Data_Sheet_1.PDF

<p>Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.</p

Immunostimulatory and adjuvant activity of TLR9 agonists derived from <i>T. cruzi</i> genome.

<p>(<b>A</b>) PBMCs derived from healthy donors were stimulated with human B-class-like CpG ODNs derived from the <i>T. cruzi</i> genome with four different concentrations (3.0, 1.0, 0.3, and 0.1 µM) and the levels of IFN-α measured in the cell culture supernatants 24 h later. The CpG ODN 2007 was used as positive controls for human B-class ODNs. PBMC experiments were performed in three different donors, yielding similar results. (<b>B</b>) Proinflammatory activity of mouse B class-like CpG motifs was evaluated in inflammatory macrophages from WT (C57BL/6), <i>TLR4</i>^−/− and <i>TLR9</i>^−/− mice. ODNs were tested at different concentrations (1.5, 0.3 and 0.06 µM) and LPS, as well as CpG ODN 7909 were used as positive controls for TLR4 and TLR9 activation, respectively. IL-12 (p40) was measured in the macrophage culture supernatants 24 h after cellular stimulation. (<b>C</b>) C57BL/6 mice received three immunization doses with alum alone, OVA plus alum or OVA plus alum associated with either CpG ODNs B344, B287, B128 or 7909 (positive control). The levels of OVA-specific total IgG, IgG1 and IgG2c were assessed by ELISA. (<b>D</b>) Amount of IFN-γ secreted by splenocytes after stimulation with OVA derived CD4⁺ T or CD8⁺ T cell epitopes was evaluated in culture supernatants 72 hours post-stimulation. Asterisks indicate that differences were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.</p

<i>T. cruzi</i> derived GIPLs are TLR4 agonists and promote high levels of antigen-specific IgG2c antibodies as well as IFN-γ production by CD4⁺ T cells.

<p>(<b>A</b>) CHO cells control (TLR2⁻/TLR4⁻) or expressing TLR2 (TLR2⁺) or TLR4 (TLR4⁺) were either left untreated (solid gray) or exposed to 100 µg/ml of GIPLs from <i>Trypanosoma cruzi</i> Tulahuen (GTH), Y strain (GY) (black line). MALP-2 (10 ηg/ml) and LPS (200 ηg/ml) were used as positive controls for activation of TLR2⁺ or TLR4⁺, respectively. (<b>B</b>) TLR4⁺ cells were activated with different preparations of GIPLs in the presence of polymyxin B (PB). LPS was used as control. (<b>C</b>) OVA specific immune responses induced by immunization with TLR2 or TLR4 agonists associated with OVA absorbed in alum. Mice were immunized with three doses on days 0, 14 and 28. The production of total IgG, IgG1 and IgG2c were assessed by ELISA using the sera from immunized mice, at day 9 after the second boost. (<b>D</b>) To assess the levels of IFN-γ production by T lymphocytes from vaccinated mice, splenocytes were collected 21 days after the third immunization dose and stimulated with either CD4⁺ T or CD8⁺ T cell epitopes from OVA. The results are representative of two independent experiments yielding similar results. Asterisks indicate that differences were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.</p

Global statistical assessment of biological replicates.

<p>Heat-map <b>(A)</b> and Principal Component Analysis (PCA) plots <b>(B)</b> of RNA-Seq data generated from the libraries mapped to the <i>T</i>. <i>cruzi</i> genome following removal of rRNA/tRNA features. Once the outlier sample was removed and data passed through normalization and surrogate variable analysis, the strong clustering by condition became evident in both analyses. In both plots, each sample is color coded by developmental stage/strain (Tryp: trypomastigotes; A60: amastigotes collected 60 hpi; A96: amastigotes collected 96 hpi).</p

Identification and Functional Analysis of <i>Trypanosoma cruzi</i> Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

<div><p>Background</p><p><i>Trypanosoma cruzi</i> is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, <i>T. cruzi</i> GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, <i>T. cruzi</i> genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.</p><p>Methodology/Principal Findings</p><p><i>In silico</i> analysis of the <i>T. cruzi</i> genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in <i>T. cruzi</i> epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. <i>T. cruzi</i> genes <i>TcDPM1</i>, <i>TcGPI10</i> and <i>TcGPI12</i> complement conditional yeast mutants in GPI biosynthesis. Attempts to generate <i>T. cruzi</i> knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding <i>TcGPI8</i>, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene.</p><p>Conclusions/Significance</p><p>Analyses of <i>T. cruzi</i> sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these <i>T. cruzi</i> genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.</p></div

Hypernociception induced by TLR agonists and the NY-ESO-1 vaccine formulations.

<p>(<b>A</b>) Different TLR agonists were injected in the footpad of mice and hypernociception evaluated at the indicated time points. (<b>B</b>) Vaccine formulations containing alum; alum plus NY-ESO-1; or alum plus NY-ESO-1 associated with TLR agonists were given to mice that were left untreated or treated with PCM orally, 30 minutes prior injection with different vaccine formulations. Asterisks mean significant difference when comparing PBS group with TLR agonists experimental groups (<i>P</i><0,05).</p

Reconfigurable Class S Power Amplifiers at RF and Microwave Frequencies

When a delta-sigma modulator (DSM) is placed before a class D switching stage the combination can be used to amplify time varying envelope signals. However a bandpass DSM is commonly employed and is required to have a sampling frequency approximately four times the carrier frequency. At RF or microwave frequencies proprietary hardware was previously needed to implement the DSM. However, it is shown here in simulation and from experimental measurement that a suitable DSM for class S power amplifiers can be implemented at RF and microwave frequencies using mid-range FPGA technology. Index Terms- Class S, efficiency, power amplifiers, sigma-delta modulatio

Comparative course of infection of human fibroblasts with <i>T</i>. <i>cruzi</i> CL Brener and CL-14.

<p>Intracellular <i>T</i>. <i>cruzi</i> life stages in mammalian cells: extracellular <i>T</i>. <i>cruzi</i> trypomastigotes actively penetrate mammalian cells where they differentiate into amastigotes and escape the vacuole before beginning to proliferate at ~24 hours post-infection <b>(A)</b>. Amastigotes replicate intracellularly in the host cell cytoplasm for 3–5 days, and then differentiate into motile trypomastigotes that are eventually released upon disruption of the host cell. Tissue-culture derived trypomastigotes of the CLB and CL-14 <i>T</i>. <i>cruzi</i> strains were similarly able to establish intracellular infection in cultured HFF <b>(B)</b> but exhibited markedly different intracellular growth dynamics as amastigotes. <b>(C)</b> Differences in the peak day of trypomastigote release from infected monolayers <b>(D)</b>.</p

Comparative global transcriptional expression patterns of <i>T</i>. <i>cruzi</i> genes encoding polymorphic cell surface proteins in CL Brener and CL-14.

<p>MA plots depicting the log2 fold change (logFC) of genes against the average expression level during the transition of CL Brener and CL-14 across developmental stages. Each dot represents one gene and colored dots represent members of the four of the six largest <i>T</i>. <i>cruzi</i> gene families analyzed: MASP (red), Mucin (blue), Trans-sialidase (purple), and GP63 (green). Dots above and below the red lines represent differentially expressed genes (logFC >1).</p

Constitutive expression of a TS gene in CL-14.

<p>The pROCKNeo vector used for transfection of CL-14 has the TS gene (Tc00.1047053509495.30) flanked by the ribosomal promoter and sequences containing signals for mRNA processing derived from the constitutively expressed housekeeping genes TcP2β (at the 5’ end) and gapdh (at the 3’ end) <b>(A)</b>. Total RNA purified from epimastigotes from WT and transgenic parasites were subjected to northern blot and hybridized with a ³²P-labelled probe that contains sequences corresponding to the C-terminal SAPA repeats present in the TS gene. Lower panel shows ethidium bromide staining of rRNAs in the same gel before transferring to the membrane <b>(B)</b>. Total protein extracts from epimastigotes from WT and transgenic parasites were evaluated for the expression of the transfected TS gene by western blotting with a monoclonal antibody anti-SAPA <b>(C)</b>. The infection profiles of four cloned cell lines derived from CL-14 parasites transfected with the TS gene or with the empty pROCKNeo vector, were compared to WT CL-14 and CL Brener in <i>in vitro</i> infection assays of Vero cells. Equal numbers of tissue culture derived trypomastigotes from each parasite cultures were added to Vero cell monolayers and the total number of trypomastigotes released in the supernatant <b>(D)</b> or the numbers of trypomastigotes released in the supernatant each day post-infection were evaluated over 8 days <b>(E)</b>. Five replicates for each infection experiment were performed.</p