The search for vaccines to prevent human leishmaniasis is an active field of investigation aimed to prevent the devastating effects of this family of diseases on human health. The design and commercialization of several vaccines against canine leishmaniasis is a hopeful advance toward the achievement of a human vaccine. Areas covered: This review includes a summary of the most relevant immunological aspects accompanying leishmaniasis in natural hosts as well as a description of the latest advances in the multiple strategies that are being followed to develop leishmanial prophylactic vaccines. We have combined citations of the latest specialized reviews with research articles presenting the most recent results. Expert commentary: Achieving safe, effective, durable and low-cost prophylactic vaccines against leishmaniasis is still a major challenge. These vaccines should control not only parasite progression, but also the accompanying pathology, which results from an imbalanced interaction between the infectious agent and the human host immune system. Different strategies for development of vaccines are currently under investigation. They range from the use of live non-pathogenic vectors to the employment of subunit vaccines combined with adjuvants and/or delivery systems inducing cell-mediated immunity.

Leishmania donovani is a unicellular parasite that causes visceral leishmaniasis, a fatal disease in humans. In this study, a complete assembly of the genome of L . donovani is provided. Apart from being the first published genome of this strain (HU3), this constitutes the best assembly for an L . donovani genome attained to date. The use of a combination of sequencing platforms enabled to assemble, without any sequence gap, the 36 chromosomes for this species. Additionally, based on this assembly and using RNA-seq reads derived from poly-A + RNA, the transcriptome for this species, not yet available, was delineated. Alternative SL addition sites and heterogeneity in the poly-A addition sites were commonly observed for most of the genes. After a complete annotation of the transcriptome, 2,410 novel transcripts were defined. Additionally, the relative expression for all transcripts present in the promastigote stage was determined. Events of cis- splicing have been documented to occur during the maturation of the transcripts derived from genes LDHU3_07.0430 and LDHU3_29.3990. The complete genome assembly and the availability of the gene models (including annotation of untranslated regions) are important pieces to understand how differential gene expression occurs in this pathogen, and to decipher phenotypic peculiarities like tissue tropism, clinical disease, and drug susceptibility.

BackgroundThe immunization with genetically attenuated Leishmania cell lines has been associated to the induction of memory and effector T cell responses against Leishmania able to control subsequent challenges. A Leishmania infantum null mutant for the HSP70-II genes has been described, possessing a non-virulent phenotype.Methodology/Principal findingsThe L. infantum attenuated parasites (LiΔHSP70-II) were inoculated in BALB/c (intravenously and subcutaneously) and C57BL/6 (subcutaneously) mice. An asymptomatic infection was generated and parasites diminished progressively to become undetectable in most of the analyzed organs. However, inoculation resulted in the long-term induction of parasite specific IFN-γ responses able to control the disease caused by a challenge of L. major infective promastigotes. BALB/c susceptible mice showed very low lesion development and a drastic decrease in parasite burdens in the lymph nodes draining the site of infection and internal organs. C57BL/6 mice did not show clinical manifestation of disease, correlated to the rapid migration of Leishmania specific IFN-γ producing T cells to the site of infection.Conclusion/SignificanceInoculation of the LiΔHSP70-II attenuated line activates mammalian immune system for inducing moderate pro-inflammatory responses. These responses are able to confer long-term protection in mice against the infection of L. major virulent parasites.

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expression.

Leishmania infantum parasites cause a severe form of visceral leishmaniasis in human and viscerocutaneous leishmaniasis in dogs. Recently, we reported that immunization with an attenuated L. infantum cell line, lacking the hsp70-II gene, protects against the development of murine cutaneous leishmaniasis. In this work, we analyzed the vaccine potential of this cell line towards the long-term protection against murine visceral leishmaniasis. This model shows an organ-dependent evolution of the disease. The infection can resolve in the liver but chronically affect spleen and bone marrow. Twelve weeks after subcutaneous administration of attenuated L. infantum, Bagg Albino (BALB/c) mice were challenged with infective L. infantum parasites expressing the luciferase-encoding gene. Combining in vivo bioimaging techniques with limiting dilution experiments, we report that, in the initial phase of the disease, vaccinated animals presented lower parasite loads than unvaccinated animals. A reduction of the severity of liver damage was also detected. Protection was associated with the induction of rapid parasite-specific IFN-γ production by CD4+ and CD8+ T cells. However, the vaccine was unable to control the chronic phase of the disease, since we did not find differences in the parasite burdens nor in the immune response at that time point.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

Leishmania amazonensis parasites are etiological agents of cutaneous leishmaniasis in the New World. BALB/c mice are highly susceptible to L. amazonensis challenge due to their inability to mount parasite-dependent IFN-γ-mediated responses. Here, we analyzed the capacity of a single administration of the LiΔHSP70-II genetically-modified attenuated L. infantum line in preventing cutaneous leishmaniasis in mice challenged with L. amazonensis virulent parasites. In previous studies, this live attenuated vaccine has demonstrated to induce long-protection against murine leishmaniasis due to Old World Leishmania species. Vaccinated mice showed a reduction in the disease evolution due to L. amazonensis challenge, namely reduction in cutaneous lesions and parasite burdens. In contrast to control animals, after the challenge, protected mice showed anti-Leishmania IgG2a circulating antibodies accompanied to the induction of Leishmania-driven specific IFN-γ systemic response. An analysis performed in the lymph node draining the site of infection revealed an increase of the parasite-specific IFN-ϒ production by CD4+ and CD8+ T cells and a decrease in the secretion of IL-10 against leishmanial antigens. Since the immunity caused by the inoculation of this live vaccine generates protection against different forms of murine leishmaniasis, we postulate LiΔHSP70-II as a candidate for the development of human vaccines.

Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named “non-JPC like”, may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability.