Pair-wise correlation analysis.

<p>Shown is pair-wise correlation analysis of antibody response to TcG_mix (A) and TcTL (B) with clinical disease category in patients enrolled in the study from Argentina-Bolivia border area. For this, seronegative/healthy subjects were labeled as 0, and patients classified as 0, I, II and III (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002018#s2" target="_blank">Materials and Methods</a>) were labeled as 1, 2, 3, and 4, respectively. Dots, individual subjects.</p

B cell epitopes in candidate antigens.

<p>Linear B cell epitopes (12 amino acid lengths) were predicted using a BCPred tool (<a href="http://ailab.cs.iastate.edu/bcpreds/" target="_blank">http://ailab.cs.iastate.edu/bcpreds/</a>).</p

Antigenicity of TcG1, TcG2 and TcG4 in inhabitants of Salta Argentina.

<p>Sera (A–C) and plasma (D–I) samples obtained in year 2010 (A–F) and year 2009 (G–I) from volunteers in Salta Argentina were identified as seropositive (+ve) or seronegative (−ve) in 1^st-phase screening by using conventional approaches. The 2^nd-phase screening for antigen-specific antibody response was conducted by an ELISA using the recombinant TcG1, TcG2 and TcG4 proteins. Data (mean of four observations from each sample) are presented as box plot. The horizontal lines of the box (bottom to top) depict the lower quartile (Q1, cuts off lowest 25% of the data), median (Q2, middle value), and upper quartile (Q3, cuts off the highest 25% of the data). The lower and upper whiskers depict the smallest and largest non-outlier observations, respectively, and solid dots represent the outliers. The spacing between the different parts of the box indicates the degree of dispersion (spread). Standard deviation for all samples was <12%.</p

Characterization of the subjects included for screening of TcG1-,TcG2-, and TcG4-specific antibody responses in this study.

a<p>Subjects in Argentina were screened for <i>T. cruzi</i>-specific antibodies by Wiener Chagatest-ELISA and Wiener Chagatest-HAI kits. Clinical exam included physical exam, electrocardiography and echocardiography. Confirmation of leishmaniasis was obtained by parasitological test, Montenegro reaction, clinical demonstration, and two PCR approaches. One of the seropositive patient presenting acute infection was referred for chemotherapeutic treatment with Benznidazole.</p>b<p>Five of the seropositive/chagasic subjects from Argentina were positive for Leishmania infection, determined by two PCR approaches.</p>c<p>Sera samples from Chiapas Mexico were screened by epimastigote antigenic lysate-based ELISA, trypomastigote-based flow cytometry, and Chagas Stat-Pak immuno-chromatograpic test.</p>d<p>Seronegative subjects from non-endemic areas were screened for <i>T. cruzi</i>-specific antibody response using <i>T. cruzi</i> trypomastigote lysate as antigen source in ELISA assays. Seronegative/cardiomyopathy patients were identified based upon blood levels of NT-proBNP to be >2000 pg/ml (normal<450 pg/ml).</p><p>N/A: not available.</p

TcG1, TcG2 and TcG4 are recognized by antibody response in human subjects from Mexico.

<p>Sera samples obtained from volunteers living in the endemic areas of Chiapas Mexico were characterized as seropositive (+ve) and seronegative (−ve) by whole-parasite antigen based serology tests in the 1^st phase. The TcG1 (A), TcG2 (B) and TcG4 (C) specific antibody response was measured by ELISA, and data are presented as box plot (details in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002018#pntd-0002018-g001" target="_blank">Fig. 1</a>).</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

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

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