Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease.

BACKGROUND: Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES: Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS: To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS: As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION: These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.

Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease

BACKGROUND Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.

Trypanosoma cruzi diversity in the Gran Chaco: mixed infections and differential host distribution of TcV and TcVI.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco.

Preponderant clonal evolution of Trypanosoma cruzi I from Argentinean Chaco revealed by Multilocus Sequence Typing (MLST).

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI.

Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems. RESULTS: While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes. CONCLUSION: Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi.