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Vaccination with
L. infantum chagasi
Nucleosomal
Histones Confers Protection against New World
Cutaneous Leishmaniasis Caused by
Leishmania
braziliensis
Marcia W. Carneiro
1
, Diego M. Santos
1
, Kiyoshi F. Fukutani
1
, Jorge Clarencio
1
, Jose Carlos Miranda
1
,
Claudia Brodskyn
1,2
, Aldina Barral
1,2
, Manoel Barral-Netto
1,2
, Manuel Soto
3
, Camila I. de Oliveira
1,2
*
1 Centro de Pesquisas Gonc¸alo Moniz, The Oswaldo Cruz Foundation, Salvador, Brazil, 2 Instituto de Investigac¸a
˜
o em Imunologia, Salvador, Brazil, 3 Centro de Biologı
´
a
Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Departamento de Biologia Molecular, Universidad Autonoma de Madrid, Madrid, Spain
Abstract
Background:
Nucleosomal histones are intracellular proteins that are highly conserved among Leishmania species. After
parasite destruction or spontaneous lysis, exposure to these proteins elicits a strong host immune response. In the present
study, we analyzed the protective capability of Leishmania infantum chagasi nucleosomal histones against L. braziliensis
infection using different immunization strategies.
Methodology/Principal Findings:
BALB/c mice were immunized with either a plasmid DNA cocktail (DNA) containing four
Leishmania nucleosomal histones or with the DNA cocktail followed by the corresponding recombinant proteins plus CpG
(DNA/Protein). Mice were later challenged with L. braziliensis, in the presence of sand fly saliva. Lesion development, parasite
load and the cellular immune response were analyzed five weeks after challenge. Immunization with either DNA alone or
with DNA/Protein was able to inhibit lesion development. This finding was highlighted by the absence of infected
macrophages in tissue sections. Further, parasite load at the infection site and in the draining lymph nodes was also
significantly lower in vaccinated animals. This outcome was associated with increased expression of IFN-c and down
regulation of IL-4 at the infection site.
Conclusion:
The data presented here demonstrate the potential use of L. infantum chagasi nucleosomal histones as targets
for the development of vaccines against infection with L. braziliensis, as shown by the significant inhibition of disease
development following a live challenge.
Citation: Carneiro MW, Santos DM, Fukutani KF, Clarencio J, Miranda JC, et al. (2012) Vaccination with L. infantum chagasi Nucleosomal Histones Confers
Protection against New World Cutaneous Leishmaniasis Caused by Leishmania braziliensis. PLoS ONE 7(12): e52296. doi:10.1371/journal.pone.0052296
Editor: Dario S. Zamboni, University of Sa
˜
o Paulo, Brazil
Received August 14, 2012; Accepted November 12, 2012; Published December 20, 2012
Copyright: ß 2012 Carneiro 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 Conselho Nacional de Desenvolvimento Cientı
´
fico e Tecnolo
´
gico and CYTED. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: camila@bahia.fiocruz.br
Introduction
Leishmaniasis is an infectious disease with significant economic
impact in several countries. Over three hundred million people are
exposed to the parasites, with 12 million infected worldwide,
predominantly in tropical and subtropical countries (World Health
Organization page: http://www.who.int/emc/diseases/leish/
leisdis1.html). Leishmaniasis can be caused by different species
of Leishmania spp. protozoans that infect macrophages in the human
host. The treatments available for all forms of leishmaniasis are
toxic, and drug resistance is on the rise, further increasing the need
for vaccine development [1].
Numerous attempts have been made to find a protective antigen
against leishmaniasis and several candidates have been tested for
this purpose [2,3], including histones. Histones are structural
proteins found in the nucleus, where they play an important role in
the organization and function of chromatin. There are five main
classes of histones; four of them (H2A, H2B, H3 and H4) form the
nucleosomal core unit of chromatin, whereas H1 joins to linker
DNA. The percentage of similarity between Leishmania nucleosome
forming histones and their mammal counterparts ranges from
49% (for the H2B) to 63% (for the H3) [4]. Differences are mainly
located in the aminoacid sequences of the nucleosome-exposed
tails of the four histones [5]. So far, no cross reactivity was found
between Leishmania histones and their mammalian counterparts.
Antibodies specific for parasite histones, obtained from dogs with
visceral leishmaniasis, react against Leishmania H2A [6], H3 [7],
H2B and H4 [8] but do not recognize mammalian histones.
Antibodies in sera from patients with cutaneous or mucocutaneous
leishmaniasisis also recognize parasite histone H1 but not the
human counterpart [9]. Regarding the T cell immunogenicity of
parasite histones, recombinant versions of H2B [10] or H2A [11]
induced IFN-c secretion upon stimulation of PBMCs obtained
from cutaneous leishmaniasis patients. The T cell response was
PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52296
specific for parasite histones, since T cell lines derived from
cutaneous leishmaniasis patients did not respond to mammalian
histones [12].
Leishmania histones are recognized by sera from cutaneous
leishmaniasis patients [13] and from dogs infected with Leishmania
[6,7,8] and it has been hypothesized that these parasite proteins
may trigger an immune response after active destruction or
spontaneous cytolysis of Leishmania amastigotes [4,14]. Immuniza-
tion with the histone H1 was able to confer protection against L.
major [15,16] and L. infantum [17]. Immunization with H2B was
able to confer protection in mice infected with L. major [18], as
seen by a decrease in parasite load and lesion size. The protective
effect against leishmaniasis was also observed upon immunization
with the four core histone proteins (H2A, H2B, H3 and H4)
[19,20,21].
In addition to antigen selection, the immunization strategy is
also important for generating protection. Various antigens have
been tested and evaluated as DNA and/or recombinant protein
vaccine candidates in murine models of leishmaniasis, resulting in
various degrees of protection [22]. DNA vaccination has also been
tested in heterologous prime-boost vaccination regimes [23], in
which the immune system is primed with DNA and boosted with
a different formulation of the corresponding antigen. This strategy
has been shown to be effective in experimental models of
cutaneous leishmaniasis [24,25,26].
Based on the protective capacity of the four nucleosomal
histones against L. infantum chagasi infection, we hypothesized that
this antigenic cocktail would confer protection against the
development of New World cutaneous leishmaniasis caused by
L. braziliensis, a species for which there are few published studies
regarding vaccine development in comparison with L. major [27].
The present study also compared immunization strategies in-
volving plasmid DNA only and plasmid DNA plus recombinant
proteins.
Materials and Methods
Mice and Ethics Statement
Female BALB/c mice, 6–8 weeks of age, were obtained from
CPqGM/FIOCRUZ animal facility where they were maintained
under pathogen-free conditions. Animals were randomly distrib-
uted into groups of five and each group was subjected to a specific
immunization strategy. All animal work was conducted according
to the Guidelines for Animal Experimentation of the Cole´gio
Brasileiro de Experimentac¸a˜o Animal and of the Conselho
Nacional de Controle de Experimentac¸a˜o Animal. The local
Ethics Committee on Animal Care and Utilization (CEUA)
approved all procedures involving animals (CEUA – Centro de
Pesquisas Gonc¸alo Muniz/FIOCRUZ - L031/08).
Preparation of DNA Plasmids and Recombinant Proteins
The recombinant plasmids [21](pcDNA3-LiH2A, pcDNA3-
LiH2B, pcDNA3-LiH3 and pcDNA3-LiH4) were prepared
using the endotoxin-free Giga-preparation Kit (Qiagen) follow-
ing the manufacturer’s instructions. The final pellet was
resuspended in sterile water and stored at 220uC until use.
Expression and purification of the His-tagged recombinant
proteins (pQEH2A, pQE-H2B, pQE-H3 and pQE-H4) were
performed as previously described [21]. After binding to a Ni-
NTA agarose column (Qiagen), recombinant proteins were
gradually refolded on the affinity column as described [28].
Recombinant proteins were eluted with 0.3 M imidazole and
dialyzed against PBS. Finally, proteins were passed through
a polymyxin-agarose column (Sigma) in order to eliminate
endotoxins. Residual endotoxin was measured with Quantitative
Chromogenic Limulus Amebocyte assay (QCL-1000, BioWhit-
taker), showing that recombinant histone preparations were
essentially endotoxin-free (less than 30 ng endotoxin per mg of
recombinant protein).
Immunization Strategies
BALB/c mice were immunized three times, with a two-week
interval between each immunization. Animals received three
intramuscular (i.m.) injections of a DNA (100 mg) cocktail
containing 25 mg of each recombinant plasmid (pcDNA3-LiH2A,
pcDNA3-LiH2B, pcDNA3-LiH3 and pcDNA3-LiH4) in a total
volume of 50 ml PBS. Control mice were immunized (i.m.) with
100 mg of WT pcDNA3. Alternatively, mice received two
inoculations (i.m.) of the recombinant DNA cocktail followed by
a third (s.c.) inoculation of a recombinant protein (20 mg) cocktail
containing 5 mg of each recombinant protein (H2A, H2B, H3 and
H4) and 25 mg of each CpG ODN (59- tcagcgttga-39 and 59-
gctagcgttagcgt-39) (E-OLIGOS). Control mice received two
immunizations (i.m.) with WT pcDNA3 followed by a third
inoculation (s.c.) of saline+CpG. DNA immunizations were
performed in the left quadriceps and protein immunizations were
performed in the left footpad.
Cytokine Detection in Immunized Mice
BALB/c mice were immunized as described above. Two weeks
after the last immunization, mice were euthanized and single-cell
suspensions of draining lymph nodes (dLN) were prepared
aseptically. Briefly, dLN were homogenized in RPMI 1640 and
cells were resuspended in RPMI supplemented with 2 mM L-
glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 10%
FCS (all from Invitrogen) and 0.05 M b-mercaptoethanol. Cell
suspensions were stimulated for 48 h with 5 mg/mL Concanavalin
A (Amersham Biosciences) or 3 mg/mL of each of the following
recombinant proteins: LiH2A, LiH2B, LiH3 and LiH4. Culture
supernatants were harvested and cytokine concentrations were
assayed using a Th1/Th2 cytokine Cytometric Bead Array (BD
Biosciences), which detects murine IFN-c, IL-4 and TNF-
a according to the manufacturer’s instructions. The data were
acquired and analyzed using a FACSort flow Cytometer (BD
Immunocytometry) and CBA Analysis Software (Becton-Dick-
inson).
Sand Flies and Preparation of SGS
Lutzomyia intermedia salivary glands were obtained as previously
described [29]. Salivary gland from adult female flies were
dissected and transferred to 10 or 20 ml Hepes, 10 mM
pH 7.0 NaCl 0.15 in 1.5 polypropilene vials, usually in groups
of 20 pairs of glands in 20 ml of Hepes saline. Salivary glands were
kept at 275uC until needed, when they were disrupted by
sonication using a Branson Sonifier 450 homogenizer (Branson,
Danbury, CT). Salivary gland sonicate (SGS) was centrifuged at
10,0006g for 4 min and the supernatants were used in the
experiments. The level of lipopolysaccharide (LPS) contamination
of SGS preparations was determined using a commercially
available LAL chromogenic kit (QCL-1000; Lonza Biologics).
LPS concentration was ,0.1 ng/ml.
Parasite Culture, Intradermal Inoculation and Lesion
Measurement
L. braziliensis promastigotes (strain MHOM/BR/01/BA788)
[30] were grown in Schneider medium (Sigma), supplemented
with 100 U/ml of penicillin, 100 mg/ml of streptomycin and 10%
Protective Capacity of Nucleosomal Histones
PLOS ONE | www.plosone.org 2 December 2012 | Volume 7 | Issue 12 | e52296
heat-inactivated fetal calf serum (Invitrogen). Two weeks after the
last immunization, mice were inoculated with L. braziliensis,as
described previously [30]. Challenges consisted of inoculation of
stationary-phase promastigotes (10
5
parasites)+SGS (1 mg, equiv-
alent to 1 pair of salivary glands), in 10 ml of saline. Lesion size was
monitored weekly for 10 weeks, using a digital caliper (Thomas
Scientific).
Parasite Load Estimate
Parasite load was determined using a quantitative limiting
dilution assay as described previously [31]. Animals were
euthanized five weeks post infection. Infected ears and draining
lymph nodes (dLNs) were aseptically excised and homogenized in
Schneider medium (Sigma). The homogenates were serially
diluted in Schneider medium supplemented with 100 U/ml of
penicillin, 100 mg/ml of streptomycin, 10% heat-inactivated fetal
calf serum (all from Invitrogen) and seeded into 96-well plates
containing biphasic blood agar (Novy-Nicolle-McNeal) medium.
The number of viable parasites was determined from the highest
to lowest dilution at which promastigotes could be grown after one
week of incubation at 25uC.
Histology
BALB/c mice were immunized and challenged as described
above. Five weeks post challenge, animals were euthanized and
ears were removed and fixed in 10% formaldehyde. Following
fixation, tissues were processed, embedded in paraffin and 5 mm
sections were stained with hematoxylin and eosin (H & E) and
analyzed by light microscopy.
RNA Isolation and Real-time PCR
Five weeks following infection with L. braziliensis+SGS, mice
were euthanized and infected ears were excised and mechanically
lysed with ceramic beads in a MagNALyzerH instrument (Roche
Molecular Systems) according to manufacturer’s instructions.
Total RNA was extracted from the resulting tissue lysates using
the RNeasy Protect Mini Kit (Qiagen) according to the
manufacturer’s instructions. RNA was eluted in 20 ml water and
used for cDNA synthesis. Real-time PCR was performed on the
ABI Prism 7500 (Applied Biosystems). Thermal cycle conditions
consisted of a two-minute initial incubation at 50uC followed by
a 10 minute denaturation at 95uC and 50 cycles at 95uC for 15
seconds and 60uC for one minute each. Each sample and the
negative control were analyzed in triplicate for each run. The
comparative method was used to analyze gene expression.
Cytokine cycle threshold (C
t
) values were normalized to GAPDH
expression as determined by the equation DC
t
=C
t (cytokine)
–C
t
(GAPDH)
. Fold change was determined by 2
–DDCt
, where
DDC
t
= DC
t (experimental)
– DC
t (control).
[32]. Primers employed
herein are described elsewhere [33].
Intracellular Cytokine Detection by Flow Cytometry
Reagents for staining cell surface markers and intracellular
cytokines were purchased from BD Biosciences. Measurement of
in vitro cytokine production was performed as described [30].
Briefly, animals were euthanized five weeks post infection. dLNs
were aseptically excised and homogenized in RPMI supple-
mented with 100 U/ml of penicillin, 100 mg/ml of streptomy-
cin, 10% heat-inactivated fetal calf serum (all from Invitrogen).
Cells were then activated using 5 mg/ml Concanavalin A
(Amersham Biosciences) and incubated with 10 mg/ml Brefeldin
A (Sigma). Cells were blocked with anti-Fc receptor antibody
(2.4G2) and were double stained with anti-mouse surface CD4
(L3T4) and CD8 (53–6.7) conjugated to FITC and Cy-Chrome,
respectively. For intracellular staining of cytokines, cells were
permeabilized using Cytofix/Cytoperm and were incubated with
the following anti-cytokine antibodies conjugated to PE: IFN-c
(XMG1.2), IL-4 (BVD4-1D11) or IL-10 (JES5-16E3). The
isotype controls used were rat IgG2b (A95-1) and rat IgG2a
(R35–95). Data were collected and analyzed using CELLQuest
software and a FACSort flow cytometer (Becton Dickinson
Immunocytometry System).
Statistical Analysis
The data are presented as the mean 6 SEM. Comparisons
between four groups (DNA, DNA/Protein and controls) were
performed by Kruskal-Wallis (non-parametric test) followed by
Dunn’s multiple comparisons test. Comparisons between two
groups (DNA vs. control or DNA/Protein vs. control) were
performed by Mann-Whitney (non-parametric t-test) using
GraphPad version 6.0a (Prism) and P-values ,0.05 were
considered significant. To evaluate disease burden in mice, ear
thickness of mice following challenge was recorded weekly for each
individual mouse. The course of disease for experimental and
control mice was plotted individually. The Area under the curve
(AUC) obtained for each mouse immunized with antigen versus
AUC obtained for each control mouse was analyzed by Mann-
Whitney (non-parametric t-test).
Results
Immunization with Nucleosomal Histones Prevents
Lesion Development in Mice Infected by L. braziliensis
Plus Sand Fly Saliva
We first analyzed the immune response induced upon
immunization with a plasmid DNA cocktail encoding histones
H2A, H2B, H3 and H4. Mice inoculated with the recombinant
DNA cocktail (rDNA) did not show a significant increase in IFN-c
(Fig. 1A), IL-4 (Fig. 1B) or TNF-a (Fig. 1C) when compared with
mice immunized with WT DNA. Similar results were observed
regarding antigen specific cytokine production in mice immunized
with the combination of plasmid DNA followed by recombinant
proteins (rDNA/rProtein+CpG) vs. control (WT DNA/CpG)
(Fig. 1D–F). Two weeks after the last immunization, mice were
challenged in the dermis of the ear by co-inoculation of L.
braziliensis plus sand fly (L. intermedia) saliva, in order to mimic the
natural context of infection by Leishmania parasites. Following
challenge, lesion development was monitored for ten weeks. Mice
immunized with rDNA did not develop disease, as shown by
maintenance of ear thickness close to baseline levels (Fig. 2A),
indicating disease prevention. Control mice (immunized with WT
DNA) developed lesions that peaked at five weeks after infection
(Fig. 2A), characteristic of the inoculation of L. braziliensis into the
ear dermis of BALB/c mice [30]. At this time point, ear thickness
reached a maximum of 1.2 mm. Mice inoculated with rDNA/
rProtein+CpG (Fig. 2B) also did not develop lesions whereas
controls immunized with WT DNA/CpG behaved as mice
inoculated with WT DNA (Fig. 2A). Importantly, disease de-
velopment, as determined by the area under the curves (AUCs)
shown in Fig. 2A and Fig. 2B (see Materials and Methods), was
significantly inhibited in mice immunized with rDNA or with
rDNA/rProtein+CpG, when compared with controls (Fig. 2C).
The AUCs of immunized mice (either rDNA only or rDNA/
rProtein+CpG) and of control mice (WT DNA only or WT DNA/
CpG) were similar. This suggests that immunization with L.
infantum chagasi histones inhibit cutaneous leishmaniasis caused by
L. braziliensis.
Protective Capacity of Nucleosomal Histones
PLOS ONE | www.plosone.org 3 December 2012 | Volume 7 | Issue 12 | e52296
We also analyzed ear sections, obtained five weeks after co-
inoculation with L. braziliensis plus sand fly (L. intermedia) saliva. As
shown in Fig. 3A, mice immunized with WT DNA developed an
intense inflammatory infiltrate with an accumulation of parasite-
infected macrophages. In mice immunized with WT DNA,
infected cells were abundant (Fig. 3A), differently from mice
immunized with rDNA (Fig. 3B). In tissue sections from mice
immunized with WT DNA/CpG, we observed a dense and
widespread inflammatory infiltrate containing infected macro-
phages displaying a foamy aspect (Fig. 3C). In rDNA/rPro-
tein+CpG-inoculated mice, amastigotes were not detected and the
inflammatory infiltrate was characterized by the presence of rare
eosinophils, plasmocytes and epithelioid macrophages (Fig. 3D).
Parasite Load and Cytokine Expression Profile at the
Infection Site
Given the significant inhibition of lesion development following
immunization with plasmid rDNA (Fig. 2A and C) or with rDNA/
rProtein+CpG (Fig. 2B and C), we also investigated the parasite
load. At five weeks post parasite inoculation, when ear thickness
was at its peak (Fig. 2A and B), mice immunized with rDNA
displayed a significantly lower parasite load at the infection site
(Fig. 4A) and at the draining lymph node (Fig. 4B). Comparable
results were observed with rDNA/rProtein+CpG-immunized mice
(Fig. 4A and B), in terms of inhibition in parasite replication. The
parasite load detected in the ear of mice immunized with histones
(rDNA vs. rDNA/rProtein+CpG) or in control animals (WT DNA
vs. WT DNA/CpG) were not significantly different (Fig. 4A and
B).
Figure 1. Antigen specific cytokine production following immunization with nucleosomal histones. BALB/c mice (5 per group) were
immunized with wild type DNA (WT DNA) or with recombinant DNA coding for nucleosomal histones (rDNA) (A–C). Alternatively, BALB/c mice (5 per
group) were immunized with wild type DNA followed by CpG (WT DNA+CpG), or with recombinant DNA followed by recombinant nucleosomal
histones+CpG (rDNA/rProtein+CpG) (D–F). Two weeks after the last immunization, dLNs were collected and cells were re-stimulated with the
recombinant proteins or with Concanavalin A (Con A). Antigen specific cytokine production in culture supernatants was determined by flow
cytometry, using a Th1–Th2 Cytometric Bead Array. Data are presented as the mean+SEM and are from two independent experiments.
doi:10.1371/journal.pone.0052296.g001
Figure 2. Lesion development in mice immunized with nucleosomal histones following infection with
L. braziliensis
plus sand fly
saliva. BALB/c mice (5 per group) were immunized with WT DNA or with rDNA (A). Alternatively, BALB/c mice (5 per group) were immunized with WT
DNA+CpG or with rDNA/rProtein+CpG (B). Two weeks after the last immunization, mice were challenged with L. braziliensis+sand fly saliva. The
course of lesion development was monitored weekly and bars represent the means and standard errors from two independent experiments. The
areas contained underneath the curves obtained in (A) and in (B) for each individual mouse from experimental and control groups were compared
(C). Data are presented as the mean+SEM.
doi:10.1371/journal.pone.0052296.g002
Protective Capacity of Nucleosomal Histones
PLOS ONE | www.plosone.org 4 December 2012 | Volume 7 | Issue 12 | e52296
Figure 3. Histological aspects of ear lesions in mice immunized with nucleosomal histones and challenged with
L. braziliensis
plus
sand fly saliva. BALB/c mice (five mice per group) were immunized with WT DNA (A) or with rDNA (B). Alternatively, mice were immunized with WT
DNA/CpG (C) or with rDNA/rProtein+CpG (D). Two weeks after the last inoculation, mice were challenged with L. braziliensis+sand fly saliva. Ears were
removed at 5 weeks post infection and stained with hematoxylin and eosin. Panels represent 1006magnification. (C) Dark symbols indicate infected
macrophages displaying a foamy aspect. (D) Red symbols indicate plasmocytes and dashed arrows indicate eosinophils.
doi:10.1371/journal.pone.0052296.g003
Figure 4. Parasite load following immunization with nucleosomal histones and challenge with
L. braziliensis
plus sand fly saliva.
BALB/c mice (5 per group) were immunized with WT DNA or with rDNA. Alternatively, BALB/c mice (5 per group) were immunized with rDNA/
rProtein+CpG. Two weeks after the last immunization, mice were challenged with L. braziliensis+sand fly saliva. Ear (A) and draining lymph node (B)
parasite loads were determined five weeks post infection via a limiting dilution assay. Data are presented as the mean+SEM and are from two
independent experiments.
doi:10.1371/journal.pone.0052296.g004
Protective Capacity of Nucleosomal Histones
PLOS ONE | www.plosone.org 5 December 2012 | Volume 7 | Issue 12 | e52296
Therefore, immunization with L. infantum chagasi nucleosomal
histones modified the course of infection in mice challenged with
L. braziliensis.
Based on these results, we analyzed cytokine expression at the
infection site. Total RNA was obtained from ear sections five
weeks after parasite inoculation, when ear thickness was at its peak
in control mice (Fig. 2) and the parasite load was significantly
different between immunized versus control groups (Fig. 4). RNA
was subjected to real-time PCR analysis, and cytokine gene
expression was normalized to GAPDH (housekeeping gene), as
described in the Materials and Methods. Ears from rDNA-
immunized mice showed a two-fold up-regulation in IFN-c
expression in comparison with control mice (Fig. 5A) whereas
the expression of IL-4 and IL-10 were down-regulated (Fig. 5A).
Mice immunized with rDNA/rProtein+CpG also showed up-
regulation of IFN-c expression (Fig. 5B). These results indicate the
predominance of a Th1-polarized response and can be correlated
with the decreased disease burden (Fig. 2) and the lower parasite
load observed at the infection site (Fig. 4) of mice immunized with
nucleosomal histones.
Cellular Immune Response in dLNs
Based on the lower parasite load observed in the dLNs of
immunized mice (Fig. 2), we also evaluated the presence of
cytokine-secreting cells therein. Five weeks after infection, cells
from lymph nodes draining the infection site were re-stimulated
in vitro with recombinant histones and the frequency of cytokine-
secreting cells was determined by flow cytometry (Figure S1). The
percentage of CD4+ IFN-c-secreting cells was similar in control vs.
rDNA-immunized mice (Fig. 6A) and in controls vs. rDNA/
rProtein+CpG-immunized mice (Fig. 6B). Also, we did not detect
significant differences in the percentage of CD4+ IL-4+-secreting
cells in (Fig. 6C–D) or in the frequency of CD4+IL-10+-secreting
cells (Fig. 6E–F), when comparing the two immunization
strategies. The percentage of CD8+ IFN-c-secreting cells was
similar in control vs. rDNA-immunized mice (Fig. 7A) but was
significantly higher in mice immunized with rDNA/rPro-
tein+CpG when compared with controls (Fig. 7B). The frequency
of CD8+IL-4+-secreting cells was also similar in control vs. rDNA-
immunized mice (Fig. 7C) but was significantly lower in mice
immunized with rDNA/rProtein+CpG when compared with
controls (Fig. 7D). As seen with CD4+ cells, the frequency of
CD8+IL-10+-secreting cells did not differ significantly in control
vs. immunized mice (Fig. 7E and F).
Discussion
In the present work, we evaluated the comparative vaccine
potential of rDNA and rDNA/rProtein+CpG using L. infantum
chagasi histones H2A, H2B, H3 and H4 in the context of eliciting
immunity against L. braziliensis. The results reported here suggest
that both strategies were able to prevent lesion development in an
experimental model of New World cutaneous leishmaniasis.
Previous studies using L. infantum chagasi nucleosomal histones
have shown the induction of a Th1-biased response, with
significant levels of protection against L. major infection in mice
[19,20,21]. To evaluate efficacy in an experimental model of New
World Cutaneous Leishmaniasis, we employed two immunization
strategies: rDNA and rDNA/rProtein+CpG. The prime-boost
strategy (rDNA/rProtein+CpG) aims at augmenting immune
responses induced by rDNA vaccination alone and it has been
successfully employed previously in leishmaniasis [34,35,36,37].
Here, recombinant proteins were formulated with CpG, given its
ability to stimulate macrophages and dendritic cells (DCs) to
synthesize cytokines, up-regulate the expression of co-stimulatory
molecules and to enhance the cross-presentation properties of DCs
[38,39]. In the present study, immunization with rDNA alone or
with rDNA followed by rProtein+CpG did not elicit a strong
immune response, as shown by the lack of a significant increase in
cytokine production in immunized mice. Herein, cell cultures were
simultaneously stimulated with the recombinant proteins corre-
sponding to histones H2A, H2B, H3 and H4 whereas in the study
by Iborra et al., cells from mice immunized with nucleosomal
histones were stimulated with each recombinant protein separately
and the authors were able to detect a significant increase in IFN-c
secretion [21].
Despite the fact that we did not detect a robust pre-challenge
immune response, both immunization strategies were able to
significantly inhibit lesion development upon intradermal in-
oculation of L. braziliensis, in the presence of L. intermedia sand fly
saliva. Although mice were challenged with a high inoculum (10
5
stationary-phase promastigote forms), significant protection was
achieved as shown by lack of lesion development and by the
significant reduction (over 2 log) in parasite load observed at the
infection site in mice vaccinated with either rDNA or rDNA/
rProtein+CpG. This level of protection is comparable to
vaccination studies published employing nucleosomal histones
and L. major infection [40]. Regarding sand fly saliva, it contains
pharmacologically active molecules that promote adequate blood
feeding and that may contribute to establishment of infection by
Leishmania (rev. in [41]). Among the salivary components
characterized to date are maxadilan [42], that modulates the
inflammatory response by inhibiting cytokines such as TNF-a;
hyaluronidase, that helps the diffusion of other pharmacological
substances through the skin matrix [43] and a 5-nucleotidase, that
exerts a vasodilator and anti-platelet aggregation role by
converting AMP to adenosine [44]. In the case of L. braziliensis,
co-inoculation of salivary gland sonicate (SGS) and parasites led to
Figure 5. Cytokine expression in the ear dermis following
immunization with nucleosomal histones and challenge with
L.
braziliensis
plus sand fly saliva. BALB/c mice (5 per group) were
immunized with rDNA (A) or with rDNA/rProtein+CpG (B). Two weeks
after the last immunization, mice were challenged with L. brazilien-
sis+sand fly saliva. Relative quantification of IFN-c, IL-4 and IL-10 at the
infection site was carried out five weeks after infection. Cytokine cycle
threshold (C
t
) values were normalized to GAPDH expression (house-
keeping) as determined by DC
t
=C
t (cytokine)
–C
t (GAPDH)
. Fold change
was determined by real-time PCR, using the 2
–DDCt
method, where
DDC
t
= DC
t (experimental)
– DC
t (control)
(see Materials and Methods). Data
(mean+SEM) are presented as fold increase in gene expression of
immunized mice over control mice and are from two independent
experiments.
doi:10.1371/journal.pone.0052296.g005
Protective Capacity of Nucleosomal Histones
PLOS ONE | www.plosone.org 6 December 2012 | Volume 7 | Issue 12 | e52296
a significant exacerbation of both lesion size and parasite load
[45,46,47]. We later showed that immunization with L. intermedia
SGS altered the course of experimental infection with L. braziliensis
[29] and stimulation of immune mice with a combination of SGS
and L. braziliensis led to a decreased CXCL10 expression and
increased IL-10 expression [33]. These results suggest that L.
intermedia saliva exerts important immunomodulatory activities
and, therefore, we judged important to include salivary compo-
nents to the challenge inoculum. Other studies also employed
salivary gland sonicate to ‘‘mimic’’ the effects of sand fly saliva in
the context of vaccination [48,49,50,51,52]. More recently, Peters
et al. showed that vaccination with autoclaved L. major anti-
gen+CpG confers protection against a needle inoculation of
Figure 6. Intracellular cytokine production by CD4+ cells from
mice immunized with nucleosomal histones and challenged
with
L. braziliensis
plus sand fly saliva. BALB/c mice (5 per group)
were immunized as described. Two weeks after the last immunization,
mice were challenged with L. braziliensis+sand fly saliva. Five weeks
later, draining lymph nodes were pooled and cells were preincubated
with Brefeldin A for four hours before staining. Data represent the
frequency of CD4+ cells positive for IFN-c (A, B), IL-4 (C, D) and IL-10 (D,
E) with signals for the particular cytokine that were greater than the
background signals established using isotype controls. Data are
presented as the mean+SEM and are from two independent experi-
ments.
doi:10.1371/journal.pone.0052296.g006
Figure 7. Intracellular cytokine production by CD8+ cells from
mice immunized with nucleosomal histones and challenged
with
L. braziliensis
plus sand fly saliva. BALB/c mice (5 per group)
were immunized as described. Two weeks after the last immunization,
mice were challenged with L. braziliensis+sand fly saliva. Five weeks
later, draining lymph nodes were pooled and cells were preincubated
with Brefeldin A for four hours before staining. Data represent the
frequency of CD8+ cells positive for IFN-c (A, B), IL-4 (C, D) and IL-10 (D,
E) with signals for the particular cytokine that were greater than the
background signals established using isotype controls. Data are
presented as the mean+SEM and are from two independent experi-
ments.
doi:10.1371/journal.pone.0052296.g007
Protective Capacity of Nucleosomal Histones
PLOS ONE | www.plosone.org 7 December 2012 | Volume 7 | Issue 12 | e52296
parasites but not against challenge with infected sand flies [53]
whereas KSAC, a polyprotein vaccine candidate [54], conferred
protection against the bite of L. major-infected sand flies [55]. To
date, a colony of L. intermedia sand flies is not available, hampering
the possibility of using infected sand flies in challenge experiments.
In this sense, we believe that addition of SGS at the time of
challenge partially addresses this limitation and allows us to
evaluate the effects of salivary molecules in the context of
vaccination.
Inhibition of lesion development in mice immunized with
nucleosomal histones (rDNA or rDNA/rProtein+CpG) was
accompanied by a significant decrease in parasite load at the
infection site. These results may be related to a significant up-
regulation of the expression of IFN-c, as seen in infected ears five
weeks after infection. Macrophages control Leishmania infection by
inducing reactive oxygen species when activated by IFN-c [56].
Therefore, we can speculate that IFN-c-secreting cells may have
migrated to the infection site, promoting macrophage activation
and parasite killing. Additionally, Iborra et al. showed 1.22% of
CD4+ IFN-c+ and 1.02% of CD8+ IFN-c+ T cells in mice
challenged with L. major [21] whereas we detected 1.7%
CD4+IFN-c+ (Figure 6) and 1.9% CD8+ IFN-c+ cells (Figure 7),
in mice immunized with rDNA and challenged with L. braziliensis.
We believe these results are comparable in terms of the immune
response detected after challenge and indicate that the protective
capacity of nucleosomal histones is associated with an expansion of
IFN-c-expressing cells.
Chenik et al. documented the participation of the carboxy-
terminal portion of the H2B histone in the activation of Treg cells
[18]. The presence IL-10 secreting cells, with a regulatory
phenotype, may have modulated potentially harmful effects
associated with the development of a Th1-immune response
[57], leading to the control of pathology at the infection site of
mice immunized with nucleossomal histones. Indeed, the presence
of Tregs has been associated with healing of experimental infection
with L. braziliensis [58]. On the other hand, control mice that did
not receive nucleosomal histomes did not develop a Th1-immune
response. In this case, the potential presence of a regulatory
response could have contributed with parasite replication instead
of a control in pathology.
During the past several decades, extensive efforts have been
made to develop an effective Leishmania vaccine [3,22]. The
majority of studies have been conducted with L. major [1]. L.
braziliensis, which is distinguished from other leishmaniasis by its
chronicity, latency and tendency to metastasize in the human host
[59], has been largely neglected in the context of vaccine
development. Moreover, candidate antigens such as the receptor
for activated C kinase protein (LACK), thiol-specific antioxidant
(TSA), Leishmania elongation and initiation factor (LeIF) and L.
major stress-inducible protein 1 (LmST1), all of which induced
protection against L. major, failed to prevent L. braziliensis infection
[60]. These findings highlight the need for continued investigation
into molecules able to confer protection against this particular
species. In conclusion, we believe our results represent an
important contribution to understanding leishmaniasis as they
extend the cross-protective effect of nucleosomal histones from L.
infantum chagasi to a model of New World cutaneous leishmaniasis
caused by L. braziliensis.
Supporting Information
Figure S1 Cytokine expression in CD4+ and in CD8+
cells in mice immunized with nucleossomal histones,
following challenge with L. braziliensis plus sand fly
saliva. BALB/c mice were immunized with DNA coding for
nucleosomal histones and two weeks after the last immunization,
mice were infected in the dermis of the ear with 10
5
L. braziliensis+
sand fly saliva, as described in Materials and Methods. Gates depict
CD4+and CD8+ T lymphocytes present in the draining lymph
node (dLN). The presence of IFN-c
+
, IL-4
+
and IL-10
+
T cells was
determined by flow cytometry in the gated populations. Data
shown are representative dot plots for IFN-c
+,
IL-4
+
and IL-10
labeling.
(TIF)
Acknowledgments
We thank Dr Almerio Noronha for help with histopathology analysis and
documentation. MWC, DMS and KFF were supported by CAPES
fellowships. MBN, AB, CB and CIO are senior investigators from CNPq.
Author Contributions
Conceived and designed the experiments: MWC DMS MS CIO.
Performed the experiments: MWC DMS JC KF JCM. Analyzed the data:
MWC DMS KF JC CB AB MBN MS CIO. Contributed reagents/
materials/analysis tools: MS AB JCM MS. Wrote the paper: MWC MS
CIO.
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