DNA barcoding reveals both known and novel taxa in the Albitarsis Group (Anopheles: Nyssorhynchus) of Neotropical malaria vectors

<p>Abstract</p> <p>Background</p> <p>Mosquitoes belonging to the Albitarsis Group (<it>Anopheles</it>: <it>Nyssorhynchus</it>) are of importance as malaria vectors across the Neotropics. The Group currently comprises six known species, and recent studies have indicated further hidden biodiversity within the Group. DNA barcoding has been proposed as a highly useful tool for species recognition, although its discriminatory utility has not been verified in closely related taxa across a wide geographic distribution.</p> <p>Methods</p> <p>DNA barcodes (658 bp of the mtDNA <it>Cytochrome c Oxidase </it>- <it>COI</it>) were generated for 565 <it>An. albitarsis </it>s.l. collected in Argentina, Brazil, Colombia, Paraguay, Trinidad and Venezuela over the past twenty years, including specimens from type series and type localities. Here we test the utility of currently advocated barcoding methodologies, including the Kimura-two-parameter distance model (K2P) and Neighbor-joining analysis (NJ), for determining species delineation within mosquitoes of the Neotropical Albitarsis Group of malaria vectors (<it>Anopheles</it>: <it>Nyssorhynchus</it>), and compare results with Bayesian analysis.</p> <p>Results</p> <p>Species delineation through barcoding analysis and Bayesian phylogenetic analysis, fully concur. Analysis of 565 sequences (302 unique haplotypes) resolved nine NJ tree clusters, with less than 2% intra-node variation. Mean intra-specific variation (K2P) was 0.009 (range 0.002 - 0.014), whereas mean inter-specific divergence were several-fold higher at 0.041 (0.020 - 0.056), supporting the reported "barcoding gap". These results show full support for separate species status of the six known species in the Albitarsis Group (<it>An. albitarsis </it>s.s., <it>An. albitarsis </it>F, <it>An. deaneorum</it>, <it>An. janconnae</it>, <it>An. marajoara </it>and <it>An. oryzalimnetes</it>), and also support species level status for two previously detected lineages - <it>An. albitarsis </it>G &<it>An. albitarsis </it>I (designated herein). In addition, we highlight the presence of a unique mitochondrial lineage close to <it>An. deaneorum </it>and <it>An. marajoara </it>(<it>An. albitarsis </it>H) from Rondônia and Mato Grosso in southwestern Brazil. Further integrated studies are required to confirm the status of this lineage.</p> <p>Conclusions</p> <p>DNA barcoding provides a reliable means of identifying both known and undiscovered biodiversity within the closely related taxa of the Albitarsis Group. We advocate its usage in future studies to elucidate the vector competence and respective distributions of all eight species in the Albitarsis Group and the novel mitochondrial lineage (<it>An. albitarsis </it>H) recovered in this study.</p

Generation and characterization of novel anti-DR4 and anti-DR5 antibodies developed by genetic immunization

International audienceDevelopment of therapeutic antibodies in oncology has attracted much interest in the past decades. More than 30 of them have been approved and are being used to treat patients suffering from cancer. Despite encouraging results, and albeit most clinical trials aiming at evaluating monoclonal antibodies directed against TRAIL agonist receptors have been discontinued, DR4 or DR5 remain interesting targets, since these receptors are overexpressed by tumour cells and are able to trigger their death. In an effort to develop novel and specific anti-DR4 and anti-DR5 antibodies with improved properties, we used genetic immunization to express native proteins in vivo. Injection of DR4 and DR5 cDNA into the tail veins of mice elicited significant humoral anti-DR4 and anti-DR5 responses and fusions of the corresponding spleens resulted in numerous hybridomas secreting antibodies that could specifically recognize DR4 or DR5 in their native forms. All antibodies bound specifically to their targets with a very high affinity, from picomolar to nanomolar range. Among the 21 anti-DR4 and anti-DR5 monoclonal antibodies that we have produced and purified, two displayed proapoptotic properties alone, five induced apoptosis after cross-linking, four were found to potentiate TRAIL-induced apoptosis and three displayed antiapoptotic potential. The most potent anti-DR4 antibody, C#16, was assessed in vivo and was found, alone, to inhibit tumour growth in animal models. This is the first demonstration that DNA-based immunization method can be used to generate novel monoclonal antibodies targeting receptors of the TNF superfamily that may constitute new therapeutic agents