Immunological characterization of a chimeric form of

Schistosoma mansoni aquaporin in the murine model

BARBARA CASTRO PIMENTEL FIGUEIREDO
1,2
, NATAN RAIMUNDO GONALVES
DE ASSIS
1,2
, SUELLEN BATISTONI DE MORAIS
1,2
, VICENTE PAULO MARTINS
2,3
,
NATASHA DELAQUA RICCI
1,2
, RODRIGO MARQUES BICALHO
1,2
, CARINA DA SILVA
PINHEIRO
2,4
and SERGIO COSTA OLIVEIRA
1,2
*
1
Departamento de Bioqumica e Imunologia do Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais,
31270-901 Belo Horizonte, MG, Brazil
2
Instituto Nacional de Cincia e Tecnologia em Doenas Tropicais (INCT-DT), CNPq MCT, 31270-901, MG, Brazil
3
Departamento de Biologia Celular do Instituto de Cincias Biolgicas, Universidade de Braslia, 70910-900 Braslia,
DF, Brazil
4
Departamento de Biointerao do Instituto de Cincias da Sade, Universidade Federal da Bahia, 40110-100, Salvador,
BA, Brazil
(Received 1 December 2013; revised 4 February and 19 February 2014; accepted 26 February 2014; first published online 1 May 2014)
SUMMARY
Aquaporin (SmAQP) is the most abundant transmembrane protein in the tegument of Schistosoma mansoni. This protein is
expressed in all developmental stages and seems to be essential in parasite survival since it plays a crucial role in
osmoregulation, nutrient transport and drug uptake. In this study, we utilized the murine model to evaluate whether this
protein was able to induce protection against challenge infection with S. mansoni cercariae. A chimeric (c) SmAQP was
formulated with Freunds adjuvant for vaccination trial and evaluation of the hosts immune response was performed. Our
results demonstrated that immunization with cSmAQP induced the production of high levels of specic anti-cSmAQP IgG
antibodies and a Th1/Th17 type of immune response characterized by IFN-, TNF- and IL-17 cytokines. However,
vaccination of mice with cSmAQP failed to reduce S. mansoni worm burden and liver pathology. Finally, we were unable to
detect humoral immune response anti-cSmAQP in the sera of S. mansoni-infected human patients. Our results lead us to
believe that SmAQP, as formulated in this study, may not be a good target in the search for an anti-schistosomiasis vaccine.
Key words: Schistosoma mansoni, tegument, aquaporin, vaccine, Th1/Th17 response.
I NTRODUCTI ON
Schistosomiasis is the most important human hel-
minth infection when it comes to morbidity and
mortality (McManus and Loukas, 2008). About 800
million people live in risk areas in 76 countries
worldwide, of which 207 million people are infected
and 20 million develop the severe disability form of
the disease (Steinmann et al. 2006; WHO, 2010). It is
estimated that 15000 people die per year, however,
mortality rate could be as high as 280000 per year in
Africa (van der Werf et al. 2003; Lustigman et al.
2012). Many countries, especially in sub-Saharan
Africa, invest in intervention strategies based on
short-term control programmes using mass drug
administration together with supply of safe water,
improvement in sanitation and snail control
(McManus and Loukas, 2008; Prichard et al. 2012).
The main goal in schistosomiasis treatment is to
achieve the reduction of disease transmission and
morbidity. Praziquantel (PZQ) is the main single
drug used in schistosomiasis treatment, but it does
not kill the larval stage form of schistosome and
does not prevent reinfection. Besides, some cases of
drug-resistant parasites should also be considered
(Gray et al. 2010; McManus, 2012). Since PZQ mass
treatment has so many limitations, many believe that
an integrative measure is to combine the traditional
PZQ treatment with an eective vaccine (Bergquist
et al. 2005). The most promising targets for vaccine
development are membrane proteins present in the
parasites outer surface or tegument identied
through bioinformatics analysis of Schistosoma man-
soni sequence databases, such as transcriptomes,
genome and proteomics (Pinheiro et al. 2011).
Proteomic analysis of S. mansoni tegument
(Braschi and Wilson, 2006; Braschi et al. 2006)
provided the schistosome vaccine eld with promis-
ing candidates for vaccine design, such as Sm29
(Cardoso et al. 2008) and TSP-2 (Tran et al. 2006).
A recent study on the abundance of S. mansoni
tegument surface proteins (Castro-Borges et al. 2011)
indicates that aquaporin is the most abundant
transmembrane protein. The S. mansoni aquaporin
(SmAQP GenBank: EU780065.1) is one small
tegumental membrane protein that transports
water and other small solutes such as glycerol
* Corresponding author: Av. Antnio Carlos, 6627
Pampulha Caixa Postal 486, Belo Horizonte, MG, Brazil
31270-901. E-mail: scozeus1@gmail.com
1277
Parasitology (2014), 141, 12771288. Cambridge University Press 2014
doi:10.1017/S0031182014000468
(Faghiri and Skelly, 2009). In addition to being very
abundant in the tegument, this protein is expressed in
all developmental stages of the parasite and plays a
crucial role in osmoregulation, nutrient transport and
drug uptake (Faghiri et al. 2010). SmAQP is vital for
schistosome survival, since the inability to control
water movement aects the parasites biochemistry,
leading to increased mortality in vitro (Faghiri and
Skelly, 2009).
Taking into account the importance of SmAQP
in parasite survival, the goal of the present study
was to test this protein as a vaccine candidate in the
murine model. In order to characterize the host
immune responses and evaluate the prophylactic
potential of this protein, we formulated a vaccine
with a chimeric form of SmAQP in Freunds
adjuvant and assessed the protection after challenge.
As described below, vaccination with cSmAQP
did not reduce worm burden in mice nor ameliorate
pathology in the liver.
MATERI ALS AND METHODS
Mice and parasites
Six- to 8-week-old female C57BL/6, TLR4 KO and
Swiss mice were purchased from the Federal
University of Minas Gerais (UFMG) animal facility.
All animal experiments were conducted in accord-
ance with the Brazilian Federal Law no. 11.794,
which regulates the scientic use of animals, and
IACUC guidelines. The protocols involving animals
used in this study were approved by the Federal
University of Minas Gerais Ethics Committee on
animal experimentation (CETEA no. 179/2010).
Cercariae of S. mansoni (LE strain) were maintained
routinely in Biomphalaria glabrata snails at Rene
Rachou Research Center (CPqRR, Fiocruz, Brazil)
and prepared by exposing infected snails to light for
2 h to induce shedding of parasites. Cercariae
numbers and viability were determined using a
light microscope prior to infection.
Antigen preparation
The plasmid pJ414 containing the cDNA sequence
for cSmAQP (pJ414::cAQUA) was manufactured by
DNA 2.0, Inc. USA (https://www.dna20.com) using
DNA 2.0 optimization algorithms for expression in
Escherichia coli. This plasmid was transformed into
E. coli Rosetta-gami (Merck KGaA, Darmstadt,
Germany) competent cells. Transformants harbour-
ing the designed plasmid were screened on LB agar
plates containing ampicillin (50 g mL
1
) and chlor-
amphenicol (34 g mL
1
) and the selected transfor-
mant was designated as Aqua-Rosetta. One litre of
Aqua-Rosetta was cultured in a 3 L Erlenmeyer ask
on a rotary shaker at 200 rpm at 37 C to an optical
density at 600 nm of approximately 0508 and gene
expression was induced by using 1 mMisopropylthio-
galactoside (IPTG). After 5 h of induction, the
bacterial cells were harvested by centrifugation at
4000 g for 20 min. Using gentle vortexing or pipet-
ting, the pellet was resuspended in 50 mL of 10 mM
Na
2
HPO
4
, 10 mM NaH
2
PO
4
, 05 M NaCl and 20 mM
imidazole. Subsequently, the cells were submitted to
three cycles of sonication lasting 30 s each and
centrifuged at 5400 g for 20 min. The cSmAQP was
recovered solubilized in the supernatant and puried
by anity chromatography on a Ni-Sepharose
column (Hitrap chelating 5 mL) using an AKTA
explorer chromatography system (GE Healthcare,
So Paulo, Brazil). After protein binding to the
Ni-Sepharose column, washes with 50 mM imidazole
were performed and the protein was eluted with
500 mM imidazole. Fractions containing the
protein were determined through Bradfords
method (Coomassie Protein Assay Kit, Pierce) and
also SDS/PAGE-12% and dialysed against PBS
pH 70. The dialysis was carried out at 4 C using
a Spectra/Por2 membrane (MWCO 6 to 8 kDa;
Spectrum Medical Industries, Inc., Laguna Hills, CA).
The recombinant protein was quantied using the
Bradfords method and used as antigen for vacci-
nation and immunological experiments. All reagents
were purchased from Sigma-Aldrich, CO (St. Louis,
MO, USA) unless otherwise specied.
Mice immunization, challenge infection and worm
burden recovery
Two groups of 10 mice each (female C57BL/6
aged 68 weeks) were subcutaneously injected in
the nape of the neck with 25 g of cSmAQP or
PBS, as a control, at days 7, 22 and 37. Both
preparations were formulated with Complete
Freunds Adjuvant (CFA) for the rst immunization
and Incomplete Freunds Adjuvant (IFA) for the
last two immunizations. All reagents were purchased
from Sigma-Aldrich, CO (St. Louis, MO, USA).
Fifteen days after the last immunization, the mice
were challenged through percutaneous exposure of
abdominal skin to water containing 100 cercariae
(LE strain) for 1 h. Forty-ve days after the
challenge, adult worms were perfused from the
portal veins of each animal, as previously described
(Fonseca et al. 2004). The immunization and
challenge protocol is demonstrated in supplementary
Fig. 1A. Three independent experiments were
performed to determine the vaccine protection
level. Protection was calculated by comparing the
number of worms recovered from the group
vaccine with cSmAQP and the control group,
as follows: PL= [(WRCGWREG)/WRCG] 100,
where PL=protection level, WRCG= worms
recovered from control group and WREG=worms
recovered from experimental group.
1278 Barbara Castro Pimentel Figueiredo and others
Measurement of anti-cSmAQP specic antibodies
Following immunizations, sera of 10 vaccinated mice
from both groups (cSmAQP or PBS) were collected
at days 0, 15, 30, 45, 60, 75 and 90. Measurements of
specic antibodies were performed using indirect
ELISA. Maxisorp 96-well microtitre plates (Nunc,
Denmark) were coated with 10 g mL
1
of cSmAQP
in carbonate-bicarbonate buer, pH 96 for 16 h at
4 C, then blocked for 2 h at room temperature with
200 Lwell
1
PBST (phosphate buered saline,
pH 72 with 005% Tween-20) plus 10% FBS (foetal
bovine sera). For each serum, 20 dilutions were
evaluated (starting at 1 : 20 and following a two-fold
serial dilution); 100 L of each dilution was added
per well and incubated for 1 h at room temperature.
Plate-bound antibody was detected after 1-h incu-
bation with peroxidase-conjugated anti-mouse IgG,
IgG1 and IgG2a diluted in PBST 1 : 5000, 1 : 10 000
and 1 : 2000, respectively. Colour reaction was
developed by addition of 100 L per well of
200 pmol OPD (o-phenylenediamine) in citrate
buer, pH 50 plus 004% H
2
O
2
for 10 min and
stopped with 50 L of 5% sulphuric acid per well.
The plates were read at 495 nm in an ELISA plate
reader (BioRad, Hercules, CA). All reagents were
purchased from Sigma-Aldrich, CO (St. Louis, MO,
USA) unless otherwise specied.
Purication of anti-cSmAQP specic antibodies
For the purication of anti-cSmAQP antibodies,
serum from the fourth bleeding was utilized (day
45 after the third immunization and before chal-
lenge). Briey, 500 g of the recombinant protein was
adsorbed to a nitrocellulose membrane. The mem-
brane was then blocked with TBST (tris buered
saline, pH 72 with 005% Tween-20) containing 5%
non-fat dry milk for 2 h at room temperature. After
three washes using TBST, the membrane was
incubated with 2 mL of pooled serum from mice
immunized with cSmAQP for 16 h at 4 C. After
another set of three washes with TBST, the
antibodies were eluted with 400 L of triethylamine
0014% for 5 min at room temperature and then
neutralized with 100 L of 10 TBS. The eluate was
dialysed against TBS pH 70. The dialysis was
carried out at 4 C using a Spectra/Por2 membrane
(MWCO 6 to 8 kDa; Spectrum Medical Industries,
Inc., Laguna Hills, CA, USA). All reagents were
purchased from Sigma-Aldrich, CO (St. Louis, MO,
USA) unless otherwise specied.
Oogram
To evaluate the eect of immunization with
cSmAQP in granuloma formation, following per-
fusion for the recovery of the schistosomes, livers
from eight mice per group were collected. Liver
fragments from each animal were separated and the
smallest part was weighed and digested with 10%
KOH overnight at 37 C. The eggs were obtained by
centrifugation at 900 g for 10 min and resuspended
in 1 mL of saline. Egg number was counted using
a light microscope. Quantitative oograms were
obtained calculating the number of eggs per gram
of liver tissue. The liver samples removed from the
central part of the left lateral lobe were xed with 10%
buered formaldehyde in PBS (phosphate buered
saline, pH 72). All reagents were purchased from
Sigma-Aldrich, CO (St. Louis, MO, USA).
Liver histopathological analysis
Histological sections were performed using a
microtome at 6 m and stained on a slide with
picrosirius-haematoxylin-eosin (PSHE). The count
of granulomas was performed at a microscope with
10objective lens. Each liver section was scanned for
calculating its whole area (mm
2
) using the ImageJ
software (http://rsbweb.nih.gov/ij/index.html). For
measurement of the total area of granulomas, a
microscope with 10 objective lens was used; images
were obtained through a JVC TK-1270/RBG micro-
camera attached to the microscope. Twenty granulo-
mas with a single well-dened egg were randomly
selected, in each liver section, and the granuloma area
was measured using the ImageJ software. All reagents
were purchased from Sigma-Aldrich, CO (St. Louis,
MO, USA).
Mice immunization and cytokine analysis
Cytokine experiments were performed using
splenocyte cultures from individual mice (C57BL/6
or TLR4 KO) immunized three times with 25 g of
cSmAQP or PBS (control) both formulated with
Complete/Incomplete Freunds Adjuvant (n = 5 for
each group). The cytokine protocol is demonstrated
in supplementary Fig. 1B. Ten days after the last
immunization, splenocytes were isolated from
macerated spleen of individual mice and washed
twice with sterile PBS. After washing, the spleno-
cytes were adjusted to 110
6
cells per well in RPMI
1640 medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 10% FBS, 100 UmL
1
of peni-
cillin G sodium and 100 g mL
1
of streptomycin
sulphate, for IL-4, IL-5, IL-10, IL-17, IFN- and
TNF- assays. Splenocytes were maintained in
culture with medium alone or stimulated with the
cSmAQP (25 g mL
1
) or with concanavalin A
(ConA) (5 g mL
1
), or LPS(1 g mL
1
), as positive
controls. The 96-well plates (Nunc, Denmark) were
maintained in an incubator at 37 C with 5% CO
2
(Fonseca et al. 2006; Pacico et al. 2006). For
C57/BL6 mice cells, polymyxin B (30 g mL
1
)
was added to the cultures since this treatment
completely abrogates the cytokine response to LPS
1279 Immunological characterization of Schistosoma mansoni aquaporin
as previously described (Cardoso et al. 2007). Culture
supernatants were collected after 24 h for IL-4 and
IL-5, after 48 h for TNF- and after 72 h for IL-17
and IFN-. The assays for the measurements of all
cytokines were performed using the Duoset ELISA
kit (R&D Diagnostic, Minneapolis, MN, USA)
according to the manufacturers directions. All
reagents were purchased from Sigma-Aldrich, CO
(St. Louis, MO, USA) unless otherwise specied.
SDS-PAGE and immunoblotting
SDS-PAGE was conducted on 15% polyacrylamide
gels prepared as previously described (Laemmli,
1970). Protein samples of cSmAQP or SWAP
(soluble worm antigen preparation), obtained from
adult worms recovered from perfused infected Swiss
mice, were run at 120 V for 23 h. The gel was
electroblotted onto nitrocellulose membrane using a
wet system (Towbin et al. 1979). The membrane was
then blocked with TBST (tris buered saline, pH 72
with 005% Tween-20) containing 5% non-fat dry
milk for 16 h at room temperature. Then, the
membrane was incubated in a 1 : 2000 dilution of
anti-HIS antibodies (GE Healthcare) or in 1 : 1000
anti-cSmAQP murine polyclonal antibodies or in
1:1000 naive mice serum in TBST for 1 h at room
temperature. After three washes using TBST, the
membrane was incubated in 1 : 2000 mouse IgG
conjugated with alkaline phosphatase (AP) treated
with AP reaction developing buer containing
nitroblue tetrazolium (NBT) and 5-bromo-4-
chloro-3-indolyl-1-phosphate (BCIP). After the re-
action was developed, the membrane was washed
using distilled water and dried on lter paper. All
reagents were purchased from Sigma-Aldrich, CO
(St. Louis, MO, USA) unless otherwise specied.
Immunolocalization
Adult male worms used in uorescence microscopy
studies were recovered from perfused infected Swiss
mice. Parasites were xed in Omnix II (Ancon
Genetics, St Petersburg, FL, USA) and sectioned in
7 m slices, and were then deparanized with xylol
series. Parasites were blocked with 1% BSA in PBST
(phosphate buered saline, pH 72 with 005%
Tween-20) for 1 h and incubated with anti-
cSmAQP serum diluted 1 : 20 in blocking buer.
Serum from non-immunized mice was used as a
negative control. Samples were washed three times
with PBST and incubated with anti-mouse IgG
antibody conjugated to FITC diluted 1 : 100 in
blocking buer containing rhodamine phalloidin to
stain actin microlaments. The samples were washed
four times and mounted in anti-fade reagent with
DAPI. The parasites were visualized using immer-
sion 40 objective in a Nikon uorescence micro-
scope at the Microscopy Center of Biological
Sciences Institute (CEMEL), Federal University of
Minas Gerais. All probes and anti-fade were pur-
chased from Molecular Probes, Life Technologies
(Grand Island, NY, USA).
Measurement of human anti-SmAQP IgG responses
in schistosomiasis patients
Sera were obtained from individuals living in
two dierent endemic areas for schistosomiasis
(Melquiades and Crrego do Ona, Minas
Gerais, and Conde, Bahia, Brazil). These indivi-
duals were classied into two groups, regarding their
infection status. Non-infected (NIN) individuals
(n = 15) were healthy people without any parasite
infection or contaminated water contact and infected
(INF) individuals (n = 15) showed stool-positive
examination and no treatment history. All patients
were negative for other helminthic infections (for
study population see supplementary Table 1). These
patients or their legal guardians gave informed
consent after explanation of the protocol that had
been previously approved by the Ethical Committee
of the Federal University of Minas Gerais, as
previously described (Cardoso et al. 2006). Sera
from these patients were used in an ELISA to
measure the levels of total IgG to SmAQP (Brito
et al. 2000). For this assay, 96-well at-bottom
microtitre plates (Nunc, Denmark) were coated
overnight at 4 C with 100 L of cSmAQP protein
or SWAP (soluble worm antigen preparation),
obtained from adult worms recovered from perfused
infected Swiss mice, at a concentration of 5 g mL
1
in 01 M carbonate bicarbonate buer (pH 96)
per well. The plates were then blocked with 10%
bovine foetal serum in PBS (pH 74) for 2 h at room
temperature. Subsequently, the plates were
washed three times with PBST. For total IgG,
serum samples were diluted 1 : 50 in PBST
(100 Lwell
1
) were added in duplicate and the
plates incubated for 1 h at room temperature.
Peroxidase-labelled anti-human IgG was added at
dilutions of 1 : 10000 (100 Lwell
1
). After 1 h at
37 C, the plates were washed and orthophenyl-
diaminobenzidine plus 005% hydrogen peroxide
in phosphate citrate buer (pH 50) was added
(100 Lwell
1
). The plates were then incubated for
30 min at room temperature, and the reaction was
stopped by addition of 5% H
2
SO
4
(50 Lwell
1
).
Absorbance was read at 492 nm using a microplate
reader (Bio-Rad, Hercules, CA). All reagents were
purchased from Sigma-Aldrich, CO (St. Louis, MO,
USA) unless otherwise specied.
Epitope analysis
The amino acids sequence of SmAQP was obtained
from SchistoDB (http://schistodb.net/schisto/).
1280 Barbara Castro Pimentel Figueiredo and others
Linear B-cell epitopes were predicted based on the
algorithm BepiPred, the score threshold parameter
ranged from 015 to 090. We also performed a
prediction with the cSmAQP sequence to evaluate
which epitopes were maintained and which ones were
lost in the construction of the chimera.
Statistical analysis
Statistical analysis was performed with the Students
t-test for comparison between two experimental
groups using the software package GraphPad Prism
(La Jolla, CA, USA). Bonferroni adjustments were
included for multiple comparisons. Pvalues obtained
by these methods were considered signicant if they
were <005.
RESULTS
Design and production of recombinant SmAQP
The S. mansoni aquaporin (SmAQP) is a large
transmembrane insoluble protein which has six
transmembrane helices. In order to overcome prob-
lems with protein size and solubility and to improve
its expression in E. coli, we developed a chimeric
protein in which the transmembrane domains are
absent. So, both the intracellular and the extracellular
hydrophilic portions were fused together, generating
what we denominated chimeric aquaporin
(cSmAQP). Chimeric SmAQP was designed through
the fusion of cDNA sequences of predicted non-
membranous SmAQP portions with a six-histidine
tag in its C-terminal. Bioinformatic analysis of the
resultant sequence revealed a soluble 169-aa protein
with a predicted molecular mass of 18705 Da and a
predicted pI of 859 (Fig. 1A). The native aquaporin
is a 304-aa protein, with 32893 Da and pI of 867
(Faghiri and Skelly, 2009). Besides, since this
chimeric protein was used in vaccine studies, we
also performed linear B-cell epitope analysis of both
cSmAQP and the native protein sequences. The
native aquaporin has 10 predicted epitopes, nine of
them in its hydrophilic portions (see supplementary
Table 2 for epitopes sequence). cSmAQP has seven
predicted epitopes, all of them with a similar
sequence to the native molecule (Fig. 1B).
The cSmAQP was expressed in E. coli and puried
by afnity chromatography. The expression and
purity of cSmAQP as a 6His-tag fusion protein
were checked by SDS-PAGE and western blotting
analysis with anti-HIS antibody, which revealed a
protein with approximately 18 kDa corresponding to
predicted mass of cSmAQP (Fig. 2).
Antibody prole following mice immunization
To evaluate the levels of total IgG, IgG1 and IgG2a
antibodies to cSmAQP, sera from 10 vaccinated
animals of each group were tested by ELISA. All
mice vaccinated with cSmAQP produced signicant
levels of specic IgG antibodies compared with
the control group vaccinated with PBS at day 45,
pre-challenge with cercariae, and at day 90, before
euthanasia (Fig. 3). The measurement of IgG
isotypes levels revealed that the cSmAQP-vaccinated
group had increased levels of IgG1 and IgG2a when
compared with the PBS-administered group.
Moreover, the IgG1/IgG2a ratio was increased at
day 45. After challenge infection with schistosome
cercariae, this elevated IgG1/IgG2a ratio was main-
tained (see supplementary Table 3).
Antibodies against cSmAQP recognize native
aquaporin in S. mansoni
In order to conrm whether polyclonal antibodies
raised against cSmAQP were able to recognize native
aquaporin, we performed western blot analysis to
detect aquaporin in skin-stage schistosomula tegu-
ment (SmTeg, kindly provided by Dr Cristina
T. Fonseca) or SWAP. Sera from nave mice were
used as a control. In both protein extract samples, the
anti-cSmAQP (1 : 1000) recognized a single band
with approximately 33 kDa, which is the predicted
size for aquaporin (Fig. 4A). This result indicates
that anti-cSmAQP were able to recognize the native
S. mansoni aquaporin. Additionally, we conrmed
that anti-cSmAQP can recognize native aquaporin in
uorescence microscopy experiments. The anti-
cSmAQP were used in immunolocalization of male
worms and they recognized native aquaporin pre-
dominantly on the worm tegument (Fig. 4B), as
demonstrated by Faghiri et al. (2010).
Cytokine prole
To determine the cytokine prole induced by
vaccination with cSmAQP, we measured the pro-
duction of IFN-, TNF-, IL-4, IL-5, IL-10 and
IL-17 in spleen cells of two mice strains, C57BL/6
and TLR4 KO immunized mice 10 days after the
third immunization. TLR4 is the major receptor for
macrophage activation by bacterial LPS (Medzhitov,
2007). Considering that the antigen utilized in this
experiment was expressed and puried frombacterial
cultures, the bacterial LPS contamination could have
aected the cytokine production. Therefore, by using
TLR4 KO we eliminated any possible eect of LPS
in cytokine synthesis. This mouse strain, together
with the samples treated with polymyxin B elimi-
nated any possible LPS contamination of the antigen.
As a result of antigen stimulation, we
detected signicant levels of IFN- in supernatant
of both cells from immunized TLR4 KO
(14937611 pg mL
1
) and C57BL/6 (15897
3229 pg mL
1
) when compared with the PBS
1281 Immunological characterization of Schistosoma mansoni aquaporin
immunized control group (Fig. 5). We also detected
high levels of TNF- in cell supernatants
of vaccinated mice stimulated with cSmAQP
compared with PBS immunized groups (11960
3352 pg mL
1
for C57BL/6 mice and
96801681 pg mL
1
for TLR4 KO mice). The
Th2 type of cytokines, IL-4 and IL-5 and the
regulatory cytokine IL-10 were produced in very
low levels with no statistical signicance when
compared with control groups (data not shown).
Upon stimulation, spleen cells of the cSmAQP
vaccinated group were also able to produce IL-17
(1474569 pg mL
1
for C57BL/6 mice and
679146 pg mL
1
for TLR4 KO mice), which
characterizes a Th17 response. These results indicate
that immunization with cSmAQP formulated
with Freunds adjuvant induced a Th1/Th17 type
of immune response characterized by the production
of IFN-, TNF- and IL-17 and the absence of Th2
cytokines.
Worm burden recovery
Protective immunity induced by vaccination
with cSmAQP was evaluated 45 days after challenge
with 100 S. mansoni cercariae. Mice vaccinated with
cSmAQP showed no statistically signicant re-
duction in worm burden recovery compared with
control animals (Table 1). We performed three
Fig. 1. Schistosoma mansoni aquaporin schematic representation. (A) Schistosoma mansoni native aquaporin is
represented on the left. The hydrophilic portions used in the chimera construction are represented as: intracellular (red),
extracellular (blue); the transmembrane portions (grey) are not present in the designed chimeric recombinant aquaporin
(cSmAQP), represented on the right. (B) The native aquaporin is represented on the top row with empty rectangles
representing the transmembrane regions and grey rectangles representing the predicted B-cell linear epitopes. The
cSmAQP, represented on the bottom row, maintained most of the original proteins epitopes.
1282 Barbara Castro Pimentel Figueiredo and others
independent trials of cercarial challenge after im-
munization.
Liver pathology
Histological analysis by digital morphometry of
PSHE-stained sections obtained from liver of mice
immunized with cSmAQP showed no eect in
granuloma volume and number compared with
mice that received only PBS. Additionally, no
dierence in the number of eggs in the liver was
observed in both groups. In general, these ndings
suggest that immunization with cSmAQP did
not alter the hepatic conditions of mice infected
with S. mansoni, as shown in Supplementary Fig. 2.
Human IgG antibody response to cSmAQP
We also tested cSmAQP as a potential tool for
diagnosing schistosomiasis patients. Before testing
human antibodies response to cSmAQP, we tested
murine polyclonal anti-SmTeg (kindly provided by
Dr Cristina T. Fonseca) against a puried sample
of cSmAQP in western blot assay. The antibodies
(1 : 1000) recognized cSmAQP (data not shown),
suggesting that both recombinant and native aqua-
porin probably have similar epitopes to B-cells.
Then, considering that anti-SmAQP might be able
to recognize the chimeric formof the protein, ELISA
was performed to investigate the presence of IgG
antibodies anti-cSmAQP in sera of infected
S. mansoni individuals. Total IgG levels in sera of
schistosomiasis patients and non-infected individuals
were evaluated. The schistosomiasis patients studied
had signicantly higher levels of IgG to SWAP and
no signicant levels of IgG to cSmAQP compared
with the non-infected group (Fig. 6). These results
demonstrate that cSmAQP is not a good antigen to
discriminate infected from healthy patients, showing
no potential as an antigen for diagnostic purposes.
DI SCUSSI ON
Tegument proteins are of great importance in the
development of schistosome vaccine because they are
the major hostparasite interface (Loukas et al. 2007).
Recently, Castro-Borges et al. (2011) identied
aquaporin as the most abundant transmembrane
protein in the tegument of S. mansoni. Schistosoma
mansoni aquaporin (SmAQP) is a well-known protein
that plays crucial roles in parasite survival, such as
osmoregulation, nutrient transport and drug uptake.
The vital importance of SmAQP was demonstrated
through RNAi experiments; the suppressed parasites
exhibit lower viability in culture relative to controls
(Faghiri et al. 2010).
Considering that SmAQP plays a vital role in
parasite survival in culture, our group decided to
evaluate its potential as vaccine candidate in prophy-
lactic treatment against S. mansoni infection using the
murine model. One of the main diculties in
working with SmAQP is that it has six trans-
membrane domains, making its expression in bacteria
complicated. To overcome this problem, we designed
a chimera protein by fusion of S. mansoni aquaporins
soluble portions. The use of chimeras is a strategy to
optimize the immunological response against schisto-
some proteins (Romeih et al. 2008; Pearson et al.
2012).
In the mouse model, immunization with re-
combinant aquaporin (cSmAQP) formulated with
Freunds adjuvant induced high levels of anti-
cSmAQP IgG compared with PBS, also formulated
with Freund, as control group. Through western blot
analysis and immunolocalization, we demonstrated
that these polyclonal antibodies were able to recog-
nize both cSmAQP and native aquaporin, suggesting
that the recombinant chimera induces an immune
response similar to the native protein. We also
Fig. 2. Heterologous expression and purication of
cSmAQP. (A) Western blot analysis of cSmAQP in the
bacterial lysate (10 g) probed with monoclonal mouse
anti-His tag antibodies. (B) 15% SDS-PAGE stained with
Coomassie brilliant blue of the puried cSmAQP (5 g).
The molecular weight protein standard (rst lane) is a
broad range pre-stained ladder from BioRad.
pre-euthanasia
Fig. 3. Kinetics of specic anti-cSmAQP IgG induced in
mice immunized with recombinant cSmAQP. Sera of
immunized mice were collected at days 0, 45 and 90 and
assayed by ELISA. Results are presented as the mean of
the antibody titres for 10 mice in each group and error
bars indicate S.D. The results shown are representative of
three independent experiments. Statistically signicant
dierences of recombinant cSmAQP vaccinated mice
compared with PBS control group is denoted by three
asterisks for P<0001.
1283 Immunological characterization of Schistosoma mansoni aquaporin
Fig. 4. Antibodies anti-cSmAQP recognized native aquaporin through western blot and immunolocalization.
(A) Twenty micrograms of SWAP or 10 g of SmTeg were applied onto 15% SDS-PAGE and transferred to a
nitrocellulose membrane. (i) The probing with polyclonal antibodies raised against cSmAQP protein demonstrated the
presence of one band with the predicted size of native aquaporin, around 33 kDa, in both samples (black arrows). These
data show that antibodies against cSmAQP are able to recognize native aquaporin. (ii) As control, serum from nave
mice was used. (B) Fluorescence microscopy images of male adult worm of S. mansoni are shown. Polyclonal anti-
cSmAQP and secondary antibody coupled to FITC (green) were used for uorescence detection of aquaporin on male
adult worm sections (i and ii). Serum from nave mice was used as negative control (iii and iv). Rhodamine phalloidin
(red) was used for the actin localization. DAPI (blue) was used for nucleus staining. (i) The aquaporin localization is
represented in green. Arrows indicate the worm tegument. (ii) aquaporin and actin staining merged. (iii) The absence of
green staining in the negative control. (iv) FITC and actin staining merged.
1284 Barbara Castro Pimentel Figueiredo and others
evaluated IgG isotypes and found high levels of
IgG1 and IgG2a in the cSmAQP vaccinated group
compared with the control group. Previous studies
have shown that high levels of IgG1 are associated
with the induction of schistosomula death by
antibody-dependent cell-mediated cytotoxicity, and
the activation of complement (Capron et al. 1975;
Khalife et al. 1989). Moreover, the stimulation of
Fig. 5. Cytokine prole of mice immunized with cSmAQP. Ten days after the nal immunization with cSmAQP or
PBS, splenocytes from ve mice C57BL/6 or TLR4 KO were isolated and assayed for the determination of cytokine
prole. (A) IFN-, (B) TNF- and C. IL-17 production in response to cSmAQP (25 g) were measured in the
supernatants of spleen cells. The results are presented as the meanS.D. for each group. The results shown are
representative of three independent experiments. Signicant dierences from mice immunized with cSmAQP and their
respective control group (either C57BL/6 or TLR4 KO) are denoted by one asterisk for P<005.
Table 1. Protective immune response induced in
mice by vaccination with cSmAQP
Worms recovered
Males Females Total
Trial 1
a
PBS+CFA/IFA 27.86.8 22.34.7 50.110.7
rSmAQP+CFA/IFA 25.64.3 18.94.2 44.57.2
b
Trial 2
a
PBS+CFA/IFA 18.48.3 19.72.4 38.110.2
rSmAQP+CFA/IFA 14.76.5 17.48.5 32.112.1
b
Trial 3
a
PBS+CFA/IFA 21.72.7 19.92.5 41.64.9
rSmAQP+CFA/IFA 20.54.3 21.25.6 42.46.7
b
a
810 animals per group, values are mean number of
recovered worms S.D.
b
There was no statistically signicant dierence of recombi-
nant cSmAQP vaccinated mice compared with PBS control
group.
Fig. 6. Levels of IgG anti-SWAP or anti-cSmAQP in
sera of schistosomiasis patients. Analysis of human IgG
antibody responses in sera of infected patients (INF) or
non-infected individuals (NIN). The results are
presented as the meanS.D. for each group (n =15). The
results shown are representative of three independent
experiments. Statistically signicant dierence of infected
compared with non-infected patients is denoted by one
asterisk for P<0001.
1285 Immunological characterization of Schistosoma mansoni aquaporin
splenocytes with cSmAQP resulted in increased
levels of IFN-, TNF- and IL-17 in both C57/
BL6 and TLR4 KO mice, the latter used as control
due to its absence of response to LPS, assuring that
the cytokine production was triggered by the antigen
itself. IFN- is important on the protective immunity
against S. mansoni, the production of this cytokine
probably is stimulated by the larval schistosomulum
when it passes through the lungs. This involves the
recruitment of macrophages and lymphocytes around
the worms, hindering their movement in the lungs
and moving them to the airways, with the consequent
elimination of these parasites (Wilson et al. 1996;
Jankovic et al. 1999). Another described mechanism
mediated by IFN- is the activation of macrophages
to kill worms inducing the production of nitric
oxide (Jankovic et al. 1999; Pearce and MacDonald,
2002). TNF- is another cytokine related to a
protective immune response against S. mansoni, it
probably operates in granuloma formation and,
together with IFN-, increases the levels of nitric
oxide for recruited macrophages (Amiri et al. 1992;
Cheever et al. 1999; Pearce and MacDonald, 2002).
The role of IL-17 in schistosoma infection has
recently been described. This cytokine, which is
the marker of a Th17 response, is important in
egg-induced inammation. The mouse strain knock-
out for both IL-17 and IFN- generated smaller
liver granulomas when compared with wild-type
mice (Rutitzky and Stadecker, 2011). In a study of
schistosomiasis co-infection with the nematode
helminth Heligmosomoides polygyrus, the switch
from a Th1/Th17-polarized response to a Th2-
polarized response, accompanied by a decrease in
IL-17, IFN- and TNF- and an increase in IL-4,
IL-5 and IL-10, signicantly reduced immuno-
pathology in mice with severe pathology (Bazzone
et al. 2008). Inthis study, the absence of productionof
IL-4, IL-5 and IL-10 seems to have aected cytokine
polarization, which maintained high levels of IL-17,
IFN- and TNF- and resulted in no amelioration of
the immunopathology of the group immunized with
cSmAQP when compared with the control group.
Despite high antibody titres and the induction
of cytokine production, vaccination of mice with
cSmAQP plus Freunds adjuvant failed to reduce the
parasite burden. Therefore, there was no apparent
correlation between the antibodies generated and
protective ecacy. Concerning the antibodies inter-
actions with the parasite, two main reasons might
justify the lack of eective protection: either the
antibodies are not binding to the native SmAQP in
live parasites or the antibody binding does not pro-
voke protein function impairment, which would not
aect the transport of water and excretion products.
When we tested the recognition of cSmAQP by
human sera from schistosomiasis patients, the sera
were unable to detect signicant levels of IgG when
compared with healthy subjects. The IgG molecules
are important in S. mansoni infection resistance since
they have functional activity of opsonization and cell-
dependent cytotoxicity, and activate the classical
complement pathway (Delgado and McLaren, 1990).
The absence of anti-cSmAQP IgG in infected
schistosomiasis patients might be an indication that
aquaporin is not important during the human
immune response to S. mansoni. However, even
considering that the two molecules share some of the
predicted epitopes for B cells, and anti-cSmAQP can
recognize the native aquaporin, no evidence is
presented on whether schistosomiasis patients anti-
bodies bind native aquaporin. At this point, what we
do know is that the chimeric protein designed in this
study cannot distinguish infected patients from
healthy ones and thus it is not a good antigen for
schistosomiasis diagnosis.
Aquaporins occur as tetramers in plasma mem-
branes, each monomer serving as a separate pore with
six membrane-spanning helices connected by ve
inter-helix loops, three external to the plasma
membrane (Gonen and Walz, 2006). Recent proteo-
mic study of S. mansoni indicated that aquaporin
represents 17% of the total tegument proteins
(Castro-Borges et al. 2011). Besides its abundance
in the tegument, this protein is almost completely
inserted in the plasma membrane, which might
impair the recognition of SmAQP by the immune
system, protecting this essential protein against host
cleavage and presentation to T cells. This is the rst
study evaluating SmAQP as a vaccine target; other
protein portions or even peptides should be tested, as
well as other adjuvants. Many methods, such as
innovative vaccine delivery procedures, can be tried
to make SmAQP immunogenic. Conversely, minor
schistosome plasma membrane components could be
better targets because fewer molecules would need to
be blocked by the host immune system. The major
protective antigens in the tegument, TSP-2 and
Sm29, are present at very low rates (243 and 031%,
respectively) (Castro-Borges et al. 2011).
Our study demonstrated that a vaccine formulated
with a chimeric form of aquaporin was not eective
against schistosomiasis in mice. The use of this
protein as a vaccine target requires more research
and use of new vaccine technologies to make it
immunogenic. Despite the results of this study, many
researchers maintain hope in aquaporin as a vaccine
antigen. However, future studies are needed to
determine whether the native protein is immuno-
genic or not. Besides vaccine studies, the develop-
ment of drugs which impair water transportation in
the parasite could also provide an eective alternative
to S. mansoni elimination.
SUPPLEMENTARY MATERI AL
For supplementary material accompanying this paper, visit
http://dx.doi.org/10.1017/S0031182014000468.
1286 Barbara Castro Pimentel Figueiredo and others
ACKNOWLEDGEMENTS
The authors thank Dr Cristina T. Fonseca for kindly
providing skin-stage schistosomula tegument (SmTeg) and
antibodies raised against this preparation (anti-SmTeg).
FI NANCI AL SUPPORT
This study was supported by Conselho Nacional de
Desenvolvimento Cientco e Tecnolgico (CNPq),
Fundao de Amparo Pesquisa do estado de Minas
Gerais (FAPEMIG), Coordenao de Aperfeioamento de
Pessoal de Nvel Superior (CAPES), INCT-Doenas
Tropicais.
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