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Acta
Tropica
120 (2011) 185–
190
Contents
lists
available
at
SciVerse
ScienceDirect
Acta
Tropica
journa
l
h
o
me
pa
g
e:
www.elsevier.com/locate/actatropica
DNA
vaccination
with
KMP11
and
Lutzomyia
longipalpis
salivary
protein
protects
hamsters
against
visceral
leishmaniasis
Robson
A.A.
da
Silvaa,b,c,
Natália
M.
Tavaresa,
Dirceu
Costaa,
Maiana
Pitomboa,
Larissa
Barbosaa,
Kyioshi
Fukutania,
Jose
C.
Mirandaa,
Camila
I.
de
Oliveiraa,
Jesus
G.
Valenzuelaf,
Aldina
Barrala,
Manuel
Sotoe,
Manoel
Barral-Nettoa,
Cláudia
Brodskynd,∗
aCentro
de
Pesquisas
Gonc¸
alo
Moniz
(CPqGM),
FIOCRUZ,
Salvador,
Bahia,
Brazil
bUniversidade
Federal
da
Bahia
-
UFBA
(Instituto
Multidisciplinar
em
Saúde,
Vitória
da
Conquista,
Bahia,
Brazil
cInstituto
de
Ciências
da
Saúde
e
Faculdade
de
Medicina,
Brazil
dInstituto
Nacional
de
Ciência
e
Tecnologia
de
Investigac¸
ão
em
Imunologia
(iii-INCT),
São
Paulo,
Brazil
eCentro
de
Biologia
Molecular
Severo
Ochoa
(SCIC-UAM),
Departamento
de
Biologia
Molecular,
Universidad
Autónoma
de
Madrid,
Spain
fVector
Molecular
Biology
Section,
Laboratory
of
Malaria
and
Vector
Research,
National
Institute
of
Allergy
and
Infectious
Diseases,
National
Institutes
of
Health,
United
States
of
America
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
10
March
2011
Received
in
revised
form
2
August
2011
Accepted
12
August
2011
Available online 22 August 2011
Keywords:
Hamster
Leishmania
chagasi
Visceral
leishmaniasis
Saliva
DNA
plasmids
Protection
a
b
s
t
r
a
c
t
It
was
recently
shown
that
immunization
of
hamsters
with
DNA
plasmids
coding
LJM19,
a
sand
fly
salivary
protein,
partially
protected
against
a
challenge
with
Leishmania
chagasi,
whereas
immunization
with
KMP11
DNA
plasmid,
a
Leishmania
antigen,
induced
protection
against
L.
donovani
infection.
In
the
present
study,
we
evaluated
the
protective
effect
of
immunization
with
both
LJM19
and
KMP11
DNA
plasmid
together.
Concerning
the
protection
against
an
infection
by
L.
chagasi,
immunization
with
DNA
plasmids
coding
LJM19
or
KMP11,
as
well
as
with
both
plasmids
combined,
induced
IFN-␥production
in
draining
lymph
nodes
at
7,
14
and
21
days
post-immunization.
Immunized
hamsters
challenged
with
L.
chagasi
plus
Salivary
Gland
Sonicate
(SGS)
from
Lutzomyia
longipalpis
showed
an
enhancement
of
IFN-␥/IL-10
and
IFN-␥/TGF-
in
draining
lymph
nodes
after
7
and
14
days
of
infection.
Two
and
five
months
after
challenge,
immunized
animals
showed
reduced
parasite
load
in
the
liver
and
spleen,
as
well
as
increased
IFN-␥/IL-10
and
IFN-␥/TGF-
ratios
in
the
spleen.
Furthermore,
immunized
animals
remained
with
a
normal
hematological
profile
even
five
months
after
the
challenge,
whereas
L.
chagasi
in
unimmunized
hamsters
lead
to
a
significant
anemia.
The
protection
observed
with
LJM19
or
KMP11
DNA
plasmids
used
alone
was
very
similar
to
the
protection
obtained
by
the
combination
of
both
plasmids.
© 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Leishmania
are
transmitted
by
sand
flies
and
are
the
etiologi-
cal
agents
of
cutaneous,
mucocutaneous
or
visceral
leishmaniasis.
Saliva
of
sand
flies
and
other
blood
feeders
contains
potent
phar-
macologic
components
that
facilitate
blood
meals
and
also
plays
a
role
in
pathogen
transmission
(Andrade
et
al.,
2007;
Ribeiro,
1995).
Small
amount
of
vector
saliva
can
also
exacerbate
parasite
infectiv-
ity
(Belkaid
et
al.,
1998;
Lima
and
Titus,
1996;
Theodos
et
al.,
1991;
Abbreviations:
DTH,
delayed-type
hypersensitivity;
GAPDH,
glyceraldehyde
3-phosphate
dehydrogenase;
SGH,
salivary
gland
homogenate;
VL,
visceral
leish-
maniasis;
i.d.,
intradermal.
∗Corresponding
author
at:
Laboratório
de
Imuno-regulac¸
ão
(LIMI),
Centro
de
Pesquisas
Gonc¸
alo
Moniz,
FIOCRUZ.
Rua
Waldemar
Falcão,
121,
Salvador,
Bahia
40296-710,
Brazil.
Tel.:
+55
71
3176
2211,
fax:
+55
71
3176
2279.
E-mail
address:
brodskyn@bahia.fiocruz.br
(C.
Brodskyn).
Titus
and
Ribeiro,
1988).
On
the
other
hand,
immune
response
to
arthropod
bites
or
to
its
saliva
precludes
establishment
of
the
pathogen
in
the
vertebrate
host
(Belkaid
et
al.,
1998;
Silva
et
al.,
2005).
Recent
reports
have
shown
the
importance
of
salivary
pro-
teins
from
sand
flies
as
potential
targets
for
vaccine
development
to
control
Leishamania
infection
(Kamhawi
et
al.,
2000;
Morris
et
al.,
2001;
Valenzuela
et
al.,
2001).
L.
chagasi
causes
VL
in
Latin
America
and
Lutzomyia
longipalpis
is
its
natural
vector.
Recently,
we
developed
a
model
for
VL
in
ham-
sters
infecting
the
animals
intradermally
in
the
ear,
with
parasites
plus
SGS
of
Lu.
longipalpis.
In
this
model,
hamsters
developed
the
main
symptoms
of
the
disease
such
as
visceral
parasite
burden,
massive
splenomegaly,
bone
marrow
dysfunction,
cachexia,
pancy-
topenia,
hypergammaglobulinaemia,
and
ultimately
death
(Melby
et
al.,
2001).
However,
hamsters
immunized
with
DNA
plasmid
coding
LJM19,
a
Lu.
longipalpis
salivary
protein,
protected
them
from
disease
development
and
the
fatal
outcome
of
visceral
leish-
maniasis
(Gomes
et
al.,
2008).
Immunization
of
hamsters
with
0001-706X/$
–
see
front
matter ©
2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2011.08.007
186 R.A.A.
da
Silva
et
al.
/
Acta
Tropica
120 (2011) 185–
190
DNA
plasmids
coding
KMP11
induced
a
mixed
Th1/Th2
T
cellular
immune
response,
with
high
levels
of
IFN-␥,
TNF-␣,
IL-4
and
IL-12
but
a
lack
of
IL-10
and
protected
the
animals
against
disease
devel-
opment
(Basu
et
al.,
2005).
It
was
also
demonstrated
that
human
CD8
T
cells
could
recognize
KMP11epitopes
associated
to
MCH
I,
pointing
out
the
importance
of
this
protein
in
the
human
immune
response
(Basu
et
al.,
2007).
Immunization
with
either
LJM19
or
KMP11
DNA
plasmid
led
to
a
marked
reduction
in
parasite
load
and
to
a
less
severe
disease,
but
neither
one
induced
parasite
cure
as
some
parasites
remained
during
the
whole
period
of
observa-
tion
(Basu
et
al.,
2005;
Gomes
et
al.,
2008).
In
the
present
study,
we
evaluated
the
combination
of
DNA
plasmids
coding
LJM19
and
KMP11
as
potent
inducers
of
protective
immunity
against
L.
chagasi
infection
in
hamsters.
2.
Methods
2.1.
Sand
flies
and
salivary
gland
lysates
Laboratory
colonies
of
Lu.
longipalpis
were
reared
at
Centro
de
Pesquisa
Gonc¸
alo
Moniz.
Salivary
gland
from
adult
female
flies
were
dissected
and
transferred
to
10
or
20
l
Hepes
10
mM
pH
7.0
NaCl
0.15
in
1.5
polypropilene
vials,
usually
in
groups
of
20
pairs
of
glands
in
20
l
of
Hepes
saline.
Salivary
glands
were
kept
at
−70 ◦C
until
used,
when
they
were
disrupted
by
sonication
using
a
Bran-
son
Sonifier
450
homogenizer
(Branson,
Danbury,
CT)
(Ribeiro
and
Modi,
2001).
Salivary
homogenates
were
centrifuged
at
10,000
×g
for
2
min
and
the
supernatants
were
used
in
the
experiments.
2.2.
Leishmania
parasites
L.
chagasi
(MCAN/BR/00/BA262)
promastigotes
were
cultivated
in
Schineider’s
insect
Medium
(Sigma
Chemical
Co.,
St
Louis,
MO,
USA)
supplemented,
with
20%
of
inactivated
FCS,
l-glutamine
(2
mM),
penicillin
(100
U/ml),
streptomycin
(100
g/ml)
at
23 ◦C
for
5–7
days
when
parasites
reached
the
stationary-phase.
These
parasites
were
washed
3
times
with
saline
at
3000
rpm
for
10
min,
resuspended
in
saline
and
adjusted
to
5
×
107–108per
ml.
2.3.
Construction
of
DNA
plasmids
coding
for
Lu.
longipalpis
salivary
proteins
and
KMP11.
Immunization
of
hamsters
Male
Golden
Syrian
Hamsters
(Mesocricetus
auratus)
at
10–12
weeks-old
from
Centro
de
Pesquisa
Gonc¸
alo
Moniz
were
used
for
experimental
purposes
with
previous
approval
of
the
Animal
Ethics
Committee
of
the
Fundac¸
ão
Oswaldo
Cruz-Fiocruz,
Bahia-
Brazil
number
17/2007.
DNA
plasmids
coding
for
Lu.
longipalpis
salivary
proteins
were
cloned
into
the
VR2001-TOPO
vector
and
purified
as
previously
described
(Oliveira
et
al.,
2006).
KMP-11
encoding
plas-
mids
were
provided
by
Dr.
Manoel
Soto
of
Universidad
Autónona
de
Madrid,
Spain
(Fuertes
et
al.,
2001).
Groups
of
hamsters
were
immunized
intradermally
(i.d.)
(Belkaid
et
al.,
1998)
in
the
left
ear
with
10
g/animal
of
LJM19
and/or
100
g/animal
of
KMP11
plas-
mids
for
three
times
at
14-day
intervals.
Control
hamsters
were
immunized
with
saline
or
empty
plasmid
constructions
VR2001-
TOPO
for
LJM19
and
pcDNA3
for
KMP11.
Each
hamster
received
plasmid
constructions
in
20
l
of
saline
in
different
situations:
I
–
injected
with
saline;
II
–
injected
with
DNA
containing
LJM19,
III
–
injected
with
DNA
containing
KMP11,
IV
–
injected
with
two
differ-
ent
plasmids
of
DNA
containing
LJM19
and
KMP11
and
V
–
injected
together
the
combination
of
two
different
empty
plasmid
(CT).
Two
weeks
after
the
last
immunization,
hamsters
were
challenged
i.d.
in
the
right
ear
with
105stationary
phase
of
promastigotes
of
L.
chagasi
plus
equivalent
of
0.5
salivary
gland
pairs
in
20
l
of
saline
(Gomes
et
al.,
2008).
2.4.
Anti-saliva
and
anti-Leishmania
antigen
serology
by
ELISA
ELISA
plates
were
coated
with
5
pairs
of
salivary
glands/ml
(approximately
5
g
protein/ml)
or
10
g/ml
soluble
Leish-
mania
antigen
(SLA)
overnight
at
4◦C.
After
three
washes
with
PBS-0.05%
Tween
20,
the
plates
were
blocked
for
1
h
at
37 ◦C
with
PBS-0.1%
Tween
20
plus
0.05%
BSA.
Sera
were
diluted
with
1:100
with
PBS-0.05%Tween
20,
then
incubated
overnight
at
4◦C.
After
further
washes,
the
wells
were
incubated
with
alkaline-phosphatase-conjugated
anti-hamster
IgG
(Jackson
Immuno
Research)
at
a
1:1000
dilution
for
45
min
at
37 ◦C.
Fol-
lowing
another
washing
cycle,
the
color
was
developed
for
30
min
with
p-nitrophenylphosphate
in
sodium
carbonate
buffer
pH
9.6
with
0.2
mM
of
MgCl2.
The
absorbance
was
recorded
at
405
nm.
2.5.
Limiting
dilution
assay
to
determine
parasites
loads
in
infected
tissues
Parasite
load
was
determined
using
the
quantitative
limiting
dilution
assay
as
described
by
Lima
et
al.
(1997).
Briefly,
infected
ears,
lymph
nodes,
liver
and
spleen
were
aseptically
removed
from
each
hamster
at
the
completion
of
the
experiments.
Tissues
were
homogenized
and
diluted
in
Schneider’s
insect
cell
culture
medium
(Sigma,
St.
Louis,
MO)
supplemented
with
10%
heat-inactivated
fetal
bovine
serum,
100
U/ml
of
penicillin
and
100
g/ml
of
strep-
tomycin.
Homogenate
samples
were
serially
diluted
in
microtiter
96-wells
and
incubated
for
one
week
at
23 ◦C.
Wells
with
positive
growth
were
noted
at
specific
dilutions
and
these
results
were
ana-
lyzed
by
ELIDA
(Lima
et
al.,
1997)
to
determine
the
parasite
burden
in
the
samples.
Results
were
expressed
as
mean
parasite
titer
±
SD.
2.6.
RNA
isolation
and
quantitative
real-time
PCR
of
cytokine
Total
RNA
was
extracted
from
the
spleen
and
liver
of
infected
hamsters
using
Trizol
reagent
(Invitrogen).
First
strand
of
cDNA
synthesis
was
performed
with
approximately
1–2
g
of
RNA
in
a
total
volume
of
20
l
using
the
SuperScriptTM III
reverse
transcriptase.
DNA
was
amplified
adding
2
g
of
RNA
in
30
l
of
a
mix
containing
primers
oligo
(dT),
2.5
M,
dNTPs,
1
mM
(Invitrogen),
buffer
1×
(Tris–HCl
20
mM,
pH
8.4,
KCl
50
mM,
MgCl22
mM),
20
U
of
ribonuclease
inhibitor
and
50
U
of
Super-
script
II
reverse
transcriptase
(Gibco).
Amplification
conditions
consisted
of
an
initial
pre-incubation
at
42 ◦C
for
50
min,
fol-
lowed
by
amplification
of
the
target
DNA
for
40
cycles
at
95 ◦C
for
5
min.
A
standard
curve
was
generated
for
each
set
of
primers
and
the
efficiency
of
each
reaction
was
determined.
The
expression
levels
of
the
genes
were
normalized
to
GAPDH
levels.
The
results
are
expressed
in
fold
change
over
control.
Oligonucleotide
primers
used
for
real
time
PCR
were:
GAPDH
(5CTGACATGCCGCCCTGGAG;
3TCAGTGTAGCCCAGGATGCC);
IFN-
␥
(5GAAGCTCACCAAGATTCCGGTAA;
3TTTTCGTGACAGGTGAGGC
AT);
IL-10
5AGACGCCTTTCTCTTGGAGCTTAT;
3GGCAACTGCAGCG
CTGTC);
TGF-
(5GCTACCACGCCAACTTCTGTC;
3TGTTGGTAGA
GGGCAAGG).
Primers
for
IL10,
TGF-,
IFN-␥
and
GAPDH
was
obtained
from
Applied
Biosystem,
EUA.
The
real
time
reaction
was
performed
in
96
well
plates
using
SYBER-Green
PCR
Master
Mix
and
Perkin-Elmer
ABI
Prism
7500
sequence
detection
system.
Forty
cycle
reactions
with
15
s
at
94 ◦C
and
1
min
at
60 ◦C
were
performed
according
with
ABI-Prism
7500
manufacturater’s
instructions.
2.7.
Hematological
analyses
Blood
samples
were
collected
from
different
animal
groups
for
hematological
evaluation,
two
and
five
months
after
chal-
lenge.
Blood
smears
were
stained
by
Giemsa
and
cell
counts
were
R.A.A.
da
Silva
et
al.
/
Acta
Tropica
120 (2011) 185–
190 187
Fig.
1.
Ear
and
dLN
parasite
loads
in
early
moments
post-infection
by
L.
chagasi.
Hamsters
were
immunized
three
times
in
the
ear
dermis.
Groups
of
animals
were
immunized
with
saline,
LJM19,
KMP11,
KMP11
plus
LJM19
and
empty
control
plas-
mids.
Two
weeks
after
the
last
immunization
hamsters
were
challenged
with
105
L.
chagasi
plus
0.5
salivary
gland
par
equivalent
in
the
lateral
ear.
Ears
and
draining
lymph
nodes
(dLN)
were
collected
7
and
14
days
after
challenge.
Parasites
loads
of
the
ears
and
dLN
were
evaluated
by
limiting
dilution
assay.
(A)
Ear
parasite
load.
(B)
dLN
parasite
load.
n
=
6.
*p
<
0.05,
**p
<
0.01.
performed
by
a
blood
cell
counter.
Healthy
hamsters
were
used
as
control
for
immunized
and
challenged
animals.
2.8.
Statistical
analysis
Results
were
expressed
as
medians
±
SD.
Comparisons
among
the
experimental
groups
were
done
by
one-way
ANOVA
(Kruskal–Wallis)
test
with
Dunn’s
post
test
using
Graphpad
5
software
program.
Differences
with
p
<
0.05
were
considered
sig-
nificant..
3.
Results
3.1.
Immunized
hamsters
showed
reduced
parasite
load
and
increased
pro-inflammatory
cytokines
profile
in
early
stages
of
infection
The
parasite
load
was
evaluated
7
and
14
days
after
the
infection
in
LJM19,
KMP11
and
KMP11
plus
LJM19
DNA
plasmid
immu-
nized
hamsters.
Seven
days
after
challenge
with
L.
chagasi
plus
Lu.
longipalpis
saliva,
KMP11
and
KMP11
plus
LJM19
DNA
plas-
mid
immunized
hamsters
showed
significantly
reduced
parasite
load
in
the
ear
(Fig.
1A).
However,
there
were
no
differences
con-
cerning
parasite
loads
in
the
draining
lymph
nodes
(dLN)
between
immunized
and
control
groups
(p
>
0.05)
(Fig.
1B).
To
examine
the
production
of
pro-inflammatory
and
anti-inflammatory
cytokines
in
immunized
and
infected
hamsters,
real
time
PCR
was
performed
in
dLN
samples
at
7
and
14
days
after
challenge.
LJM19
DNA
plasmid
immunized
hamsters
showed
a
significantly
higher
IFN-
␥/IL-10
and
IFN-␥/TGF-
ratios
than
control
animals
(p
<
0.05)
at
both
time
points.
KMP11
or
LJM19
plus
KMP11
DNA
plasmid
immu-
Fig.
2.
Cytokines
expression
by
dLN
of
hamsters
immunized
with
DNA
plasmids
coding
KMP11
and
LJM19
and
challenged
with
L.
chagasi
plus
saliva.
Hamsters
were
immunized
for
three
times
in
the
ear
dermis.
Groups
of
animals
were
immunized
with
saline,
LJM19,
KMP11,
KMP11
plus
LJM19
and
empty
control
plasmids.
Two
weeks
after
the
last
immunization
hamsters
were
challenged
with
105L.
chagasi
plus
0.5
salivary
gland
par
equivalent
in
the
lateral
ear.
Retromandibulars
dLN
were
collected
7
and
14
days
after
challenge.
IFN-␥,
IL-10
and
TGF-
expression
was
evaluated
by
real
time
PCR.
The
relative
quantification
(RQ)
was
obtained
using
non-
immunized
control
hamsters
and
the
ratio
was
obtained
dividing
IFN-␥
expression
by
IL-10
or
TGF-
expression.
(A)
IFN-␥/IL-10.
(B)
IFN-␥/TGF-.
n
=
6.
*p
<
0.05.
nized
hamsters
showed
an
enhancement
of
3
and
2.5
times
in
the
IFN-␥/IL-10
and
IFN-␥/TGF-
ratios
respectively,
compared
to
con-
trol
unimmunized
animals.
However,
these
ratios
in
the
dLN
did
not
show
significant
differences
compared
to
the
control
groups
(Fig.
2A
and
B).
Another
alternative
is
that
immunization
LJM19
DNA
plasmid
induced
a
faster
recall
response,
whereas
KMP11
did
not.
At
the
same
time,
it
seems
that
immunization
with
KMP11
DNA
plasmid
could
exert
an
inhibitory
effect
since
the
immunization
with
the
combination
of
KMP11
plus
LJM19
DNA
plasmids
resulted
in
lower
IFN-␥/IL-10
and
IFN-␥/TGF-
ratios.
These
results
sug-
gest
that
in
the
initial
events
after
the
infection,
other
mechanisms
such
as
innate
immunity
might
be
responsible
for
the
reduction
in
the
parasite
load
observed
at
the
site
of
infection
and
not
only
the
adaptive
immune
response
against
parasite
or
saliva.
3.2.
Late
stages
of
infection
showed
reduced
visceral
parasite
load
in
immunized
hamsters
Parasite
loads
in
the
liver
and
spleen
were
quantified
two
and
five
months
after
infection
by
L.
chagasi
plus
SGS.
Two
months
after
infection
hamsters
immunized
with
LJM19,
KMP11
or
LJM19
plus
KMP11
DNA
plasmid
showed
a
105fold
reduction
in
the
parasite
load
in
the
spleen
compared
with
control
unimmunized
group
(p
<
0.05)
(Fig.
3A).
The
liver
of
LJM19
or
LJM19
plus
KMP11
DNA
plasmid
immunized
hamsters
did
not
show
parasites
after
two
months
of
infection
(Fig.
3B).
Parasite
loads
in
the
spleen
and
liver
remained
lower
five
months
after
infection
in
immu-
nized
hamsters.
Spleens
and
livers
from
LJM19
and
LJM19
plus
KMP11
DNA
plasmid
immunized
hamsters
showed
a
107fold
reduction
at
this
time
point
and
were
significantly
lower
than
saline
and
empty
plasmid
control
groups
(p
<
0.01
and
p
<
0.05,
188 R.A.A.
da
Silva
et
al.
/
Acta
Tropica
120 (2011) 185–
190
Fig.
3.
Liver
and
spleen
parasite
loads
of
hamsters
immunized
with
DNA
plasmids
coding
KMP11
and
LJM19
and
challenged
with
L.
chagasi
plus
saliva.
Hamsters
were
immunized
for
three
times
in
the
ear
dermis.
Groups
of
animals
were
immunized
with
saline,
LJM19,
KMP11,
KMP11
plus
LJM19
and
control
plasmids.
Two
weeks
after
the
last
immunization
hamsters
were
challenged
with
105L.
chagasi
plus
0.5
salivary
gland
par
equivalent
in
the
lateral
ear.
Two
and
five
months
after
challenge
spleen
and
liver
were
collected
and
weighted.
Parasites
loads
of
the
spleen
and
liver
were
evaluated
by
limiting
dilution
assay.
(A).
Spleen
parasite
load.
(B).
Liver
parasite
load.
n
=
5–9.
*p
<
0.05.
respectively)
(Fig.
3).
At
two
months
post-infection
no
significant
differences
were
observed
in
IFN-␥/IL-10
ratio
in
immunized
and
non-immunized
hamster
groups
(Fig.
4A).
However,
the
group
of
KMP11
DNA
plasmid
immunized
hamsters
showed
a
significant
increased
IFN-␥/TGF-
ratio
(Fig.
4B).
At
five
months
post-infection
immunized
groups
showed
an
increase
in
IFN-␥/IL-10
and
IFN-
␥/TGF-
ratio.
However,
control
group
(saline)
also
showed
an
increase
in
this
ratio
as
well
as
empty
vectors,
suggesting
that
the
high
number
of
parasites
at
this
point
could
be
responsible
for
this
alteration
in
cytokine
profile.
3.3.
Immunized
hamsters
do
not
develop
hematological
disorders
after
challenge
with
L.
chagasi
It
is
well
known
that
patients
with
visceral
leishmaniasis
develop
hematological
disorders
(Fernandez-Guerrero
et
al.,
2004).
Hamsters
can
reproduce
many
of
these
clinical
features
and
develop
anemia,
leukopenia
and
pancytopenia.
To
examine
if
immunization
with
DNA
plasmids
could
preclude
anemia
devel-
opment,
blood
samples
from
different
groups
of
hamsters
were
evaluated
two
and
five
months
after
challenge.
We
observed
that
immunization
of
hamsters
with
DNA
coding
plasmids
abro-
gated
the
development
of
anemia.
Healthy
hamsters
were
used
as
controls.
Compared
with
healthy
hamsters,
our
control
ani-
mals
showed
a
decreased
number
of
red
blood
cells
(p
<
0.05),
a
decreased
hematocrit
(p
<
0.05)
and
a
reduced
amount
of
hemoglobin
(p
<
0.05).
However,
immunized
hamsters
did
not
develop
reduction
in
these
hematological
parameters
and
did
not
show
significant
differences
compared
to
the
healthy
control
ani-
mal
group
(p
>
0.05)
(Table
1
).
Fig.
4.
Spleen
cytokine
expression
in
immunized
hamsters
challenged
with
L.
cha-
gasi
plus
saliva.
Hamsters
were
immunized
for
three
times
in
the
ear
dermis.
Groups
of
animals
were
immunized
with
saline,
LJM19,
KMP11,
KMP11
plus
LJM19
and
empty
control
plasmids.
Two
weeks
after
the
last
immunization
hamsters
were
challenged
with
105L.
chagasi
plus
0.5
salivary
gland
par
equivalent
in
the
lat-
eral
ear.
Two
and
five
months
after
challenge
spleen
samples
were
collected.
IFN-␥,
IL-10
and
TGF-
production
was
evaluated
by
real
time
PCR.
The
relative
quantifi-
cation
(RQ)
was
obtained
using
non-immunized
control
hamsters
and
the
ratio
was
obtained
dividing
IFN-␥
expression
by
IL-10
or
TGF-
expression.
(A)
IFN-␥/IL-10.
(B)
IFN-␥/TGF-.
n
=
5–9.
*p
<
0.05,
**p
<
0.01.
4.
Discussion
Previous
studies
from
other
groups
as
well
as
ours
have
demon-
strated
protection
against
experimental
visceral
leishmaniasis
using
plasmids
coding
KMP11,
a
parasite
product,
or
LJM19,
a
prod-
uct
from
the
vector
saliva
(Basu
et
al.,
2005;
Gomes
et
al.,
2008).
In
this
report,
we
show
that
such
effects
are
not
addictive
as
the
pro-
tection
obtained
with
a
combination
of
KMP11
+
LJM19
was
similar
to
the
levels
obtained
with
either
product
used
separately.
Accord-
ingly,
the
production
of
IFN-␥,
TGF-
as
well
as
IL-10
was
similar
in
all
immunized
groups.
Although
no
significant
differences
in
para-
site
load
were
found
in
the
draining
lymph
nodes
in
the
early
stages
of
infection
(7
and
14
days),
hamsters
immunized
with
KMP11
and
LJM19
plus
KMP11,
did
not
showed
a
significant
reduction
in
para-
site
load
at
7
and
14
days
after
infection
in
the
ears.
This
reduction
persisted
in
the
spleen
and
liver
in
later
stages
of
infection,
possi-
bly,
reflecting
the
initial
control
in
the
number
of
parasites
through
innate
immunity
and/or
increase
in
levels
of
IFN-␥produced
in
the
early
stages
of
the
infection.
An
enhancement
of
IFN-␥
early
production
in
lymph
nodes
could
be
important
for
parasite
control
at
the
inoculation
site
and
could
be
related
with
lower
visceralization
in
immunized
groups.
Many
studies
show
the
important
role
of
IL-10
as
anti-
inflammatory
cytokine,
deactivating
macrophages,
diminishing
NO
production
and
facilitating
Leishmania
survival
inside
phagocytes
(Olivier
et
al.,
2005).
Similarly,
TGF-
is
an
important
cytokine
for
parasite
survival
and
disease
progression
(Gantt
et
al.,
2003).
Interestingly,
our
control
hamsters
showed
higher
ratios
of
IFN-
␥/IL-10
and
IFN-␥/TGF-
in
the
spleen
five
months
after
challenge.
These
ratios
are
in
agreement
with
previous
studies
using
cells
from
human
VL
patients,
which
show
elevated
parasite
load
even
in
a
pro-inflammatory
environment
(Nylen
et
al.,
2007).
The
level
R.A.A.
da
Silva
et
al.
/
Acta
Tropica
120 (2011) 185–
190 189
Table
1
Eritrogram
from
hamsters
immunized
with
DNA
plasmids
coding
KMP11
and
LJM19
and
challenged
with
L.
infantum
chagasi
plus
saliva
of
Lutzomyia
longipalpis.
Healthy Saline Ljm19 Kmp11 Kmp11
+
Ljm19 CT
2
Months
5
months
2
Months
5
months
2
Months
5
months
2
Months
5
months
2
Months
5
months
Red
blood
cells
(106)
8.5 ±
0.1
8.1 ±
0.1
6.8 ±
0.4*8.4 ±
0.2
8.4 ±
0.1
8.2 ±
0.1
7.7 ±
0.4
7.8 ±
0.2
8.5 ±
0.3
7.8 ±
0.2
6.9 ±
0.4*
Hemoglobin
(g%) 16.1 ±
0.4
14.9
±
0.3
13.2
±
1.1*16.2
±
0.4
14.9
±
0.5
16.1
±
0.2
14.1
±
0.7
14.4
±
0.4
15.8
±
0.4
14.5
±
0.7
13.1
±
0.6*
Hematocrit
(%) 47.5
±
0.6
45.1
±
0.5
37.3
±
1.7*45.8
±
1.0
47.6
±
1.6
46.4
±
0.6
47.7
±
2.1
43.7
±
1.2
47.9
±
1.9
43.6
±
1.2
37.6
±
2.2*
n
=
6.
*p
<
0.05.
of
IFN-␥/TGF-
probably
increased
as
a
response
to
an
excessive
pathology
caused
by
sustained
infection
(Gomes
et
al.,
2008).
Inter-
estingly,
immunized
hamsters
did
not
show
sterilizing
infection,
however
they
did
not
develop
clinical
manifestations
of
disease
neither
hematological
alterations,
The
role
of
phlebotomine
sand
fly
saliva
has
been
shown
both
in
animal
and
human
models
(Barral
et
al.,
2000;
Costa
et
al.,
2004;
Guilpin
et
al.,
2002)
The
formal
demonstration
that
mice
immunized
with
DNA
plasmids
coding
SP15,
a
P.
papatasi
salivary
component,
and
hamsters
immunized
with
DNA
plasmid
coding
LJM19,
a
Lu.
longipalpis
salivary
pro-
tein,
protect
these
animals
against
L.
major
and
L.
chagasi
infection
respectively,
pointing
out
for
new
topics
in
vaccination
against
Leishmania
infection
(Gomes
et
al.,
2008;
Valenzuela
et
al.,
2001).
These
studies
showed
that
DTH
development
at
the
site
bites
pro-
mote
protective
immunity
against
Leishmania.
Recently,
it
was
demonstrated
that
immunization
with
DNA
plasmid
coding
KMP11,
a
conserved
protein
in
different
parasite
species,
protected
hamsters
against
L.
donovani
infection
(Basu
et
al.,
2005;
Fuertes
et
al.,
2001;
Ramirez
et
al.,
2001).
This
pro-
tection
was
related
with
an
enhancement
of
inducible
nitric
oxide
synthetase,
IFN-␥
and
IL-4
production,
a
reduction
of
IL-10
and
anti-Leishmania
antibodies
production
(Basu
et
al.,
2005).
Our
data
confirmed
the
protection
conferred
by
KMP11,
using
L.
chagasi
plus
saliva
as
challenge.
Blood
disorders
are
frequently
observed
both
in
humans
and
animal
models
for
visceral
leishmaniasis
(Caldas
et
al.,
2006;
Fernandez-Guerrero
et
al.,
2004;
Moreno
et
al.,
2007).
Similarly
to
human
patients,
hamsters
develop
anemia
and
leucopenia
(Melby
et
al.,
1998).
Parasitism
and
higher
cytokine
production
can
induce
alterations
in
hematopoetic
cell
behavior
conducting
to
pancytope-
nia
(Pastorino
et
al.,
2002;
Yarali
et
al.,
2002).
Interestingly,
KMP11
and/or
LJM19
DNA
plasmid
immunized
hamsters
did
not
show
hematological
disorders
even
5
months
after
challenge
when
com-
pared
with
healthy
controls,
suggesting
that
the
control
of
parasite
number
also
correlated
to
the
better
condition
of
the
animals.
Interestingly,
in
our
study
the
combination
of
DNA
plasmids
coding
KMP11
and
LJM19
did
not
enhance
the
protection
observed
in
immunized
hamsters.
Different
immunization
protocols
can
be
used
to
reach
higher
protection.
Heterelogous
prime/booster
immunization,
using
DNA
and
protein,
has
shown
excellent
results
in
vaccine
development
protocols
(Dondji
et
al.,
2005).
Similarly,
Leishmania
antigens
entrapment
in
liposomes
and
nanoparticles
has
demonstrated
to
induce
Th1
profiles
in
different
animal
mod-
els,
contributing
to
the
control
of
intracellular
microoganisms
(Greenland
and
Letvin,
2007;
Sharma
et
al.,
2006).
In
summary,
we
have
confirmed
the
ability
of
KMP11
DNA
plas-
mid
to
induce
a
protective
immune
response
against
L.
chagasi
infection
in
the
hamster
model.
We
confirm
the
previous
results
showing
protection
against
this
Leishmania
specie
in
hamsters
immunized
with
LJM19
DNA
plasmid.
This
protection
is
followed
by
an
enhancement
of
IFN-␥
production
in
the
dLN
and
a
reduction
in
parasites
load
in
the
liver
and
spleen.
Interestingly,
we
demon-
strated
for
the
first
time
that
the
immunization
with
these
plasmids
can
abrogate
hematological
disorders.
However,
the
combination
of
DNA
plasmids
coding
KMP11
or
LJM19
does
not
enhance
these
protective
effects
probably
because
the
immune
response
elicited
by
these
plasmid
immunization
could
be
acting
more
in
the
initial
phase
of
infection
(presence
of
saliva)
and
in
promastigotes
that
are
more
abundant
in
the
early
infection
stages,
conferring
protection
in
this
model.
Acknowledgements
We
thank
Edivaldo
Passos
for
technical
assistance.
This
research
was
supported
by
FAPESB,
CNPq
and
CAPES.
CB,
AB,
CIO
and
MB-N
190 R.A.A.
da
Silva
et
al.
/
Acta
Tropica
120 (2011) 185–
190
are
senior
investigators
of
CNPq-Brasil.
RAAS
received
a
fellowship
from
CNPq.
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