APCS TMC CR MAC
MLOP. Analyzed the data: CJ ATG TMC MAC LMP JOP FQC RTG.
Are shown on the left representative dot blots of tetramer staining for each experimental group. Graph on the right shows the average (n=4) of the
percentage of CD3+/CD8+/NY-ESO-1+for each experimental group.
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PLoS ONE | www.plosone.org11May 2012 | Volume 7 | Issue 5 | e36245
Contributed reagents/materials/analysis tools: MLOP LMP JOP GR.
Wrote the paper: CJ ATG RTG.
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