Leishmania infantum UBC1 in Metacyclic Promastigotes from Phlebotomus perniciosus, a Vaccine Candidate for Zoonotic Visceral Leishmaniasis

Leishmania parasites cause outstanding levels of morbidity and mortality in many developing countries in tropical and subtropical regions. Numerous gene expression profiling studies have been performed comparing different Leishmania species' life-cycles and stage forms in regard to their distinct infective ability. Based on expression patterns, homology to human orthologues, in silico HLA-binding predictions, and annotated functions, we were able to select several vaccine candidates which are currently under study. One of these candidates is the Leishmania infantum ubiquitin-conjugating enzyme E2 (LiUBC1), whose relative levels, subcellular location, in vitro infectivity in the U937 myeloid human cell model, and protection levels in Syrian hamsters against L. infantum infection were studied herein. LiUBC1 displays a low level of similarity with the mammalian orthologs and relevant structure differences, such as the C-terminal domain, which is absent in the human ortholog. LiUBC1 is present in highly infective promastigotes. Knock-in parasites overexpressing the enzyme increased their infectivity, according to in vitro experiments. Syrian hamsters immunized with the recombinant LiUBC1 protein did not show any parasite burden in the spleen, unlike the infection control group. The IFN-γ transcript levels in splenocytes were significantly higher in the LiUBC1 immunized group. Therefore, LiUBC1 induced partial protection against L. infantum in the Syrian hamster model.This work was financed by a contract with CZ Vaccines, Porriño, Spain, and partially defrayed by a grant from the Fundación Ramón Areces. JL thanks CZ Vaccines for the fellowship.S

A non-replicative antibiotic resistance-free DNA vaccine delivered by the intranasal route protects against canine leishmaniasis

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

The antibiotic resistance-free vaccine based on the non-replicative pPAL vector is fully protective against SARS-CoV-2 in the murine model

1 p.Background. The main objective of this work is the development of a DNA vaccine against the SARS-CoV-2 virus based on the non-replicative antibiotic resistance marker gene-free the plasmid vector pPAL.Methods. We designed pPAL-Sfs and pPAL-structural protein constructs. A PCR cloning procedure was carried out to obtain the pPAL-based recombinant vaccine and laboratory-scale batches of pPAL-based SARS-CoV-2 vaccine constructs were produced. Transfection was performed on the human HEK293 cell line with the pPAL-based recombinant vaccine. Expression was evaluated by Western blot. Evaluation of protection experiments against a lethal dose of 105 pfu of SARS-CoV-2 (Wuhan-Hu-1 and Delta strains) in K18-hACE2 female mice vaccinated intramuscularly with a prime/boost regimen was carried out by assessing both humoral and cellular immune responses. ELISA was used to evaluate humoral immunity, namely total IgG, as well as IgG1 and IgG2c subclasses. The cellular immune response was evaluated by quantifying the rate of IFN-γ producing splenocyte clones used ELISpot. In addition, characterization of the cellular response was carried out by intracellular staining (ICS) to identify of the rate of IFN-γ and TNF-α producing TCD4+ lymphocytes, as well as the proportion of TCD8+ lymphocytes. Determination of viral load in the main target organs was done by RT-PCR (lungs, heart, and brain). Virus replication capacity was also evaluated in target organs tissues. In vitro assays were performed out to determine the levels of neutralizing antibodies against SARS-CoV-2 virus.Results. The results show 100% protection of vaccinated animals in terms of symptomatology, animal weight, level of neutralizing antibodies against the virus and the rate of IFN-γ and TNF-α producing splenocyte clones. The analysis of IgG subclasses shows a predominance of IgG2c over IgG1, indicating the activation of a specific and cytotoxic Th1 protective cellular immune response and immunological memory. Finally, a reduction of viral load has been observed in vaccinated animals, with a clear reduction of virus replication in the main target organs. Furthermore, there is a synergistic effect increasing protection using the two plasmids p-PALSfs + pPAL-structural protein (under patent).Conclusions. The DNA vaccine pPAL-Sfs + pPAL-structural protein is fully protective in the mouse model in terms of maintenance of body weight, absence of significant clinical signs, viral load clearance in target organs and immune response. The immune response included neutralizing antibodies, predominance of IgG2c over IgG1 ratio, a Th1 response, and a multifunctional cytotoxic cellular response.Peer reviewe

Non-replicative antibiotic resistance-free DNA vaccine encoding S and N proteins induces full protection in mice against SARS-CoV-2

17 p.-8 fig.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.This work was funded by PTI-Salud Global (CSIC), Center for Technological and Industrial Development (CDTI), REACT-ANTICIPA-UCM (Comunidad de Madrid), and European Research Council (Advanced Grant VERDI, ERC2015AdG grant number 694160).Peer reviewe

Research Priorities for Neglected Infectious Diseases in Latin America and the Caribbean Region

Dujardin, J. C. et al. 5 p.-1 tab.Global priorities for research in neglected infectious diseases (NIDs) can be assessed in different ways, but it is important to realize that regional priorities may significantly differ one from another. The region of Latin America and the Caribbean (LAC) is—along with Africa and Asia—more affected by NIDs than other regions of the world. Some of the Latin American NIDs are common to other continents, while others are very specific or disproportionately affect the Latin American region [1– 3] (Table 1). Because of its huge ecological diversity, ongoing environmental changes, and massive migrations, LAC is also a catalyst for the (re-)emergence and spreading of NIDs, both inside and outside the subcontinent. Following a colloquium on NIDs in LAC held in Lima, Peru, between 12 and 14 November 2009, a thematic workshop was organized with the support of the European Commission (EC). It involved 29 scientists (16 from the Americas, two from the Democratic Republic of Congo and India, respectively, and nine from Europe) working on different NIDs and representing several research areas from basic to applied. 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 LACThis work was supported by the Directorate-General for Development Cooperation of the Belgian Government (framework agreement 03, project 95502) and the European CommissionPeer reviewe

Research priorities for neglected infectious diseases in Latin America and the Caribbean Region

Global priorities for research in neglected infectious diseases (NIDs) can be assessed in different ways, but it is important to realize that regional priorities may significantly differ one from another. The region of Latin America and the Caribbean (LAC) is—along with Africa and Asia—more affected by NIDs than other regions of the world. Some of the Latin American NIDs are common to other continents, while others are very specific or disproportionately affect the Latin American region [1– 3] (Table 1). Because of its huge ecological diversity, ongoing environmental changes, and massive migrations, LAC is also a catalyst for the (re-)emergence and spreading of NIDs, both inside and outside the subcontinent. Following a colloquium on NIDs in LAC held in Lima, Peru, between 12 and 14 November 2009, a thematic workshop was organized with the support of the European Commission (EC). It involved 29 scientists (16 from the Americas, two from the Democratic Republic of Congo and India, respectively, and nine from Europe) working on different NIDs and representing several research areas from basic to applied. 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. (Párrafo extraído del texto a modo de resumen)Facultad de Ciencias Médica

Caracterización de la proteína conjugadora de ubiquitina Ubc1 de Leishmania infantum en promastigotes wild type y knock-in

111 p.-31 fig.-8 tab.[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 has been studied in wild type and knock-in promastigotes. It has been found to have a location in subcellular structures (nuclear membrane, kinetoplast and flagellar pocket). These structures match the regulatory function assigned to the protein. A challenge experiment against infection in the Mesocricetus auratus model has shown that inoculation of the LiUbc1 protein induces protection in animals against unvaccinated controls. These experiments confirm that the LiUbc1 protein could be a vaccine candidate.[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 infectiva del parásito. Se ha estudiado la localización de la proteína LiUbc1 en ambos aislados y además en los promastigotes knock-in. Se ha visto que tiene una localización en estructuras subcelulares (membrana nuclear, kinetoplasto y bolsillo flagelar). Estas estructuras concuerdan con la función de regulación asignada a la proteína. En un experimento de reto frente a la infección en el modelo de Mesocricetus auratus se ha mostrado que la inoculación de la proteína LiUbc1 induce protección en los animales frente a los controles no vacunados. Estos experimentos confirman que la proteína LiUbc1 puede ser un candidato vacunal.Peer reviewe

Characterization of ubiquitin-activating (E1) and ubiquitinconjugating (E2) genes over-expressed in the infective stage of Leishmania infantum

2 p. Abstracts of the 9th European Congress on Tropical Medicine and International HealthPeer reviewe

Desarrollo de una vacuna de ADN frente a la infección por el virus SARS-CoV-2

1 p.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.Peer reviewe

The non-replicative antibiotic resistance genefree plasmid vector pPAL for the development of DNA vaccines

1 p.-3 fig.Background: The pPAL vaccination platform consists of the pPAL plasmid, whose production has been fine-tuned under good manufacturing practice (GMP). The main objective is to use this plasmid, which doesn´t contain antibiotic resistance genes, for the development of DNA vaccines and thus its worldwide distribution. We have generated the Neoleish vaccine against natural infection of canine leishmaniasis, which elicits protection in 60% of vaccinated dogs.Leishmaniasis is a vector-borne parasitic disease that, causes more than 50,000 deaths per year in its visceral form.Methods: The generation of the pPAL plasmid was carried out from the pCIneo expression mammalian cells plasmid.The procedure consisted of replacing the antibiotic resistance genes npt and bla by the fab I gene from a modified Escherichia coli strain (SURE) with a reduced of genetic material, and its cor responding promoter. A PCR cloning procedure was applied to obtain the recombinant pPAL-LACK vaccine using XbaI and EcoRI target sequences. In vitro expression of the LACK gene in the human HEK293 cell line was verified by Western blot. Evaluation of the humoral immune response of the NeoleishR vaccine in Beagle dogs against canine leishmaniasis was carried out by detecting the level of total IgG and IgG1 and IgG2 subclasses by ELISA. The evaluation of the parasite load in the main target organs (bone marrow, spleen and liver) was performed by qPCR. In addition, the percentage of CD4+ cells and the level of cytokines IFNγ and IL-10 were determined by lymphoblastic transformation test (LTT) in PBMC.Results: We have developed an antibiotic resistance gene-free plasmid, which requires the use of triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol) instead of an antibiotic as a selection agent (Alcolea et al., 2019). Selection of transformants that carry our plasmid of interest is based on the tolerance to this molecule, which toxic to the bacteria.The fab I gene, encodes the enoyl-ACP reductase (ACP being the acyl carrier protein), an enzyme essential for fatty acid synthesis in E. coli and is sensitive to triclosan.This plasmid has been used in the development of a naked DNA vaccine using a homologous prime- boost regimen via intranasal route (NeoleishR), against canine leishmaniasis, achieving 60% protection. This vaccine is based on the LACK gene (activated protein kinase C receptor analog) of Leishmania infantum, which causes zoonotic visceral leishmaniasis. The expression of the LACK gene in HEK293 cells was confirmed by Western blotting. Protection by this vaccine is characterized by a reduction 60 of parasite load and symptomatology, including a robust cellular immune response with a high production of IFNγ in protected dogs, which is an essential factor in the development of vaccines against an intracellular parasite.Conclusions: The pPAL vaccination platform could be used in the development of DNA vaccines against other infectious diseases. The non-replicative pPAL plasmid which doesn’t contain antibiotic resistance genes, therefore allowing its worldwide distribution.Peer reviewe