T cell and cytokine response in vaccinated dogs.

<p>(<b>A</b>) Spleen cells were obtained from vaccinated and non-vaccinated dogs at one year after challenge infection with <i>T. cruzi</i>. 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>B</b>) The circulatory IFN-γ level was measured by an ELISA. Data are presented as mean ± SD (*<i>p</i><0.05).</p

The antibody response to <i>T. cruzi</i> infection was polarized to type 1 in vaccinated dogs.

<p>Dogs were vaccinated as above, and infected with <i>T. cruzi</i>. An ELISA was performed to evaluate sera levels (1∶100-dilution) of <i>T. cruzi</i>-specific IgM (<b>A</b>), IgG (<b>B</b>), IgG1 (<b>C</b>), and IgG2 (<b>D</b>) antibodies at 0, 15, 30, 45, and 60 days post-infection.</p

Morphological alterations in the heart.

<p>Dogs were vaccinated and infected with <i>T. cruzi</i> as above. Shown are representative morphologic alterations of the heart during the acute infection phase (60 dpi) in dogs injected with vector only (<b>B&B1</b>) or immunized with TcVac1 (<b>C&C1</b>). Images of normal heart (<b>A&A1</b>) 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.</p

Histological analysis of hearts.

<p>Dogs were vaccinated, and challenged with <i>T. cruzi</i>. 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>B,B1,B2</b>) or immunized with TcVac1 (<b>C,C1,C2</b>). Micrographs from normal/uninfected dogs (<b>A,A1,A2</b>) are shown for comparison. Vertical arrows show amastigote nests and horizontal arrows show lymphocyte infiltration, and cardiomyocytes destruction.</p

Shown 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.

<p>Data are presented as mean ± SD (*<i>p</i><0.05).</p

TcVac1-induced antibody response in dogs.

<p>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 (<b>A</b>), IgG (<b>B</b>), IgG1 (<b>C</b>) and IgG2 (<b>D</b>). 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 (*<i>p</i><0.05, vaccinated versus non-vaccinated, n = 6 per experiment).</p

Figure 5

<p>(<b>A</b>) Blood parasitemia was determined by light microscopy. Data are presented as mean ± SD (*<i>p</i><0.05). (<b>B</b>) Percent survival from infection.</p

Electrocardiographic evaluation of TcVac4-immunized dogs post challenge infection with <i>T</i>. <i>cruzi</i>.

<p>Dogs were vaccinated and challenged with <i>T</i>. <i>cruzi</i> as described in Materials and Methods. Cardiac hemodynamics were monitored by electrocardiography, and graded as 0–10, 10 being most severe. HAD, High axis deviation; LVC, Low Voltage Complex; ICP, Interventricular conduction Problems; RP, Repolarization Problems. Type II (Mobitz) AVB, 2^nd degree Atrio-Ventricular Block; ADR, Axis deviation to the right; Small QRS, reduced QRS wave; ND, Not determined (animals not included for the study of these parameters).</p><p>Electrocardiographic evaluation of TcVac4-immunized dogs post challenge infection with <i>T</i>. <i>cruzi</i>.</p

Macroscopic and microscopic alterations in the heart of <i>T</i>. <i>cruzi</i>-infected dogs (±TcVac4).

<p><b>(A)</b> Morphologic alterations of heart in acute infection phase (±TcVac4). Shown are representative heart pictures from dogs included in pcDNA3.1/no <i>Tc</i> (A), pcDNA3.1/<i>Tc</i> (B), TcVac4/<i>Tc</i> (C) and TrIE/<i>Tc</i> (D) groups, harvested at day 60 pi. Yellow arrows indicate right ventricular dilation. Red arrows indicate the pale/striated areas of fibrotic tissue. <b>(B)</b> Histological analysis of heart tissue-sections in acutely-infected dogs (±TcVac4). Heart tissue sections (5-μM) from left ventricle, septum, and right ventricle were obtained at 60 days pi, and stained with hematoxylin-eosin. Shown are representative micrographs of heart from dogs injected with pcDNA3.1/<i>Tc</i> (B, B1, B2), TcVac4/<i>Tc</i> (C, C1, C2), and TrIE/<i>Tc</i> (D, D1, D2). Micrographs from pcDNA3.1/no <i>Tc</i> (A, A1, A2) are shown as negative controls. Black arrows show amastigotes nests. White arrows show inflammatory infiltrate in the myocardium. <b>(C)</b> Morphologic alterations of heart in chronic disease phase (±TcVac4). Shown are representative heart pictures from dogs included in pcDNA3.1/no <i>Tc</i> (A), pcDNA3.1/<i>Tc</i> (B), TcVac4/<i>Tc</i> (C) and TrIE/<i>Tc</i> (D) groups, harvested at day 365 pi. Black arrows point out the right ventricular dilation. <b>(D)</b> Histological analysis of heart tissue-sections in chronically-infected dogs (±TcVac4). Heart tissue sections (5-μm) from left ventricle, septum, and right ventricle were obtained at 365 days pi, and stained with hematoxylin-eosin. Shown are representative micrographs of chronically-infected dogs injected with pcDNA3.1 only (B, B1, B2) or immunized with TcVac4 (C, C1, C2). Micrographs from pcDNA3.1/no <i>Tc</i>, i.e. normal/healthy controls (A, A1, A2) are shown for comparison. White arrows mark infiltrating inflammatory cells in myocardium.</p

Anatomopathological and histopathological abnormalities in dogs during the acute and chronic phases of <i>T</i>. <i>cruzi</i> infection and disease development (± TcVac4 vaccine).

<p>Anatomopathological and histopathological evaluations were conducted during acute (day 60 pi) and chronic (day 365 pi) phases of infection and disease development. Classification of abnormalities was conducted according to [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003625#pntd.0003625.ref025" target="_blank">25</a>] and [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003625#pntd.0003625.ref039" target="_blank">39</a>], and represented as-, None; +, slight; ++, moderate; and +++, severe; ND, Not determined (animals not included for the study of these parameters).</p><p>Anatomopathological and histopathological abnormalities in dogs during the acute and chronic phases of <i>T</i>. <i>cruzi</i> infection and disease development (± TcVac4 vaccine).</p