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Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis (ZVL). The disease is endemic in Central and South America, Central and South East Asia, and the Mediterranean basin. Dogs are the main reservoir, with an estimated prevalence of approximately 2.5 million dogs in Southern Europe. Current treatments cause side effects, disease recurrence, and drug resistance. Therefore, the development of vaccines against canine leishmaniasis is necessary. We have generated a DNA vaccine based on the non-replicative antibiotic resistance marker-free plasmid vector pPAL that contains the encoding gene for the L. infantum activated protein kinase C receptor analog (LACK). Homologous pPAL-LACK prime-boost intranasal administration confers efficacious protection in Beagle dogs with a reduction of clinical signs and a statistically significant reduction of the parasite burden in the bone marrow of more than 90% of dogs after experimental infection with highly infective promastigotes. This DNA vaccine elicits a robust cellular immune response skewed towards the Th1 profile.

Vaccines represent one of the most significant advancements in public health since they prevented morbidity and mortality in millions of people every year. Conventionally, vaccine technology focused on either live attenuated or inactivated vaccines. However, the application of nanotechnology to vaccine development revolutionized the field. Nanoparticles emerged in both academia and the pharmaceutical industry as promising vectors to develop future vaccines. Regardless of the striking development of nanoparticles vaccines research and the variety of conceptually and structurally different formulations proposed, only a few of them advanced to clinical investigation and usage in the clinic so far. This review covered some of the most important developments of nanotechnology applied to vaccine technologies in the last few years, focusing on the successful race for the preparation of lipid nanoparticles employed in the successful anti-SARS-CoV-2 vaccines.

The compartmentalization of untranslated mRNA molecules in granules occurring in many eukaryotic organisms including trypanosomatids involves the formation of complexes between mRNA molecules and RNA-binding proteins (RBPs). The putative ATP-dependent DEAD/H RNA helicase (DEVH1) from Leishmania infantum (Kinetoplastida: Trypanosomatidae) is one such proteins. The objective of this research is finding differentially expressed genes in a stable episomal transfectant L. infantum promastigote line over-expressing DEVH1 in the stationary phase of growth in axenic culture to get insight into the biological roles of this RNA helicase in the parasite. Interestingly, genes related to parasite survival and virulence factors, such as the hydrophilic surface protein/small hydrophilic endoplasmic reticulum protein (HASP/SHERP) gene cluster, an amastin, and genes related to reactive oxygen species detoxification are down-regulated in DEVH1 transfectant promastigotes.

SARS-CoV-2 vaccines currently in use have contributed to controlling the COVID-19 pandemic. Notwithstanding, the high mutation rate, fundamentally in the spike glycoprotein (S), is causing the emergence of new variants. Solely utilizing this antigen is a drawback that may reduce the efficacy of these vaccines. Herein we present a DNA vaccine candidate that contains the genes encoding the S and the nucleocapsid (N) proteins implemented into the non-replicative mammalian expression plasmid vector, pPAL. This plasmid lacks antibiotic resistance genes and contains an alternative selectable marker for production. The S gene sequence was modified to avoid furin cleavage (Sfs). Potent humoral and cellular immune responses were observed in C57BL/6J mice vaccinated with pPAL-Sfs + pPAL-N following a prime/boost regimen by the intramuscular route applying in vivo electroporation. The immunogen fully protected K18-hACE2 mice against a lethal dose (105 PFU) of SARS-CoV-2. Viral replication was completely controlled in the lungs, brain, and heart of vaccinated mice. Therefore, pPAL-Sfs + pPAL-N is a promising DNA vaccine candidate for protection from COVID-19.

El impacto en la salud y en la economía de la pandemia Covid-19 ha llevado al rápido desarrollo de varias vacunas frente a su agente etiológico, el virus SARS-CoV-2. Estas vacunas están basadas fundamentalmente en la proteína de la corona del virus: spike (S). Un antígeno con gran tendencia a mutar, lo que hace que constantemente aparezcan distintas cepas del mismo con características variables. Nuestro equipo de trabajo ha desarrollado una vacuna de ADN sintético que inicialmente ha producido en la infección experimental, protecciones superiores al 50% en ratones transgénicos para el receptor de la enzima convertidora de angiotensina humana (KhACE2). Para mejorar los porcentajes de protección frente a la infección se han medido los efectos en la misma de la vía de administración: intramuscular, intranasal e intradérmica. Así como, la dosis introducida y la formulación, mediante la adición al plásmido inicial de moléculas cargadas positivamente que aumenten su captación por las células del huésped mamífero. Se han probado también otras proteínas más estables del virus, para obtener una vacuna efectiva frente a diversas cepas del mismo.

New drugs against visceral leishmaniasis with mechanisms of action differing from existing treatments and with adequate cost, stability, and properties are urgently needed. No antitubulin drug is currently in the clinic against Leishmania infantum, the causative agent of visceral leishmaniasis in the Mediterranean area. We have designed and synthesized a focused library of 350 compounds against the Leishmania tubulin based on the structure-activity relationship (SAR) and sequence differences between host and parasite. The compounds synthesized are accessible, stable, and appropriately soluble in water. We assayed the library against Leishmania promastigotes, axenic, and intracellular amastigotes and found 0, 8, and 16 active compounds, respectively, with a high success rate against intracellular amastigotes of over 10%, not including the cytotoxic compounds. Five compounds have a similar or better potency than the clinically used miltefosine. 14 compounds showed a host-dependent mechanism of action that might be advantageous as it may render them less susceptible to the development of drug resistance. The active compounds cluster in five chemical classes that provide structure-activity relationships for further hit improvement and facilitate series development. Molecular docking is consistent with the proposed mechanism of action, supported by the observed structure-activity relationships, and suggests a potential extension to other Leishmania species due to sequence similarities. A new family of diarylsulfonamides designed against the parasite tubulins is active against Leishmania infantum and represents a new class of potential drugs with favorable cost, stability, and aqueous solubility for the treatment of visceral leishmaniasis (VL). These results could be extended to other clinically relevant species of Leishmania spp.

Este libro surge como una iniciativa para compilar una serie de artículos alumbrados a partir de la génesis del Grupo CURIE (Científic@s Unid@s por la Reactivación de la Investigación en España), semilla de la Asociación Española para el Avance de la Ciencia (AEAC). Durante meses sus miembros estuvieron intercambiando reflexiones y debatiendo sobre la distancia entre la ciencia y la sociedad, y la necesidad de crear una asociación que llenase ese vacío entre los científicos y los ciudadanos, que apostase por llevar la ciencia y los conocimientos generados por la misma a toda la sociedad, tal y como establecen la Declaración de Derechos Humanos y la Constitución Española, en su artículo 44.

BACKGROUND:Leishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression-profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly-and culture-derived promastigotes., FINDINGS:Transcriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly-derived promastigotes have been found. Sand fly-derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein (HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly-derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut., CONCLUSIONS:Transcriptome analysis has been demonstrated to be technically efficacious to study differential gene expression in sand fly gut promastigote forms. Transcript and protein levels are not well correlated in these organisms (approximately 25% quantitative coincidences), especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view (increase, decrease, or no variation). Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly-derived promastigotes so far.

DNA vaccines require a vector to replicate genes and express encoding antigens. Antibiotic resistance genes are often used as selection markers, which must not be released to the environment upon final product commercialization. For this reason, generation of antibiotic resistance-free vectors is imperative. The pPAL vector contains the cytomegalovirus enhancer and promoter for expression in mammalian cells and the E. coli fabI chromosomal gene as a selectable marker. The fabI gene encodes the enoyl-ACP reductase (FabI). The bacteriostatic compound triclosan is an inhibitor of this enzyme. Therefore, the selection of positive clones depends on the enzyme:inhibitor molar ratio. According to western blot analysis, the pPAL vector is functional for expression of the Leishmania infantum (Kinetoplastid: Trypanosomatidae) gene encoding for the protein kinase C receptor analog (LACK/p36) in the HEK293T human cell line transfected with pPAL-LACK. The fabI gene sequence contains a 210 bp CpG island, suggesting a potential role as an adjuvant of the antibiotic resistance-free pPAL vector. In fact, Th1 response induction levels against canine leishmaniasis only using pPAL-LACK was shown to be as strong as in previous strategies using a recombinant vaccinia virus in combination with standard mammalian expression plasmid vectors. In summary, the pPAL plasmid contains the essential elements for manipulation and expression of any cloned DNA sequence in prokaryotic and mammalian cells using an E. coli endogenous gene as a selectable marker, which also provides a long CpG island. This element enhances Th1 immune response against L. infantum infection in dogs using the gene encoding for the LACK antigen. Therefore, this antibiotic resistance-free plasmid is a vaccine vector actively participating in protection against canine leishmaniasis and may be potentially tested as a vaccine vector with other antigens against different pathogens.

Leishmania infantum is responsible for human and canine leishmaniasis in the Mediterranean basin, where the major vector is Phlebotomus perniciosus. Because isolation of sufficient parasites from the sand fly gut is technically challenging, axenic cultivation of promastigotes is routinely used to obtain material for biochemical and genetic analyses. Here, we report the use of Spliced Leader RNA-seq (SL-seq) to compare transcript abundance in cultured promastigotes and those obtained from the whole midgut of the sand fly 5 days after infection. SL-seq allows for amplification of RNA from the parasite avoiding contamination with RNA from the gut of the insect. The study has been performed by means of a single technical replicate comparing pools of samples obtained from sand fly-derived (sfPro) and axenic culture promastigotes (acPro). Although there was a moderate correlation (R2 = 0.83) in gene expression, 793 genes showed significantly different (≥2-fold, p <0.05) mRNA levels in sand fly-derived promastigotes and in culture, of which 31 were up-regulated ≥8-fold (p < 10-8 in most cases). These included several genes that are typically up-regulated during metacyclogenesis, suggesting that sand fly-derived promastigotes contain a substantial number of metacyclics, and/or that their differentiation status as metacyclics is more advanced in these populations. Infection experiments and studies evaluating the proportion of metacyclic promastigotes in culture and within the sand fly gut, previously reported by us, support the last hypothesis.

La irrupción del coronavirus SARS-CoV-2 y de la pandemia de covid-19 ha planteado un enorme desafío a la ciencia. Buscar soluciones a un virus que ha causado, hasta el momento, 130 millones de infectados y ha provocado casi 3 millones de muertes. Para desentrañar las claves de esta nueva amenaza global, el Consejo Superior de Investigaciones Científicas, el CSIC, puso en marcha en marzo de 2020 la Plataforma Temática Interdisciplinar Salud Global. Su objetivo es coordinar a cientos de equipos de diversas disciplinas en busca de soluciones a la pandemia, creando nuevas redes de colaboración con organismos públicos de investigación, universidades, la clínica y el sector empresarial. La plataforma ha reunido a expertos de la investigación en biomedicina (virólogos, inmunólogos, genetistas y biotecnólogos), junto a químicos y físicos, además de sociólogos y demógrafos. Su trabajo es determinar las características del virus y de la covid-19, y diseñar estrategias que permitan hacer frente a esta y futuras pandemias. Tras un año de investigación, la Plataforma Salud Global ha obtenido conocimientos fundamentales en todos los aspectos de la pandemia: prevención, contención, diagnóstico, terapia y vacunas, e impacto social.

The kinetoplastid parasite Leishmania infantum is responsible for zoonotic visceral leishmaniasis in the mediterranean basin, where dogs are the reservoir. Differential gene expression analysis by whole genome DNA microarray hybridization revealed up-regulation of genes involved in infectivity of metacyclic promastigotes in axenic culture, together with two unidentified genes that had not been annotated in the parasite's genome sequences. Sequence analysis revealed that these genes encode for guide RNAs (gRNAs), which are located in the kinetoplast and participate in the kinetoplastid-specific uridine insertion/deletion RNA editing process. Northern blot assays confirmed that both gRNA genes are up-regulated in metacyclic promastigotes, thus suggesting that uridine insertion/deletion RNA editing contributes to metabolic shifts at this stage. A screening strategy described herein has revealed an uncharacterized 16S-like rRNA transcript as a target of one of the aforementioned gRNAs.

The first completed genome project of any living organism, excluding viruses, was of the gammaproteobacteria Haemophilus influenzae in 1995. Until the last decade, genome sequencing was very tedious because genome survey sequences (GSS) and/or expressed sequence tags (ESTs) belonging to plasmid, cosmid, and artificial chromosome genome libraries had to be sequenced and assembled in silico. No genome is completely assembled because gaps and unassembled contigs are always remaining. However, most represent an organism's whole genome from a practical point of view. The first genome sequencing projects of trypanosomatid parasites Leishmania major, Trypanosoma cruzi, and T. brucei were completed in 2005 following those strategies. The functional genomics era developed on the basis of microarray technology and has been continuously evolving. In the case of the genus Leishmania, substantial information about differentiation in the digenetic life cycle of the parasite has been obtained. More recently, next generation sequencing has revolutionized genome sequencing and functional genomics, leading to more sensitive and accurate results by using much fewer resources. Though this new technology is more advantageous, it does not invalidate microarray results. In fact, promising vaccine candidates and drug targets have been found by means of microarray-based screening and preliminary proof-of-concept tests.

Secuencia de polinucleótidos que comprende: a. una secuencia de ácidos nucleicos que consiste en SEC ID Nº: 1 o una secuencia con al menos el 85 % de identidad con la secuencia de ácidos nucleicos de SEC ID Nº: 1; o b. una secuencia de ácidos nucleicos que codifica una secuencia de aminoácidos que consiste en SEC ID Nº: 2 o una secuencia de aminoácidos con al menos el 90 % de identidad con SEC ID Nº: 2, para su uso como un adyuvante de vacuna. [ES], The invention relates to the fields of vaccines and vaccine adjuvants, and generally relates to polynucleotide adjuvants, polynucleotide vaccines and vaccine compositions. More specifically, the invention relates to said polynucleotides and vaccine compositions for use in inducing or enhancing a prophylactic or therapeutic immune response in a mammalian subject. Furthermore, it relates to said polynucleotides and vaccine compositions for use in the prophylactic or therapeutic treatment of an infectious disease, such as in the prophylactic or therapeutic treatment of leishmaniasis. [EN]

Tres de los sueros españoles más prometedores, desarrollados a partir de viruela atenuada, ADN recombinante y replicones de ADN, cuentan además con la ventaja de una conservación relativamente sencilla. En medio de los anuncios en cascada de grandes farmacéuticas sobre sus avances en la vacuna contra la COVID-19, varios grupos científicos españoles prosiguen sus investigaciones para conseguir un suero eficaz contra la epidemia en el que no se dependa del exterior.

El mundo está pendiente de cualquier noticia relativa a las vacunas frente al Covid-19 y, en la Unión Europea, se sigue con especial atención el desarrollo de los ensayos en Fase III de la vacuna de Oxford y AstraZeneca, la primera sobre la que se cerró un acuerdo de compra centralizada y cuyas primeras dosis estarían disponibles en diciembre, una fecha que se aleja después de que el laboratorio interrumpiera el ensayo este miércoles, por un caso de mielitis transversa en uno de los voluntarios. La vacuna “verá retrasada su aparición en el mercado”, tal y como señala el Dr. Vicente Larraga, científico del Centro de Investigaciones Biológicas-Margarita Salas del CSIC. Sin embargo, Larraga, que también desarrolla una vacuna del SARS-CoV-2, cree que est

El equipo de Vicente Larraga comienza con la última fase de experimentación en roedores. En un mes tendrán los resultados. En caso de ser óptimos serán enviados a la Agencia Española del Medicamento que es quien autorizaría el ensayo de la vacuna en humanos

El por-venir está todavía por-hacer. Y la democracia está en peligro. El futuro que anhelamos emergerá de la conciencia global, de la ciudadanía mundial, con una equidad progresiva, capaz por fin de expresarse y dejar de ser invisible, silenciosa, sumisa. Por fin, la ciudadanía podrá, presencialmente y en el ciberespacio, manifestarse sin cortapisas. Por fin, la fuerza de la razón en lugar de la razón de la fuerza. Por fin, todos y no unos cuantos. Por fin, la implicación ciudadana. Por fin, la palabra esclareciendo los hoy sombríos caminos del mañana

This report summarizes the consensus comments of the expert group after oral and written consultation. It is envisaged that this document should stimulate a debate within the scientific community and serve as a recommendation for future actions by international or regional funding agencies in the area of NIDs in LAC., This work was supported by the Directorate-General for Development Cooperation of the Belgian Government (framework agreement 03, project 95502) and the European Commission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[EN]Leishmania infantum is a protozoan parasite that causes leishmaniasis in the northern Mediterranean basin. It presents a complex biological cycle with an extracellular phase in the digestive tract of the insect vector and an intracellular phase within the mammalian host. Gene expression throughout the extracellular phase presents variations that seem to be aimed at preparing the parasite to resist the activity of the host immune system in what has been called pre-adaptation theory. In this pre-infection phase, the gene corresponding to the ubiquitin conjugate protein E2 (LinJ.33.2910) appears over-expressed. Given the importance of the functionality of this protein (LiUbc1) along the evolutionary scale, the characterization of this protein in a primitive eukaryotic such as L. infantum is of interest. We have compared this protein with its homologous proteins in other protozoa of the same family and of other lower and higher eukaryotes. It has been seen that homology remains within the genus, decreases in the same family and falls with respect to those of other higher eukaryotes. Molecular modeling of the protein suggests that it is a homodimer similar to that of the structure of Saccharomyces cerevisiae and shows partial homology with that of Homo sapiens. We have proceeded to the cloning of the gene and its expression, purifying the recombinant protein. Its expression has been studied in two different isolates of the parasite and differences between them have been detected. The existence of a dimeric and a monomeric form has been confirmed as predicted by molecular models. Knock-in promastigotes have been obtained that over-express the LiUbc1 protein and its infective capacity has been studied in vitro. It has been detected that over-expression favors the survival of the amastigote phase in the first 24h of the infection in vitro. This suggests that the LiUbc1 protein may be related to the infective capacity of the parasite. The location of the LiUbc1 protein h, [ES]Leishmania infantum es protozoo parásito causante de leishmaniasis en la cuenca norte Mediterránea. Presenta un ciclo biológico complejo con una fase extracelular en el aparato digestivo del insecto vector y una fase intracelular dentro del hospedador mamífero. La expresión génica a lo largo de la fase extracelular presenta variaciones que parece que van dirigidas a preparar al parásito para resistir la actividad del sistema inmune del huésped en lo que se ha llamado teoría de la pre-adaptación. En esta fase previa a la infección, el gen correspondiente a la proteína conjugadora de ubiquitina E2 (LinJ.33.2910), aparece sobre expresado. Dada la importancia de la funcionalidad de esta proteína (LiUbc1) a lo largo de la escala evolutiva, resulta de interés la caracterización de esta proteína en un eucariota primitivo como es L. infantum. Se ha procedido a comparar dicha proteína con sus proteínas homólogas en otros protozoos de la misma familia y de otros eucariotas inferiores y superiores. Se ha visto que la homología se mantiene dentro del género, disminuye en la misma familia y desciende respecto a las de otros eucariotas superiores. El modelado de la proteína sugiere que se trata de un homodímero similar al de la estructura de Saccharomyces cerevisiae y muestra una homología parcial con la de Homo sapiens. Se ha procedido al clonaje del gen y a la expresión del mismo, purificando la proteína recombinante. Se ha estudiado su expresión en dos aislados distintos del parásito y se han detectado diferencias entre los mismos. Se ha confirmado la existencia de una forma dimérica y otra monomérica como predecían los modelos moleculares. Se han obtenido promastigotes knock-in que la sobre-expresan la proteína LiUbc1 y se ha estudiado su capacidad infectiva in vitro. Se ha detectado que la sobre-expresión favorece la supervivencia de la fase amastigote en las primeras 24h de la infección. Esto sugiere que la proteína LiUbc1 puede estar relacionada con la capacidad infect

Purpose:Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil. Domestic and stray dogs are the main reservoirs. The life cycle of the parasite involves two stages. Promastigotes are extracellular and develop within the sand fly gut. Amastigotes survive inside the harsh environment of the phagolysosome of mammalian host phagocytes, which display the nitric oxide defense mechanism. Surprisingly, we were able to isolate promastigotes that are also resistant to NO. This finding may be explained by the preadaptative hypothesis. An insight into the proteome of NO‐sensitive and resistant promastigotes is presented herein., Experimental design: Total protein extracts were prepared from promastigote cultures of an NO-sensitive and a resistant strain at early-logarithmic, mid-logarithmic and stationary phase. A population enriched in metacyclic promastigotes was also isolated by Percoll gradient centrifugation. In vitro infectivity of both strains was compared. Differential protein abundance was analyzed by 2DE-MALDI-TOF/TOF. The most striking results were tested at the mRNA level by qRT-PCR. Three biological replicates were performed in all cases., Results: NO-resistant L. chagasi promastigotes are more infective than NO-sensitive ones. Among the differentially abundant spots, 40 proteins could be successfully identified in the sensitive strain and 38 in resistant promastigotes., Conclusions and clinical relevance: The increase of G6PD and the decrease of ARG and GPX transcripts and proteins contribute to NO resistance in L. chagasi promastigotes. These proteins may be studied as potential drug targets and/or vaccine candidates in the future.