International Symposium: The parasitic diseases at the post genome era
Madrid, 23 y 24 de abril de 2013
Madrid, April 23-24, 2013
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- Vaccine development against relevant protozoan parasites in cattle: neosporosis as an example. Luis Ortega
- “Real-time’ genome evolution in natural populations of Leishmania donovani across two successive drug eras s. Jean Claude Dujardin
- Leishmania transcription and modified nucleotide baseJ. Peter Myler
- The use of functional genomics in Leishmania on the development of a recombinant DNA vaccine. Pedro J. Alcolea
- Genome and therapeutic targets. Jacob Lorenzo-Morales
- Functional genomics towards vaccine development against vectors and parasites. Ana Domingos
- Host-Pathogen interaction and the control of infectious diseases. Michael Parkhouse
- Vaccine against helminths: Do we need to know their genomes? Antonio Muro
- The development of a vaccine against parasitic nematodes. Antonio Osuna
- Biomarkers of pathology of Chagas disease and treatment efficacy.Ana. I. Fernández
- Chagas Disease: transcriptomics of the host-parasite interaction. Carlos Robello
- Congenital Chagas disease. Why do not prevent? Manuel Segovia
- Identifying and characterizing Plasmodium vivax vaccine candidates. Manuel Alfonso Patarroyo
- Malaria research in the post-genomics era: an insight into the patho-biology of Plasmodium vivax, a neglected human malaria parasite. Hernando del Portillo
Vaccine development against relevant protozoan parasites in cattle: neosporosis as an example. Luis Ortega
Bovine neosporosis, caused by the protozoan apicomplexan parasite Neospora caninum, is one of the leading causes of cattle abortion worldwide. A structured analysis of the published literature has provided a first global economic impact of neosporosis arriving at an estimate in excess of one billion dollars annually from just nine countries alone. In this scenario, vaccination is considered the best option for the cost-effective control of this disease. In pregnant animals, Neospora caninum reaches and crosses the placenta and infects the foetus, multiplying in different vital organs. Such foetal infections can lead to abortion, stillbirth, the birth of weak calves or, alternatively, the birth of clinically healthy but persistently infected calves that can transmit the pathogen to their progeny in the case of females. Consequently, an effective vaccine for bovine neosporosis must demonstrate protection against congenital transmission, and the two key parameters that must be evaluated are foetal death and vertical transmission. Several approaches have been assessed for the development of safe and effective vaccine products for bovine neosporosis. These approaches include live-attenuated vaccines, killed whole-parasite vaccines and sub-unit vaccines, among others. Recent knowledge may help in the development of protective vaccines in the future. First, the genome and transcriptome of N. caninum have been sequenced and a comparative analysis with another well-known close-related protozoan (Toxoplasma gondii) has been undertaken showing remarkable differences in key genes involved in parasite virulence and host cell interactions. In addition, proteomic techniques have allowed to the detection of differences in the abundance of several proteins with main roles in tachyzoite invasion and intracellular proliferation potentially associated to the isolate virulence. Second, a great variability in virulence has been demonstrated in both in vitro and in vivo among different Neospora isolates. This has allowed the selection of low virulent isolates that have been used in vaccination studies in the target species with good results. Finally, one key factor in testing the safety and efficacy of vaccines is the availability of standardised and accurate in vitro and in vivo experimental models. These models are essential to study host-pathogen interactions, to investigate host immunity at the local and systemic level, and to evaluate vaccine candidates and therapeutics. At present, there is a need of consensus guidelines, including the isolates/strains of N. caninum used, the challenge dose, the time and route of challenge, the preparation of the inoculum, the animal model (mice versus cattle; sheep versus cattle), and other parameters.
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“Real-time’ genome evolution in natural populations of Leishmania donovani across two successive drug eras. Jean Claude Dujardin
The parasite population of Leishmania donovani in the Indian Subcontinent (ISC) underwent a major evolutionary bottleneck in the 60’s during the DDT spraying campaigns and caused a massive epidemic after their interruption. During this period, this parasite was submitted to drug pressure, (i) initially with antimonials (SSG), up to their replacement because of resistance and (ii) nowadays with miltefosine (MIL) in the frame of a regional control program. L. donovani from the ISC thus constitutes a unique model for studies of ‘real-time’ evolution of parasites and its clinical implications. In the frame of two collaborative projects (see Kaladrug-R and Gemini in we followed cohorts of VL patients from the SSG and MIL eras, both in India and Nepal, collected and documented 196 strains and sequenced their complete nuclear genome (mean 51x coverage). SNP analysis revealed a main and relatively homogeneous group within our sample (in average 173 SNPs/strain in 94 % of the strains), consistent with a post-DDT bottleneck clonal expansion. A few strains originating from Himalayan valleys of Nepal (hence called ‘Yeti’ strains) were shown to be highly divergent (about 55,000 SNPs away from core group) from the main population and could represent survivors of the pre-DDT bottleneck diversity. The scattering of parasites with a lower susceptibility to SSG or MIL across the SNP-based phylogenetic trees suggests (i) multiple and independent events of drug resistance emergence, likely involving different adaptive mechanisms or (ii) the occurrence of recombination events. In contrast with the SNP homogeneity observed in the main ISC L.donovani population, a high diversity was observed at genome structure level, essentially in the form of massive aneuploidy, episomal amplification and expansion of tandem arrays. Gene dosage appears to be a major and fast adaptive strategy in natural populations of Leishmania; considering the affected genes, effects are predicted at the level of translation machinery (mini-exon and rDNA genes), regulation (MAPK1 and secreted acid phosphatase), virulence (gp63) and drug resistance (MRPA). This pioneer study lays the groundwork for more sensitive and powerful approaches to collating genomic diversity, as well as comprehensively describing population-level Leishmania variability in a clinical context.
This work was supported by the European Commission (Kaladrug-R, EC-FP7-222895) and EWI (SOFI-B Gemini)
Selected topics for Abstract submission:
Genomics, transcriptomics, proteomics, metabolomics
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Leishmania transcription and modified nucleotide base J. Peter Myler
β-glucosylhydroxymethyluracil (base J) is a modified DNA base found only in Euglenozoa, including the parasitic kinetoplastid genera, Trypanosoma and Leishmania. Base J synthesis requires two enzymes (JBP1 and JBP2) that catalyze hydroxylation of thymine, and the resultant HOMeU is modified by an as-yet-unidentified glucosyltransferase. JBP1 is essential in Leishmania, while knock-out of JBP2 causes gradual loss of J, leading to transcriptional read-through at transcription termination sites that contain internal J (iJ), and false-starts at transcription initiation sites. Bromodeoxyuridine (BrdU) treatment of JBP2KO parasites caused further reduction in iJ levels, and the eventual death of the cells. We have used RNA-seq and Nanostring technology to quantify mRNA levels in BrdU-treated WT L.tarentolae and a new JPB2KO line, revealing consistent and substantial changes in mRNA levels for several genes near iJ sites. These results suggest that loss of iJ induces de-repression of genes deleterious to cell growth, and/or down-regulation of essential genes (due to accumulation of antisense RNA at convergent strand-switch regions). To investigate the signals responsible for J insertion, we cloned several J-containing sequences into plasmids and grew them as episomes in L.tarentolae. Plasmids containing cSSR-25.2 sequence or the telomeric hexamer sequence ([GGGTTA]10) accumulated J when grown in WT (but not JBP2KO) cells, while those containing the atypical cSSR-28.2 (which lacks iJ in WT cells) accumulated no detectable J. SMRT sequencing of the J-containing plasmids revealed that most J sites occurred in pairs at 13-nt intervals on opposite DNA strands. Thus, we hypothesize that JBP2 recognizes the signal for de novo J insertion, while JBP1 is responsible for J maintenance (following DNA replication) by recognizing this J and inserting a new J downstream on the opposite strand.
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The use of functional genomics in Leishmania on the development of a recombinant DNA vaccine. Pedro J. Alcolea
Canine leishmaniasis is a public health problem in Spain and other Mediterranean areas caused by Leishmania infantum. The successful development of a vaccine is essential for the control of the disease. The use of the canine model is ideal for future research on vaccines against human visceral leishmaniasis. The CIB Molecular Parasitology Laboratory has been working on the development of a DNA vaccine against canine leishmaniasis for more than 10 years. This vaccine consists of two immunizations with the LACK (homologous of the receptor of the activated protein kinase C) antigen-encoding gene cloned in the pCI-neo vector (homologous regime) or this construction in the prime dose followed by the boost dose with the same gene carried in a recombinant attenuated vaccinia virus (heterologous regime). This vaccine confers partial protection in dogs eliciting a Th1 response and the heterologous regime works better than the homologous one. The following step has been the improvement of the construction to substitute the antibiotic resistance genes by other selection system to comply with the FDA and EMEA recommendations. Safety and efficacy of the new construction are being evaluated in the laboratory. The use of commercial canine oligonucleotide microarrays is leading to higher accuracy in the evaluation of the immune response. Gene expression profiling experiments in L. infantum have provided valuable information about the basic biology of the parasite and are contributing to the selection of new candidate genes in an attempt to generate an improved multigenic vaccine.
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Genome and therapeutic targets. Jacob Lorenzo-Morales
Free Living amoebae (FLA) belonging to Acanthamoeba and Sappinia genera as well as Balamuthia mandrillaris and Naegleria fowleri species are aerobic, mitochondriate, eukaryotic protists that occur worldwide and can potentially cause infections in humans and other animals. These amoebae have the ability to exist as free-living organisms in nature and only occasionally invade a host and live as parasites. All these four mentioned amoebae above are able to cause infections of the central nervous system (CNS) that are normally lethal. Several species of Acanthamoeba, the only known species of Balamuthia, B. mandrillaris, two species of Sappinia genus and only one species of Naegleria, N. fowleri are known to cause disease in humans and other animals. Acanthamoeba species are also causative agents of a vision threating infection of the cornea, known as Acanthamoeba keratitis, which mostly affects immunocompetent contact lens wearers. Currently there are not effective therapies against these parasitic amoebae mainly due to the existence of a highly resistant cyst stage in their life cycle. Our laboratory is developing novel therapies based on the recently sequenced genome of Acanthamoeba and the use of siRNAs in order to validate different cellular targets and searching for chemical substitutes or further developing a RNAi-based technology. The obtained results so far in our Institution are being presented in this symposium.
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Functional genomics towards vaccine development against vectors and parasites. Ana Domingos
Diseases transmitted by arthropod vectors such as mosquitoes and ticks have a great impact in human and animal health. Vaccines are an environmentally friendly solution for vector-borne diseases control. Functional analysis of genes expressed in response to infection allows assessing the potential use of proteins in the development of a vaccine targeting both the vector and pathogen. The potential role of tick genes differentially expressed in both feeding and transmission were evaluated after parasite infection. Eight hundred randomly selected clones were sequenced and analyzed. Differentially expressed genes with putative functions in tick-pathogen interactions were selected for validation by real-time RT-PCR. Functional analysis of differentially expressed genes by dsRNA-mediated RNAi showed that knockdown of selected genes reduced pathogen infection levels and on tick weight and oviposition, when compared to controls. Our results demonstrate the importance of gene knockdown by RNA interference on tick development and gene expression confirming the usefulness of this methodology on vaccine antigens selection. Transcriptomicof mosquitoes Anopheles sp.salivary glands under Plasmodium sp. infection towards malaria control is, as well, under study in our laboratorypointing to new gene targets, potential vaccine antigens. Data on vaccination effect encourages search for newantigens targeting both arthropods and multiple pathogens which may greatly contribute for vectors and parasites control.
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Host- Pathogen interaction and the control of infectious diseases. Michael Parkhouse
The pathogenesis of an infection depends on interaction between the immune system of the host and life style of the pathogen. This is not a one-sided interaction as pathogens have evolved mechanisms to evade and modify host immune responses, ideally achieving an equilibrium between the long-term survival of both the pathogen and the host. Thus, recently emerging infections are often highly pathogenic, whereas well adapted pathogens have evolved strategies to survive and replicate without necessarily inducing severe pathological consequences.
Pathogen strategies for host evasion provide an exploitable source for (1) Novel approaches for the control of both infectious and non-infectious diseases, (2) Reagents to define novel cellular process and (3) Readymade tools for gene manipulation/therapy. In the past, the majority of such virus “host evasion” genes have been identified through their homologies. It is clear, however that some of these evasion molecules do not have structural homologues, but are functionally similar to essential components of the vertebrate immune system. These can only be identifiable through appropriate functional assays.
Viruses and bacteria have been particularly efficient in evolving such strategies, which impinge and modify the cell biology and immune responses of their hosts. Host evasion mechanisms of parasites have been considerably less well studied at the molecular level. One recent example, is a secreted/surface protein of the infective larva, or oncosphere, of the human tapeworms Taenia saginata and Taenia solium. This molecule, known a HP6, was found to be structurally and functionally homologous to the mammalian adhesion molecule fibronectin, conceivably with a role in promoting the adhesion of oncospheres and thus perhaps aiding parasite invasion. Therefore, HP6 is an excellent vaccine candidate as it combines membrane localization with the relevant functional properties of a secreted protein with proven adhesion molecule characteristics, and indeed vaccination of Taenia saginata infected cattle with recombinant HP6 resulted in 100% protection.
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Vaccine against helminths: Do we need to know their genomes? Antonio Muro
We are still far from having a vaccine applicable against diseases caused by helminths, flukes specifically. Complex life cycles, sexual and asexual reproduction, unknown genomes and elucidation of the immune response are some of the most important reasons. However, we must be optimistic, as new biotechnological tools will allow us to move quickly. In 2009, the first version of the genome of a fluke, Schistosoma mansoni was published. Now, Schistosoma spp. genomes are available as SchistoDB. Genomes of different helminths have been sequenced, annotated and published just this year. So, there is an ambitious project of hundred species of helminths led by Sanger Institute. In addition, new methods of high-throughput sequencing are helping us to know in detail the mechanisms of host-parasite relationship, taking as reference models genomes of different animal species using Ensembl. At the present time, a grant funded by “Fundación Areces”, our group is driving studies about the development of a vaccine against Fasciola hepatica based on bioinformatic tools. Because of the lack of Fasciola genome sequences, we have used sequences available at NCBI. Programs such as Bepipred, SYFPHEITI Antheprot have been used to identify peptides containing B and T epitopes and having conserved catalytic sites. Twenty-four selected peptides were synthesized and characterized by mass spectrometry (MALDI-TOF) and have been carried out to study its potential immunological response. The selected molecules belong to the families of amoebapore, cathepsin, cytochrome oxidase, NADH dehydrogenase and vitelline. Protection has been evaluated in mice and rat models, obtaining high rates of survival and worm reduction. Future studies will be conducted to assay this new vaccine on natural infection models.
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The development of a vaccine against parasitic nematodes. Antonio Osuna
We describe the capacity to induce an immune response against parasitic nematodes of a recombinant antigen deriving from a 345 bp clone known as CT2-2, corresponding to the catalytic region of threonine serine phosphatase PP2A. The antigen showed high immunization capacity against Angiostrongylus costaricensis and in pilot assays when used in vaccinating lambs against an infectious cocktail of the parasites Trichostrongylus columbriformis, Haemonchus contortus and Teladosargia circunscinta (Solano-parada et al. 2010), and more recently against Trichinella spiralis.
It shows high homology with the sequence of the same catalytic region of PP2A in other nematode parasites.
We have tested new adjuvants capable of stimulating Th1 and Th17 responses, found very recently to be involved in immunomodulation against parasite nematodes (Solano-parada et al. 2010; Hang L, et al. 2011), for which reason we suggest that the antigen be encapsulated with the new generation of adjuvants in the form of 40 nm diameter nanocapsules, capable of activating dendritic cells, and of cholera toxin, all being activators of the responses mentioned above.