Testing the Efficacy of a Multi-Component DNA-Prime/DNA-Boost Vaccine against Trypanosoma cruzi Infection in Dogs

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

Plant origin authentication of Sonoran Desert propolis: an antiproliferative propolis from a semi-arid region

The main chemical composition of Sonoran propolis (SP), as well as its antiproliferative activity on cancer cells through apoptosis induction, has been reported. The chemical constitution of SP remained qualitatively similar throughout the year, whereas the antiproliferative effect on cancer cells exhibited significant differences amongst seasonal samples. The main goal of this study was to provide phytochemical and pharmacological evidence for the botanical source of SP and its antiproliferative constituents. A chemical comparative analysis of SP and plant resins of species found in the surrounding areas of the beehives was carried out by HPLC-UV-DAD, as well as by 1H NMR experiments. The antiproliferative activity on cancerous (M12.C3.F6, HeLa, A549, PC-3) and normal cell lines (L-929; ARPE-19) was assessed through MTT assays. Here, the main polyphenolic profile of SP resulted to be qualitatively similar to Populus fremontii resins (PFR). However, the antiproliferative activity of PFR on cancer cells did not consistently match that exhibited by SP throughout the year. Additionally, SP induced morphological modifications on treated cells characterised by elongation, similar to those induced by colchicine, and different to those observed with PFR treatment. These results suggest that P. fremontii is the main botanical source of SP along the year. Nevertheless, the antiproliferative constituents of SP that induce that characteristic morphological elongation on treated cells are not obtained from PFR. Moreover, the presence of kaempferol-3-methyl-ether in SP could point Ambrosia ambrosioides as a secondary plant source. In conclusion, SP is a bioactive poplar-type propolis from semi-arid zones, in which chemical compounds derived from other semi-arid plant sources than poplar contribute to its antiproliferative activity

Immune protection against Trypanosoma cruzi induced by TcVac4 in a canine model.

Chagas disease, caused by Trypanosoma cruzi, is endemic in southern parts of the American continent. Herein, we have tested the protective efficacy of a DNA-prime/T. rangeli-boost (TcVac4) vaccine in a dog (Canis familiaris) model. Dogs were immunized with two-doses of DNA vaccine (pcDNA3.1 encoding TcG1, TcG2, and TcG4 antigens plus IL-12- and GM-CSF-encoding plasmids) followed by two doses of glutaraldehyde-inactivated T. rangeli epimastigotes (TrIE); and challenged with highly pathogenic T. cruzi (SylvioX10/4) isolate. Dogs given TrIE or empty pcDNA3.1 were used as controls. We monitored post-vaccination and post-challenge infection antibody response by an ELISA, parasitemia by blood analysis and xenodiagnosis, and heart function by electrocardiography. Post-mortem anatomic and pathologic evaluation of the heart was conducted. TcVac4 induced a strong IgG response (IgG2>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2/IgG1 response in chronic phase. In comparison, dogs given TrIE or empty plasmid DNA only developed high IgG titers with IgG2 predominance in response to T. cruzi infection. Blood parasitemia, tissue parasite foci, parasite transmission to triatomines, electrocardiographic abnormalities were significantly lower in TcVac4-vaccinated dogs than was observed in dogs given TrIE or empty plasmid DNA only. Macroscopic and microscopic alterations, the hallmarks of chronic Chagas disease, were significantly decreased in the myocardium of TcVac4-vaccinated dogs. We conclude that TcVac4 induced immunity was beneficial in providing resistance to T. cruzi infection, evidenced by control of chronic pathology of the heart and preservation of cardiac function in dogs. Additionally, TcVac4 vaccination decreased the transmission of parasites from vaccinated/infected animals to triatomines

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

TcVac4-induced antibody response in dogs (± <i>T</i>. <i>cruzi</i>).

<p>Dogs were vaccinated with TcVac4 or TrIE only and infected with <i>T</i>. <i>cruzi</i>, as described in Materials and Methods. Shown are sera levels of <i>T</i>. <i>cruzi</i>-specific IgG <b>(A)</b>, IgG1 <b>(B)</b>, and IgG2 <b>(C)</b> antibody subtypes, determined by an ELISA. Dogs given pcDNA3.1/no infection and dogs given pcDNA3.1/<i>T</i>. <i>cruzi</i> were included as negative and positive controls, respectively. The serology time points are described as day -65 = basal response before immunization, day 0 representing antibody response after last immunization but before challenge infection, day 60 post challenge equivalent to acute infection phase, and day 365 post challenge equivalent to chronic disease phase. Each bar represents the absorbance mean value ± standard deviation. Within the same time point, statistical differences (p < 0.05) among groups are shown with different characters above the bars according to Tukey’s test.</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

Frequency of parasite transmission from TcVac4-vaccinated 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. Triatomines (<i>Meccus longipennis</i>) were fed on dogs at day 36 pi and infection of insects was evaluated 60 days post-feeding.</p><p>Frequency of parasite transmission from TcVac4-vaccinated dogs post challenge infection with <i>T</i>. <i>cruzi</i>.</p

Blood parasitemia control in dogs immunized with TcVac4.

<p>Dogs were immunized with TcVac4 or TrIE, and infected with <i>T</i>. <i>cruzi</i> as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003625#pntd.0003625.g001" target="_blank">Fig 1</a>. Blood samples were evaluated for parasitemia by light microscopy at alternate days post-infection. Shown are data from day 16 to 46 after challenge infection, presented as mean values (n = 6/group). Different letters above lines show statistical differences (p< 0.05) among treatments within the same day of sampling according to Tukey’s test.</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