Immunization of dogs with DNA-prime/DNA-boost vaccine (TcVac1) enhanced the
Trypanosoma cruzi-specific type 1 antibody and
CD8+ T cell responses that resulted in an early control of
acute parasitemia and a moderate decline in pathological symptoms during chronic
phase. Further improvement of vaccine-induced immunity would be required to
achieve clinical and epidemiological benefits and prevent transmission of
parasites from vaccinated/infected dogs to triatomines.
Background
Trypanosoma cruzi, the etiologic agent of Chagas Disease, is
a major vector borne health problem in Latin America and an emerging
infectious disease in the United States.MethodsWe tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine
(TcVac1) against experimental T. cruzi infection in a
canine model. Dogs were immunized with antigen-encoding plasmids and
cytokine adjuvants, and two weeks after the last immunization, challenged
with T. cruzi trypomastigotes. We measured antibody
responses by ELISA and haemagglutination assay, parasitemia and infectivity
to triatomines by xenodiagnosis, and performed electrocardiography and
histology to assess myocardial damage and tissue pathology.ResultsVaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG
(IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs,
as compared to non-vaccinated controls dogs, responded to T.
cruzi with a rapid expansion of antibody response, moderately
enhanced CD8+ T cell proliferation and IFN-γ production,
and suppression of phagocytes’ activity evidenced by decreased
myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled
the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and
exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized
dogs did not control the myocardial parasite burden and electrocardiographic
and histopatholgic cardiac alterations that are the hallmarks of acute
Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a
moderate decline in cardiac alterations determined by EKG and
anatomo-/histo-pathological analysis while
chronically-infected/non-vaccinated dogs continued to exhibit severe EKG
alterations.ConclusionsOverall, these results demonstrated that TcVac1 provided a partial resistance
to T. cruzi infection and Chagas disease, and provide an
impetus to improve the vaccination strategy against Chagas disease.
IntroductionAmerican trypanosomiasis (Chagas disease) is a disease of humans and caused by the
protozoan Trypanosoma cruzi of the family trypanosomatidae [1]. Approximately
30–40% of the infected patients develop a chronic debilitating illness
of the cardiac system, characterized by clinically irreversible and progressive
tissue destruction, and myocardial hypertrophy, eventually leading to heart failure
and the patient’s death [2], [3].Several investigators have shown the potential utility of T. cruzi
surface antigens as vaccine candidates in murine experimental models [4], [5](reviewed in
[6], [7]). We have
shown the protective efficacy of amastigote surface proteins ASP-1 and ASP-2, and
trypomastigote surface antigen TSA-1 as DNA vaccines in mice [8]. Vaccination with ASP-2 provided
maximal immunity to T. cruzi infection in mice that was further
enhanced by co-delivery of cytokine adjuvants [8]. In recent studies, we have
identified additional potential vaccine candidates by computational screening of
T. cruzi sequence database [9]. Of these, TcG1-TcG8 were
phylogenetically conserved in clinically important strains of T.
cruzi and expressed in the infective and intracellular stages of the
parasite [9]. When
delivered as a DNA vaccine in mice, TcG1, TcG2 and TcG4 elicited a significant
trypanolytic antibody response and Th1 cytokine (IFN-γ) response, a property
associated with immune control of T. cruzi
[10]. These novel
vaccine candidates, thus, increased the pool of protective vaccine candidates
against T. cruzi.In this study, we proceeded to examine the prophylactic and transmission-blocking
efficacy of the multi-component vaccine constituted of TcG1, TcG2 and TcG4 in dogs.
We chose dogs for our studies because dogs provide an excellent model for studying
the human disease [11]–[13]. Experimentally and naturally infected young dogs
(2–3 months) elicit reproducible and comparable acute infection associated
with increase in blood parasitemia, IgG and IgM antibodies [14], [15] and T cell response
[16]. The
presence of myocarditis with a moderate or small number of parasitized cells and
extensive and frequent focal necrosis turns the disease in dogs similar to acute
Chagas disease in humans [17], [18]. A few infected dogs
(10–20%; 5% humans) develop severe acute myocarditis and may die
of cardiac arrest. More than 80% of dogs recover from acute parasitemia as
parasites become undetectable in the blood, and remnant mild myocardial changes with
scattered microscopic foci of fibrosis and lymphocytic infiltration [19] present a
picture similar to that of human infections [20], [21]. At 12–18 months
post-infection (pi), ∼50% of infected dogs exhibit symptomatic chronic
cardiac disease (20% develop severe myocarditis), associated with progressive
cardiomegaly; arrhythmia, including RBBB with left anterior hemiblock [22], [23]; diffused
myocarditis with focal and interstitial fibrosis, and self-perpetuating myofibril
destruction - a picture reminiscent of the chronic form of Chagas disease observed
in humans. Thus, testing the vaccine efficacy in dogs would provide a strong basis
for developing a human vaccine against T. cruzi and Chagas
disease.Further, dogs are an important reservoir host for domestic transmission of T.
cruzi. The prevalence rate of T. cruzi infection in
dogs may reach up to 84% in endemic areas (e.g. rural Argentina, Chiapas,
Mexico), determined by serological procedures and xenodiagnosis [24], [25]. Dogs are also
the most frequent blood meal source for the domestic triatomines, i.e., T.
barberi and T. pallidipennis in Mexico [15] and
T. infestans in Argentina [25], [26]. Likewise, a high prevalence
of seropositive dogs and infected triatomines is routinely noted in rural and urban
development in the southern US [27], [28], and suggested to maintain T. cruzi
transmission in the human habitat. Triatomines are several times more likely to take
their blood meal from dogs than from humans [26]. The ratio of dog blood meals
to human blood meals in the engorged guts of triatomines is estimated to be
2.3–2.6 times higher than the ratio of the number of dogs to the number of
humans in a household [29]. Thus, the probability of infecting an insect in one
blood meal from dogs is estimated to be 200-times higher as compared to the
probability from adult humans [25]. These studies demonstrate that dogs are an important
host blood source for domiciliary triatomines, and the risk of T.
cruzi infection in humans is increased by the presence of infected
dogs. Strategies that can limit T. cruzi infection in domestic
reservoir host may, thus, prove to be effective in interrupting the parasite
transmission to the vector, and consequently, to the human host.We immunized dogs with DNA-prime/DNA-boost vaccine (TcVac1). We examined the efficacy
of TcVac1 in eliciting antigen-and parasite-specific antibody and T cell immunity,
and determined if vaccination with TcVac1 modulated the host immune response towards
protective type 1 upon T. cruzi infection. We also examined the
efficacy of TcVac1 in controlling acute parasitemia, blocking the parasite
transmission to triatomines, and preventing clinical severity of chronic
disease.Materials and MethodsAnimalsTwelve mongrel dogs (6 males and 6 females, 3–4 months old) were acquired
locally and kept at the animal facility at the UAEM Research Center until they
were included in the experiment, at eight months of age (8–12 kg body
weight). Dogs were confirmed free of T. cruzi infection by
microscopic examination of blood smears and serological evaluation of
anti-T. cruzi antibodies using an indirect
haemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA)
[15], [18]. Before inclusion
in experimental studies, dogs were treated with anti-helminthes and vaccines
against regional infectious diseases (Canine distemper, Parvovirus infection,
Canine hepatitis, Leptospirosis, and Rabies). All dogs received water ad
libitum, and commercial dog food fed twice a day according to their
age and development requirements. Experimental protocols were conducted under
the technical specifications for the production, care, and use of lab animals
from the Norma Official Mexicana (NOM-062-ZOO-1999), and the Council for
International Organizations of Medical Science [30], [31]. The research protocols
were approved by the Laboratory Animal Care Committee at the Universidad
Nacional Autonoma de Mexico.Immunization and challenge infectionTcVac1 vaccine was constituted of antigen-encoding plasmids (pCDNA3.TcG1,
pCDNA3.TcG2 and pCDNA3.TcG4) and IL-12- and GMCSF-expression plasmids, described
previously [8],
[32]. The
eukaryotic expression plasmids encoding dog cytokines (IL-12 and GM-CSF) were a
kind gift from Dr. Peter Melby [33]. All recombinant
plasmids were transformed into E. coli DH5-α competent
cells, grown in L-broth containing 100 µg/ml ampicillin, and purified by
anion exchange chromatography using the Qiagen maxi prep kit (Qiagen,
Chatsworth, CA) according to the manufacturer’s specifications.
Trypomastigotes of T. cruzi (SylvioX10/4) were maintained and
propagated by continuous in vitro passage in C2C12 cells.Dogs (n = 6/group, 3 males and 3 females) were
intramuscularly immunized with TcVac1 (200 µg each plasmid DNA/dog),
delivered four-times at 2-week intervals. Dogs vaccinated with empty vector
(pcDNA3 only) were used as controls. Two-weeks after the last immunization, dogs
were challenged with culture-derived T. cruzi SylvioX10/4
(3.5×103 trypomastigotes/kg body weight, i.p.). The
selected dose of the parasites was sufficient to produce acute parasitemia
within 1–2 weeks of inoculation, and symptomatic clinical disease within
6–8 weeks post-infection [18]. Dogs were
observed daily for general physical condition, at weekly intervals for clinical
condition, and at 2-week intervals for cardiac function, monitored by
electrocardiography (EKG). Sera samples were obtained before each immunization
and at two-week intervals thereafter. After challenge infection, in addition to
sera samples, blood samples for parasitemia diagnostics were collected beginning
day 5 pi, on alternate days up to 50 dpi and at two-week intervals
thereafter.Parasitological measuresWe measured blood parasitemia using hemacytometer counts of 5 µl blood
mixed with equal volume of ACK red blood cell lysis buffer. Xenodiagnostic
analysis was performed as described [25], [34], [35]. Briefly,
stage 4 naive triatomine (T. pallidipenis) nymphs (6 per dog)
were fed on vaccinated and control dogs on day 30 and day 60 pi. Fecal samples
were collected from triatomines at day 60 after feeding, and analyzed by light
microscopy to detect epimastigote and metacyclic trypomastigotes. At least 10
microscopic fields were analyzed for each fecal sample, and triatomines were
considered T. cruzi positive when >1 parasites were
detected.SerologyThe cDNAs for TcG1, TcG2 and TcG4 were cloned
in pET-22b plasmid (Novagen) such that the encoded proteins would be expressed
in-frame with a C-terminal His-tag. All cloned sequences were confirmed by
restriction digestion and sequencing at the Recombinant DNA Core Facility at
UTMB. For the purification of recombinant proteins, plasmids were transformed in
BL21 (DE3) pLysS competent cells, and recombinant proteins purified using the
polyhistidine fusion peptide-metal chelation chromatography system
(Novagen).Blood samples were obtained by venopuncture of the cephalic vein, and immediately
processed to separate sera, using standard methods [15], [18].
Sera samples (1∶50–1∶100 dilution) were analyzed for IgM and
IgG by using the Chagas diagnostic kits for ELISA (Laboratorio-Lemos SRL, Buenos
Aires, Argentina). The horseradish peroxidase (HRP)-labeled anti-human-IgG in
ELISA kit was replaced with HRP-conjugated goat-anti-dog IgM- or IgG-specific
secondary antibody (Bethyl Laboratories) [15], [18].
In some experiments, instead of T. cruzi lysate, plates were
coated with recombinant antigens (TcG1, TcG2 or TcG4, 10-µg protein/ml) to
capture the antigen-specific antibodies. Sera samples from chronically infected
dogs with confirmed T. cruzi infection and from healthy
domestic dogs were used as positive and negative controls, respectively (cut off
value: ELISA, mean OD450nm from negative dogs ±2 SD; IHA,
positive titer at ≥1∶16 serum dilution).To identify the antibody sub-types (IgG1 and IgG2), plates were coated with
T. cruzi antigen, and, then sequentially incubated at room
temperature with sera samples (1∶50 dilution) for 2 h, biotin-conjugated
goat anti-dog Ig subtypes (IgG1 and IgG2) for 2 h, and streptavidin-horseradish
peroxidase conjugate for 30 min. All antibodies and conjugates were from Bethyl
Laboratories, and used at a 1∶3000 dilution in PBST-0.5% NFDM
(100-µl/well). Color was developed with 100-µl/well Sure Blue TMB
substrate (Kirkegaard & Perry Labs), reaction was stopped with 2N sulfuric
acid, and antibody response was monitored at 450 nm using a SpectraMax M5
microplate reader.Spleen cell phenotypeSplenic level of CD4+ and CD8+ cell population in
immunized/challenged dogs was determined by flow cytometry. Briefly, splenocytes
were suspended in PBS (1×106 cells/100 µl) and incubated
for 30 min with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 antibodies
(1∶50 dilution, from ABD Serotec). Following incubation, cells were fixed
with 2% paraformaldehyde, washed and re-suspended in 500 µl PBS,
and analyzed on a FACScan apparatus (BD Biosciences). Cells stained with PE- and
FITC- conjugated rat IgGs (isotype matched) were used as negative controls. Flow
data were analyzed by Cell Quest software (BD Biosciences).Serum cytokine levelsCytokine levels in sera of vaccinated dogs were measured by sandwich ELISA.
Briefly, 96 well plates were coated overnight with anti-IFN-γ or anti-IL-10
antibodies (500-ng/ml in PBS), washed with PBS/0.05% Tween-20 (PBST), and
incubated for 2 h with 1% BSA. Plates were then sequentially incubated at
room temperature with sera samples (50-µl/well) for 2 h, biotinylated
anti-dog IFN-γ antibody (0.5-µg/ml) or anti-dog IL-10 antibody
(2-µg/ml) for 2 h, and streptavidin conjugated horse radish peroxidase
(1∶3000 dilution) for 45 min. All antibodies were from R&D systems.
Colorimetric reaction was performed as above. Cytokine concentrations were
calculated using a standard curve derived using recombinant IFN-γ or IL-10
(1–4000 pg/ml).Myeloperoxidase (MPO) activityMPO activity was determined by a dianisidine-H2O2 method
[36],
modified for 96-well plates [37]. Briefly, plasma samples (10-µg protein) were
added in triplicate to 0.53 mM o-dianisidine dihydrochloride
(Sigma) and 0.15 mM H2O2 in 50 mM potassium phosphate
buffer (pH 6.0). After incubation for 5 min at room temperature, the reaction
was stopped with 30% sodium azide and the change in absorbance was
measured at 460 nm (ε = 11,300 M−1
cm−1). Results were expressed as units of MPO/mg protein,
whereby one unit of MPO was defined as the amount of enzyme degrading one n mol
H2O2 per min at 25°C.Nitrite levelThe nitrite/nitrate content, indicative of inducible nitrite oxide synthase
(iNOS) activity, was monitored by the Greiss reagent assay, as described [37]. In 96-well
plates, reduced plasma samples (10 µg protein) were mixed with 100
µl Greiss reagent, consisting of 1% sulfanilamide in 5%
phosphoric acid and 0.1% N-(1-napthyl)ethylenediamine
dihydrochloride (1∶1, v/v), and incubated for 10 min. The change in
absorbance was monitored at 545 nm (standard curve, 0–200 n mol sodium
nitrite).ElectrocardiographyChanges in cardiac rhythm and conduction in all dogs was monitored before
inclusion in the study, and after challenge infection, at 2-week intervals up to
8-weeks and at monthly intervals thereafter. We used electrocardiograph (Stylus,
EK-8, USA) setting at 120 V, 60 Hertz, 20 amps, and 25 Watt in all experiments.
Six leads of the electrocardiogram were considered at 25 mm/sec at 1-mV,
standardized to 1 cm for the present study.Necropsy and histological studiesNecropsy was performed the day animals died due to infection or after
humanitarian sacrifice at day 60 (acute phase) and day 365 (chronic phase) post
challenge infection. Dogs were sedated with xylazine (1–3 mg/kg body
weight) and then euthanized according to the Mexican Norma Official Mexicana
[30],
[31],
using protocols approved by the Laboratory Animal Care Committee at the
Universidad Nacional Autonoma de Mexico. A macroscopic and microscopic analysis
of affected organs was performed. Postmortem studies were conducted using
standard protocols with emphasis on macroscopic findings related to Chagas
disease in heart tissue [15]. For histological
analysis, tissue samples were fixed in 10% buffered formalin for 24 h,
dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin.
Tissue sections (5-µm thick) were stained with hematoxylin-eosin, and
evaluated by light microscopy at 100× and 400× [15], [18].
Tissues were scored 0 to 4 in blind studies, according to the extent of
inflammation and tissue damage from normal to total wall involvement [38].Statistical analysisData are expressed as means ± SD, and derived from duplicate experiments
(n≥6 animals/group/experiment) with at least duplicate observations per
sample. Results were analyzed for significant differences using ANOVA procedures
and Student’s t-tests. The level of significance was accepted at
*p<0.05 (vaccinated versus non-vaccinated).ResultsThe development of an antibody response induced by TcVac1 was determined by an ELISA.
All dogs were seronegative before vaccination was initiated. The T.
cruzi-specific IgM and IgG antibody response was detectable in sera
(1∶50 dilution) of vaccinated dogs after the first immunization, and
moderately increased upon delivery of booster vaccine doses (Fig. 1A&B). The level of antigen-specific
antibody response was detected in the order of TcG1>TcG2>TcG4, and was
additive in nature (Fig. 1E).
The vaccine-induced antibody response was predominantly of the Th1 type with
IgG2/IgG1 ratios >1 (Fig.
1C&D). Control dogs immunized with plasmid vector alone exhibited no
parasite- and antigen-specific antibody response (Fig. 1).10.1371/journal.pntd.0001050.g001Figure 1TcVac1-induced antibody response in dogs.Dogs were vaccinated with TcVac1 or injected with vector only, and sera were
collected two weeks after each immunization dose. An ELISA was performed to
monitor the sera levels (1∶50 dilution) of parasite-specific IgM
(A), IgG (B), IgG1 (C) and IgG2
(D). The vaccine-induced antigen-specific (TcG1, TcG2 and
TcG4) antibody response in sera collected 14 days after last dose of vaccine
was determined using recombinant antigens. Data are presented as mean
± SD (*p<0.05, vaccinated versus
non-vaccinated, n = 6 per experiment).After challenge infection with T. cruzi, sera samples were analyzed
at 2-week intervals (1∶100 dilution) (Fig. 2). Non-vaccinated/infected dogs exhibited a
slight increase in parasite-specific IgM levels (Fig. 2A). All dogs, irrespective of vaccination
status, responded to T. cruzi infection by a gradual increase in
anti-parasite IgG levels (Fig.
2B). The TcVac1-immunized dogs exhibited a faster increase in IgG
antibody response to T. cruzi infection (Fig. 2B, p<0.05) as compared
to that detected in non-vaccinated/infected dogs. Likewise, the vaccine-induced
dominance of IgG2 antibodies (compared to IgG1 subtype) was significantly expanded
after infection (Fig. 2C&D).
Together, the results presented in Fig.
1 and Fig. 2
suggested that vaccination of dogs with TcVac1 skewed the antibody response towards
Th1 type that was further expanded upon challenge infection with T.
cruzi.10.1371/journal.pntd.0001050.g002Figure 2The antibody response to T. cruzi infection was
polarized to type 1 in vaccinated dogs.Dogs were vaccinated as above, and infected with T. cruzi.
An ELISA was performed to evaluate sera levels (1∶100-dilution) of
T. cruzi-specific IgM (A), IgG
(B), IgG1 (C), and IgG2 (D)
antibodies at 0, 15, 30, 45, and 60 days post-infection.Next, we measured vaccine’s efficacy in activation of phagocytic (neutrophils
and macrophages) response to T. cruzi by evaluating the plasma
level of MPO activity and nitrite contents (Fig. 3). Vaccinated and non-vaccinated dogs
exhibited no detectable level of MPO activity before challenge infection (Fig. 3A). After exposure to
T. cruzi, all dogs responded by a rise in MPO activity. During
day 15–30 pi, non-vaccinated/infected and vaccinated/infected dogs exhibited a
2-fold and 25% increase in MPO activity in response to T.
cruzi infection (Fig.
3A). After day 30 pi, all dogs exhibited a similar decline in circulatory
MPO activity. Likewise, the nitrite levels, indicative of iNOS activation and NO
production, were increased by 2-fold in non-vaccinated/infected dogs at 30 dpi,
while vaccinated/infected dogs exhibited ∼22% increase in plasma nitrite
contents (Fig. 3B). These data
suggested that immunization with TcVac1 suppressed the T.
cruzi-mediated activation of phagocytes evidenced by decreased plasma
levels of MPO and nitrite in vaccinated/acutely-infected dogs as compared to
non-vaccinated controls.10.1371/journal.pntd.0001050.g003Figure 3Shown are plasma levels of myeloperoxidase activity (A) and nitrite
content (B) in dogs immunized with vector only or TcVac1 vaccine during the
course of 0–60 days post-infection.Data are presented as mean ± SD (*p<0.05).A predominance of CD8+ T cells and type 1 cytokines (IFN-γ) is
shown to be essential for control of T. cruzi infection [32]. All dogs,
irrespective of vaccination regimen, responded to T. cruzi
infection by a strong increase in parasite-specific lymphocyte activation (Fig. 4). The vaccinated/infected
dogs exhibited a moderately stronger CD8+T cell response as compared
to the non-vaccinated/infected dogs that was maintained during acute infection and
chronic disease phase (Fig. 4A).
The circulatory cytokine levels (IFN-γ and IL-10) were below detection limit
before and after immunization with TcVac1. The sera level of IL-10 remained
undetectable after challenge infection with T. cruzi in all dogs.
In comparison, all dogs responded to infection by a rapid increase in circulatory
IFN-γ level that was significantly higher in vaccinated/infected dogs as
compared to that noted in non-vaccinated/infected dogs (Fig. 4B). These results indicated that
TcVac1-immunized dogs were moderately better than non-vaccinated dogs in responding
to T. cruzi infection by elicitation of higher level of type 1
biased CD8+T cell response.10.1371/journal.pntd.0001050.g004Figure 4T cell and cytokine response in vaccinated dogs.(A) Spleen cells were obtained from vaccinated and
non-vaccinated dogs at one year after challenge infection with T.
cruzi. Spleen cells were incubated for 30 min with
FITC-conjugated anti-CD8 and PE-conjugated anti-CD4 antibodies and
CD4+ and CD8+ T cell subsets monitored
by flow cytometry. (B) The circulatory IFN-γ level was
measured by an ELISA. Data are presented as mean ± SD
(*p<0.05).Detectable parasitemia that peaked during day 30–35 pi was noted in all dogs
(Fig. 5A). Dogs vaccinated
with TcVac1 exhibited an early rise in parasitemia that was controlled by day 37 pi.
In comparison, non-vaccinated/infected dogs exhibited a slight delay in peak
parasitemia; however, blood parasitemia persisted beyond day 37 pi. No signs of
clinical illness were apparent in vaccinated/infected and non-vaccinated/infected
dogs during the physical exam, yet 33% of the TcVac1-vaccinated/infected dogs
succumbed during 40–42 dpi (Fig.
5B).10.1371/journal.pntd.0001050.g005Figure 5(A) Blood parasitemia was determined by light microscopy. Data
are presented as mean ± SD (*p<0.05).
(B) Percent survival from infection.Xenodiagnostic studies were performed to determine if dog’s infectivity to
triatomines is altered by vaccination. Triatomines were fed on dogs during acute
phase (30 dpi) and after control of acute parasitemia (60 dpi), and feces were
analyzed 30 days post-feeding for the detection of parasites by microscopy. In
agreement with the peak parasitemia, all triatomines fed at day 30 pi on vaccinated
and non-vaccinated dogs became T. cruzi positive. Of the 36
triatomines fed on each group of dogs at day 60 pi, 47% (17 out of 36)
insects fed on TcVac1-vaccinated/infected dogs and 30% (11 out of 36) insects
fed on non-vaccinated/infected dogs died during the incubation period. Of those
surviving, we detected T. cruzi in feces of 52.63% (10/19)
and 84.6% (21/25) of the insects fed on vaccinated/infected and
non-vaccinate/infected dogs, respectively. These results indicated that TcVac1 was
not effective in preventing infection or early rise in acute parasitemia, and was
moderately effective in reducing the time-course of parasitemia and dogs’
infectivity to triatomines after day 37 post-infection.Normal electrocardiographic readings were noted in all dogs included in the study,
before and after immunization. After challenge infection, vaccinated/infected and
non-vaccinated/infected dogs exhibited no cardiac alterations up to 30 dpi. By day
60 pi, 67% of non-vaccinated dogs displayed electrocardiographic alterations,
including reduced P-R interval, reduced R wave voltage, axis rotated to the right,
S-T segment line elevation from the isoelectric line (>0.2 mV), long QT segment,
J wave elevation, and sinus tachycardia that were diagnostic of myocarditis,
pericarditis, and high degree of myocardiocyte necrosis. Two vaccinated/infected
dogs died by day 42 pi due to high electrical conductance problems and arrhythmia.
Of remaining, 50% of the vaccinated/infected dogs exhibited at 60 dpi no
electrocardiographic alterations, and other 50% exhibited a moderate level of
EKG abnormalities including low voltage complex, a positive deviation of S-T with
elevation of J-wave, left axel rotation, and tachycardia that were diagnostic of
ventricular dilation, myocarditis, and arrhythmia. At one-year post-challenge
infection, electrocardiographic analysis revealed a spectrum of cardiac dysfunction
in chronically infected dogs. Among the unvaccinated/chronically-infected dogs,
66% exhibited major electrical conduction problems (right axel rotation and
LBBB), and 33% exhibited lateral re-polarization problems. Among the
vaccinated/chronically-infected dogs, 33% exhibited electrical conduction
problems (right axel rotation and LBBB) similar to that noted in
unvaccinated/infected dogs, 33% showed minor axel rotation problems, and
33% dogs exhibited normal EKG. On a scale of 0 (normal) to 10 (severe EKG
alterations), 66% of non-vaccinated/chronically infected dogs were graded as
10 and 33% as normal (zero EKG alterations). In comparison, 33% of
vaccinated/chronically infected dogs were graded normal (0), 33% moderate
(score: 5), and 33% with severe electrical conduction problems (score: 10) at
one year post-infection.Next, we evaluated the pathology of the heart in dogs. Anatomo-pathological analysis
of the heart, performed at day 60 pi, showed dilated cardiomyopathy (bi-ventricular
dilation) and focal and diffused myocarditis in vaccinated dogs as well as in dogs
injected with vector alone (Fig.
6). Irrespective of vaccination status, some animals exhibited whitish
zones and rounded edges in the spleen, dilation of esophagus, and pinkish ampoules
at the cecum. At one-year post-challenge infection, all dogs had round shaped
hearts. Sixty six percent of non-vaccinated/chronically infected dogs exhibited
severe right ventricle dilation. In comparison, 66% of
vaccinated/chronically-infected dogs exhibited moderate level of right ventricle
dilation. Epicardial hemorrhages were seen in 66% dogs from the control group
and 33% dogs in the TcVac1 group.10.1371/journal.pntd.0001050.g006Figure 6Morphological alterations in the heart.Dogs were vaccinated and infected with T. cruzi as above.
Shown are representative morphologic alterations of the heart during the
acute infection phase (60 dpi) in dogs injected with vector only
(B&B1) or immunized with TcVac1
(C&C1). Images of normal heart (A&A1) are
shown for comparison. Horizontal arrows show right ventricle wall thinning
characteristic of ventricle dilation. Vertical arrows show pale striated
epicardium and myocardium, characteristic of necrosis produced after
inflammatory response to infection.Histopathology studies on day 60 pi demonstrated some differences between two groups
(Fig. 7). In epicardium of
dogs vaccinated with TcVac1, non-suppurative moderate to severe myocarditis with
focal or zonal mononuclear and polymorphonuclear inflammatory infiltrate associated
with presence of amastigotes nests and severe active necrosis was generally noted.
In non-vaccinated/infected dogs, histopathological findings were similar to that
noted in vaccinated dogs with the exception that inflammatory infiltrate tended to
be mainly constituted of mononuclear rather than polymorphonuclear cell type (Fig. 7). In the myocardium,
vaccinated and non-vaccinated dogs exhibited multiple coagulative necrosis foci with
mononuclear infiltration and some necrotic areas with polymorphonuclear and
neutrophil infiltration. The diffused multi-focal and zonal mononuclear inflammatory
infiltrate and hemorrhagic areas in myocardium appeared to be larger in
vaccinated/infected dogs (Fig. 7C,
C2) as compared to non-vaccinated/infected dogs (Fig. 7B, B2). Vaccinated/infected dogs also
showed abundant cellular detritus in myocardium. Mononuclear infiltration was
moderate in ventricles and septum of all dogs. Folding of myocardial fibers in right
ventricle was observed in both vaccinated/acutely-infected and
non-vaccinated/acutely-infected dogs. Mural multifocal endocarditis with mononuclear
infiltration was also noted in dogs from the two groups. Abundant amastigote nests
(range 18–21 per microscopic field (mf)) were found in each region (right and
left ventricles, and septum) of the heart of TcVac1-vaccinated/acutely-infected
dogs. Non-vaccinated/infected dogs exhibited, in general, lesser number of parasite
foci in equivalent studied areas. Statistical analysis showed that overall, the
number of myocardial necrotic foci, lymphocyte infiltration foci and number of
amastigote nests were more abundant in TcVac1-vaccinated dogs than was observed in
non-vaccinated/infected dogs at 60 dpi (p<0.05) (Fig. 7).10.1371/journal.pntd.0001050.g007Figure 7Histological analysis of hearts.Dogs were vaccinated, and challenged with T. cruzi. Heart
tissue sections (5-ìM) from left ventricle, septum, and right
ventricle were obtained at 60 days post-infection (acute phase), and stained
with hematoxylin-eosin. Shown are representative micrographs of dogs
injected with vector only (B,B1,B2) or immunized with TcVac1
(C,C1,C2). Micrographs from normal/uninfected dogs
(A,A1,A2) are shown for comparison. Vertical arrows show
amastigote nests and horizontal arrows show lymphocyte infiltration, and
cardiomyocytes destruction.At one-year post-challenge infection, severe myocardial inflammation persisted in
66% of the non-vaccinated/infected dogs while remaining 33% exhibited
slight inflammatory infiltrate in the heart. In comparison, 66% of
vaccinated/chronically infected dogs exhibited moderate level of myocardial
inflammatory infiltrate. Slight to moderate presence of connective tissue was
apparent in all chronically infected dogs; however, it was more evident in
TcVac1-vaccinated/chronic dogs. Folding of myocardial fibers and vacuolization of
Purkinje fibers was observed in 33% of vaccinated/chronically infected
dogs.DiscussionThe objective of the present study was to test the efficacy of a multi-component DNA
vaccine (TcVac1) in dogs. The antigenic candidates included in TcVac1 were
identified by computational analysis of T. cruzi sequence database
and selected because they were conserved among several clinically relevant
T. cruzi strains, expressed in infective and intracellular
stages of T. cruzi
[9], and
recognized by the antibody and T cell response in infected mice [32] and humans
(unpublished data). When delivered as a DNA vaccine in mice, TcG1, TcG2 and TcG4
elicited trypanolytic antibody response and Th1 cytokines (e.g. IFN-γ) [9] that resulted in
significant protection from acute infection and chronic disease severity. We
utilized IL-12 and GM-CSF expression plasmids as adjuvants as these cytokines induce
type 1 B and T cell responses [33], and shown to significantly enhance the protective
immunity elicited by the vaccine candidates in mice [8], [39] and dogs [33]. To the best of our
knowledge, this is the first report testing the prophylactic and
transmission-blocking efficacy of DNA vaccine against T. cruzi in
dogs.Immunization of dogs with TcVac1 resulted in elicitation of antigen-specific and
parasite-specific antibody response that was dominated by IgG2 subtype. The delivery
of booster doses of vaccine resulted in no significant increase in antibody response
that could be explained, at least partially, by the fact that DNA delivery system,
used in this study, is known to drive a low level of antigen expression. Several
investigators have reported that needle delivery of DNA vaccines in muscle induce
low immune response in large animals and humans, even when 1000-fold higher doses of
DNA than those proved to be effective in rodents were given [40], [41]. Other DNA vaccine delivery
systems such as gene gun (biolistic gun) [42], [43], adenovirus or vaccinia virus
delivery vectors [44], replicating attenuated strains of intracellular
microorganisms, such as Salmonella
[45] have shown
promising results in eliciting antigen expression. Additionally, heterologous
prime/boost approaches are noted to be more effective in eliciting stronger,
long-term immunity against intracellular pathogens [46], [47], to be tested in future
studies.Despite low vaccine-induced antibody response, vaccinated dogs, upon challenge
infection with T. cruzi, exhibited an early expansion in antibody
response. The Ig (G+M) response during acute phase was of higher magnitude in
vaccinated/infected dogs than that observed in dogs injected with vector alone. The
IgG response in vaccinated/infected dogs was primarily of the Th1 type with
IgG2/IgG1 ratios being >1, known to provide protection from acute infection in
dogs [48], [49]. The higher
level and rapid expansion of antibody titers indicates that TcVac1 primed the B cell
response that was expanded upon exposure to T. cruzi. Previously,
we have shown that TcG1-, TcG2- and TcG4-specific antibodies,
elicited in vaccinated mice, were lytic in nature, and efficiently killed
trypomastigotes in a complement-dependent manner [9]. In this study, our observation
of a shorter detectable parasitemic period of 37 days in vaccinated/infected dogs
than that noted in control dogs (44 days) indicate that antibody response primed by
vaccination with TcVac1 was lytic in nature and contributed to a control of blood
parasitemia. Yet, immunization with TcVac1 failed to prevent peak parasitemia. This
was likely because other components of immune system, i.e., innate response
constituted by phagocytes and type 1 biased CD8+ T cell response,
were not strongly primed by vaccine or expanded upon challenge infection in
vaccinated dogs. It is well documented that phagocytes, through activation of NADPH
oxidase, MPO, and iNOS activities and production of cytotoxic reactive oxygen and
nitrogen species, play an important role in control of T. cruzi
[37], [50]-[52]. Numerous
studies have also demonstrated that an efficient control of acute parasitemia
requires concerted activities of Th1 helper cells, and cytotoxic
CD8+ T lymphocytes (CTLs) (reviewed in [53], [54]). Vaccination with TcVac1
resulted in a suppression of phagocytes’ response to challenge infection as
was evidenced by decreased activation of MPO and iNOS activity in
vaccinated/infected dogs as compared to that noted in non-vaccinated/acutely
infected dogs. Equally, immunization with TcVac1 resulted in a significant but only
moderately better expansion of CD8+ T cells and IFN-γ levels
upon challenge infection when compared to that noted in non-vaccinated/infected
dogs. Consequently, it was not surprising to find no significant decline in
infectivity of vaccinated dogs to triatomines during the acute period of infection.
All triatomines, fed on vaccinated/infected or non-vaccinated/infected dogs when
dogs were exhibiting peak parasitemia (day 30 pi), and analyzed at day 60
post-feeding, were infected evidenced by fecal presence of T.
cruzi. However, at 60 dpi, vaccinated dogs exhibited a better control of
parasitemia and moderately reduced infectivity to triatomines (52.63% versus
84.6% infected). The mathematical modeling of transmission dynamics [55] and other
studies using insecticide-treated dog collars [56], [57] indicate that a decline in
infectivity to <20% would be required to block vectorial transmission of
T. cruzi to humans. Thus, we surmise that current formulation
of TcVac1, though provided a decline in dogs’ infectivity to triatomines after
peak parasitemia, would not be effective in blocking the transmission cycle, and
further improvement in vaccination strategy is required.Infection of dogs with SylvioX10/4 strain of T. cruzi produced
reproducible acute phase and chronic pathology as we have previously reported [18],
causing sudden death in some of the infected dogs, and cardiomyopathic changes in
most of the infected animals during acute stage. EKG alterations were found in more
than half of the acutely infected dogs and ranged from electrical conduction
problems, ventricular dilatations, pericarditis, myocarditis, high lateral necrosis,
and arrhythmia. Most of these changes were validated by necropsy and histopathology
findings, thus, confirming that this T. cruzi strain is highly
pathogenic in dogs. Despite a similar or higher infiltration of inflammatory
infiltrate in the heart, vaccinated dogs exhibited a significantly higher number of
amastigote nests (P<0.05) in cardiac tissue than was observed in non-vaccinated
control dogs (Fig. 7). Others
have reported a direct correlation between in vitro infectivity and
blood parasitism kinetics with heart parasitism intensity during long-term infection
of Beagle dogs [58], [59]. Because of high inflammatory infiltrate and tissue
parasite burden, two of the vaccinated dogs exhibited myocarditis and died suddenly
due to arrhythmia.Chronically infected/vaccinated dogs were better equipped in controlling the disease
symptoms. EKG findings demonstrated mild-to-moderate cardiac alterations in animals
given TcVac1 vaccine while severe EKG alterations persisted in dogs injected with
vector alone. These findings were supported by anatomo-pathological analysis
performed at one-year post-challenge infection. Anatomo-pathological lesions and
epicardial hemorrhages were fewer and moderate in TcVac1-vaccinated/chronically
infected dogs as compared to non-vaccinated/chronic dogs that exhibited severe right
ventricle dilation and extensive epicardial hemorrhages. These findings were
observed despite no decline in inflammatory infiltrate in the heart in chronically
infected dogs. These data indicate that TcVac1-induced immunity was at least
partially effective in controlling the clinical progression of cardiac disease
severity in chagasic dogs.Summarizing, in this study, we tested a multi-component DNA vaccine against
T. cruzi infection in dogs. Our data showed that TcVac1 geared
a modest parasite- and antigen-specific type 1 antibody and CD8+ T
cell response that was effective in providing an early control of acute parasitemia
and moderately decreased the infectivity of dogs to triatomines. However, tissue
parasite burden was not controlled in vaccinated dogs, likely due to suppression of
phagocytic cell response, evidenced by decreased myeloperoxidase and nitrite (iNOS)
levels in immunized dogs. Despite this, vaccinated dogs exhibited a moderate decline
in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis
during chronic stage of disease development. Overall, our data demonstrated that
TcVac1-elicited immunity provided a partial protection from chronic Chagas disease
and provided an impetus to further improve the vaccination strategy against Chagas
disease.
Cardiovascular Disorders/Myopathies Infectious Diseases/Neglected Tropical Diseases