A Tailored Approach to Leishmaniases Vaccination: Comparative Evaluation of the Efficacy and Cross-Protection Capacity of DNA vs. Peptide-Based Vaccines in a Murine Model.

Zoonotic leishmaniases are a worldwide public health problem for which the development of effective vaccines remains a challenge. A vaccine against leishmaniases must be safe and affordable and should induce cross-protection against the different disease-causing species. In this context, the DNA vaccine pHisAK70 has been demonstrated to induce, in a murine model, a resistant phenotype against L. major, L. infantum, and L. amazonensis. Moreover, a chimeric multiepitope peptide, HisDTC, has been obtained by in silico analysis from the histone proteins encoded in the DNA vaccine and has showed its ability to activate a potent CD4+ and CD8+ T-cell protective immune response in mice against L. infantum infection. In the present study, we evaluated the plasmid DNA vaccine pHisAK70 in comparison with the peptide HisDTC (with and without saponin) against L. major and L. infantum infection. Our preliminary results showed that both formulations were able to induce a potent cellular response leading to a decrease in parasite load against L. infantum. In addition, the DNA candidate was able to induce better lesion control in mice against L. major. These preliminary results indicate that both strategies are potentially effective candidates for leishmaniases control. Furthermore, it is important to carry out such comparative studies to elucidate which vaccine candidates are the most appropriate for further development.

Transcriptomic Profile of Canine DH82 Macrophages Infected by Leishmania infantum Promastigotes with Different Virulence Behavior.

Zoonotic visceral leishmaniosis caused by Leishmania infantum is an endemic disease in the Mediterranean Basin affecting mainly humans and dogs, the main reservoir. The leishmaniosis outbreak declared in the Community of Madrid (Spain) led to a significant increase in human disease incidence without enhancing canine leishmaniosis prevalence, suggesting a better adaptation of the outbreak's isolates by other host species. One of the isolates obtained in the focus, IPER/ES/2012/BOS1FL1 (BOS1FL1), has previously demonstrated a different phenotype than the reference strain MCAN/ES/1996/BCN150 (BCN150), characterized by a lower infectivity when interacting with canine macrophages. Nevertheless, not enough changes in the cell defensive response were found to support their different behavior. Thus, we decided to investigate the molecular mechanisms involved in the interaction of both parasites with DH82 canine macrophages by studying their transcriptomic profiles developed after infection using RNA sequencing. The results showed a common regulation induced by both parasites in the phosphoinositide-3-kinase-protein kinase B/Akt and NOD-like receptor signaling pathways. However, other pathways, such as phagocytosis and signal transduction, including tumor necrosis factor, mitogen-activated kinases and nuclear factor-κB, were only regulated after infection with BOS1FL1. These differences could contribute to the reduced infection ability of the outbreak isolates in canine cells. Our results open a new avenue to investigate the true role of adaptation of L. infantum isolates in their interaction with their different hosts.

Enhancing Control of Leishmania infantum Infection: A Multi-Epitope Nanovaccine for Durable T-Cell Immunity.

Canine leishmaniosis (CanL) is a growing health problem for which vaccination is a crucial tool for the control of disease. The successful development of an effective vaccine against this disease relies on eliciting a robust and enduring T-cell immune response involving the activation of CD4+ Th1 and CD8+ T-cells. This study aimed to evaluate the immunogenicity and prophylactic efficacy of a novel nanovaccine comprising a multi-epitope peptide, known as HisDTC, encapsulated in PLGA nanoparticles against Leishmania infantum infection in the murine model. The encapsulation strategy was designed to enhance antigen loading and sustain release, ensuring prolonged exposure to the immune system. Our results showed that mice immunized with PLGA-encapsulated HisDTC exhibited a significant reduction in the parasite load in the liver and spleen over both short and long-term duration. This reduction was associated with a cellular immune profile marked by elevated levels of pro-inflammatory cytokines, such as IFN-γ, and the generation of memory T cells. In conclusion, the current study establishes that PLGA-encapsulated HisDTC can promote effective and long-lasting T-cell responses against L. infantum in the murine model. These findings underscore the potential utility of multi-epitope vaccines, in conjunction with appropriate delivery systems, as an alternative strategy for CanL control.

Detection and Antimicrobial Resistance of Enterobacteriaceae other than Escherichia Coli in Raccoons from the Madrid Region of Spain.

Introduction: Raccoons are an invasive alien species widely distributed in the Madrid region of Spain. These animals can carry a variety of enteric bacteria with associated antimicrobial resistance, which can infect humans and livestock. However, to our knowledge, the presence of non-E. coli Enterobacteriaceae in raccoons has not been previously studied. Material and Methods: We conducted a study to examine the species distribution of Enterobacteriaceae isolates other than E. coli, as well as their antimicrobial resistance, in the faeces of 83 raccoons in the Madrid region. Results: We detected 12 Enterobacteriaceae isolates other than E. coli belonging to seven different species: Citrobacter freundii (1 isolate), Citrobacter gillenii (3 isolates), Citrobacter murliniae (1 isolate), Citrobacter portucalensis (2 isolates), Enterobacter hormaechei subsp. hoffmannii (1 isolate), Hafnia paralvei (2 isolates) and Raoultella ornithinolytica (2 isolates). These isolates were found in 7 of the 83 (8.4%) animals studied. To our knowledge, this study is the first report of the presence of non-E. coli Enterobacteriaceae in raccoon faeces. All isolates but one were resistant to at least one of the 14 antimicrobials tested. Resistance to ampicillin (83.3%), amoxicillinclavulanic acid (50%) and cefoxitin (33.3%) was the most frequent. Conclusion: Our study indicates that raccoons are a potential source of infection with Enterobacteriaceae other than E. coli for humans and livestock in the Madrid region.

Properties of virulence emergence of Leishmania infantum isolates from Phlebotomus perniciosus collected during the human leishmaniosis outbreak in Madrid, Spain. Hepatic histopathology and immunological parameters as virulence markers in the mouse model.

Recent anthropic activity related to the construction of the Bosquesur Green Park in a large urban setting in Madrid (Spain) has resulted in the largest reported community outbreak of human leishmaniosis in Europe. Previous phylogenetic and molecular-typing studies of parasite isolates have implicated the Leishmania infantum ITS-Lombardi genotype in this outbreak. In an unusual scenario, visceral leishmaniosis (VL) is affecting a significant number of individuals, suggesting that an increase in parasite virulence has occurred. In this work, using an in vivo BALB/c model of VL, we aimed to investigate the properties of emergent virulence of the L. infantum POL2FL7 and BOS1FL1 isolates obtained from Phlebotomus perniciosus collected in the outbreak area and compare them with those of the well-characterized strain BCN150 MON-1 isolated from a dog. The P. perniciosus specimens were collected during an entomological survey conducted in the transmission season of 2012. We observed a range of virulence phenotypes from moderately to highly aggressive after 5 weeks of infection. IV challenge of mice with outbreak isolates from sand flies induced higher splenic and liver parasite burdens, higher serological titres of specific anti-Leishmania antibodies and impaired capacities to control infection, as revealed by the arginine metabolism and low ratios of Th1/Th2 cytokine profiles analysed, compared with the corresponding measures evaluated in mice infected with the BCN150 strain. The BOS1FL1 isolate showed the highest degree of virulence among the isolates, superior to that of POL2FL7, as evidenced by the analysed biomarkers and the histopathological severity of liver lesions. These results provide insight into how L. infantum isolates from sand flies collected in the outbreak area have been able to affect not only immunosuppressed patients but also middle-aged people with normal immunocompetence in the largest human VL outbreak in Europe.

Raccoons (Procyon lotor) in the Madrid region of Spain are carriers of antimicrobial-resistant Escherichia coli and enteropathogenic E. coli.

The role of wildlife in the epidemiology of antimicrobial resistance is unclear. Raccoons in North America can carry a variety of enteric bacteria, with associated antimicrobial resistance, that could infect humans and livestock. The potential for raccoons to carry these bacteria in Europe, where they are an invasive species, has not been explored. Our objectives were to determine the prevalence of Escherichia coli with associated antimicrobial resistance in raccoons from the Madrid region of Spain and to determine whether they are carriers of potential human pathogens, including verotoxin-producing E. coli (VTEC) and enteropathogenic E. coli (EPEC). In total, we tested 237 E. coli isolates from the faeces of 83 euthanized raccoons for susceptibility to 14 antimicrobial agents and the presence of VTEC and EPEC. Antimicrobial resistance to at least one antimicrobial was detected in the faeces of 51% (42/83; 95% CI, 40.1-61.1) of the raccoons tested. A high percentage of raccoons carried, in their faeces, E. coli isolates resistant to ampicillin (33%), streptomycin (33%), tetracycline (30%), sulphafurazole (31%) and trimethoprim-sulphamethoxazole (23%). We detected one isolate of extended-spectrum ß-lactamase-producing E. coli from the faeces of one raccoon. We detected VTEC in the faeces of one raccoon, and EPEC in the faeces of 12% (10/83) of the raccoons. Of the raccoons that carried EPEC in their faeces, 60% (6/10) carried EPEC isolates that exhibited characteristics associated with pathogenicity in humans. Raccoons in Madrid can carry pathogenic and antimicrobial-resistant E. coli in their faeces and may be a risk to public health because of their potential to contaminate food and the environment with their faeces.

A further investigation of the leishmaniosis outbreak in Madrid (Spain): low-infectivity phenotype of the Leishmania infantum BOS1FL1 isolate to establish infection in canine cells.

Human leishmaniosis caused by Leishmania infantum is a zoonotic disease, with dogs as the main reservoir in Mediterranean Basin countries. The largest European outbreak of human leishmaniosis declared in the southwestern Madrid region (Spain) is characterized by unusual epidemiological and clinical features, such as the emergence of new wild reservoirs (hares and rabbits), whereas the seroprevalence, infection, and severity of canine leishmaniosis have not substantially changed since the first studies conducted in Madrid before the outbreak. Previous studies reported that L. infantum isolates from the Madrid leishmaniosis focus displayed elevated virulence in in vivo models of infection and increased infectivity in murine target cells. With the aim of studying whether changes in the host-parasite interaction and virulence profile have developed, we first assessed the behaviour of one circulating isolate of the outbreak, IPER/ES/2012/BOS1FL1 (BOS1FL1), compared to that of a well-characterized strain from canine leishmaniosis, MCAN/ES/1996/BCN150 (BCN150), in terms of infection capacity (percentage of infected cells, representing infectivity, and number of amastigotes per infected cell, representing the intensity of infection) in canine monocytes and macrophages. BCN150 displayed significantly higher infectivity (76.82 ±â€¯4.40 vs 38.58 ±â€¯2.19; P <  0.0001) and intensity of infection (3.64 ± 0.13 vs 1.83 ±â€¯0.12; P <  0.0001) than BOS1FL1 when interacting with canine cells. Our ROS induction results did not differ significantly between the two isolates or with the responses previously described for other L. infantum isolates. Paradoxically, increased resilience to hydrogen peroxide exposure was observed for BOS1FL1 (% viability 40.62 ±â€¯5.54 vs 26.37 ±â€¯2.93; P = 0.039). Finally, we demonstrated that a decreased intracellular load of BOS1FL1 was associated with increased IFN-γ (261.21 ±â€¯26.29 vs 69.80 ±â€¯9.02; P = 0.0151) and decreased IL-10 production (165.06 ±â€¯23.87 vs 264.41 ±â€¯30.58; P = 0.0002). In this study, we provide the first detailed insight into the differences between the isolate BOS1FL1 from the outbreak in Madrid and the well-characterized strain BCN150 MON-1 obtained from a dog in their response to interacting with canine cells. However, further studies are necessary to shed light on the immune mechanisms resulting in BOS1FL1 exhibiting less virulent behaviour in canine cells than in cells derived from other host species.

Epitope Selection for Fighting Visceral Leishmaniosis: Not All Peptides Function the Same Way.

Visceral leishmaniosis (VL) caused by Leishmania infantum is a disease with an increasing prevalence worldwide. Treatments are expensive, toxic, and ineffective. Therefore, vaccination seems to be a promising approach to control VL. Peptide-based vaccination is a useful method due to its stability, absence of local side effects, and ease of scaling up. In this context, bioinformatics seems to facilitate the use of peptides, as this analysis can predict high binding affinity epitopes to MHC class I and II molecules of different species. We have recently reported the use of HisAK70 DNA immunization in mice to induce a resistant phenotype against L. major, L. infantum, and L. amazonensis infections. In the present study, we used bioinformatics tools to select promising multiepitope peptides (HisDTC and AK) from the polyprotein encoded in the HisAK70 DNA to evaluate their immunogenicity in the murine model of VL by L. infantum. Our results revealed that both multiepitope peptides were able to induce the control of VL in mice. Furthermore, HisDTC was able to induce a better cell-mediated immune response in terms of reduced parasite burden, protective cytokine profile, leishmanicidal enzyme modulation, and specific IgG2a isotype production in immunized mice, before and after infectious challenge. Overall, this study indicates that the HisDTC chimera may be considered a satisfactory tool to control VL because it is able to activate a potent CD4+ and CD8+ T-cell protective immune responses.

Immunization with the HisAK70 DNA Vaccine Induces Resistance against Leishmania Amazonensis Infection in BALB/c Mice.

Leishmania amazonensis is the aetiological agent of a broad spectrum of leishmaniosis in South America. It can cause not only numerous cases of cutaneous leishmaniosis but also diffuse cutaneous leishmaniosis. Considering the diversity of parasite species causing different forms of the disease that coexist in the same region, it is desirable to develop a vaccine capable of eliciting cross-protection. We have previously described the use of HisAK70 DNA vaccine for immunization of mice to assess the induction of a resistant phenotype against Leishmania major and infantum infections. In this study, we extended its application in the murine model of infection by using L. amazonensis promastigotes. Our data revealed that 14 weeks post-infection, HisAK70-vaccinated mice showed key biomarkers of protection, such as higher iNOS/arginase activity, IFN-γ/IL-10, IFN-γ/IL-4, and GM-CSF/IL-10 ratios, in addition to an IgG2a-type response when compared to the control group. These findings correlated with the presentation of lower footpad swelling and parasite burdens in the immunized compared to the control mice. Overall, this study suggests that immunization with HisAK70 may be considered a suitable tool to combat leishmaniosis as it is able to induce a potent cellular immune response, which allows to control the infection caused by L. amazonensis.

Strength and medium-term impact of HisAK70 immunization in dogs: Vaccine safety and biomarkers of effectiveness for ex vivo Leishmania infantum infection.

HisAK70 candidates have successfully been tested in cutaneous (CL) and visceral leishmaniosis (VL) mouse models. Here, we analyse different biomarkers in dog trials after a heterologous immunization strategy with a HisAK70 candidate (plasmid DNA plus adoptive transfer of peripheral blood-derived dendritic cells (DCs) pulsed with the same pathoantigen and CpG ODN as an adjuvant) to explore the antileishmanial activity in an ex vivo canine co-culture system in the presence of Leishmania infantum parasites. In the canine model, the heterologous HisAK70 vaccine could decrease the infection index in the DC-T cell co-culture system by up to 54% after 30 days and reach almost 67% after 100 days post-immunization, respectively, compared to those obtained in the control group of dogs. The observed security and potential to fight ex vivo L. infantum infection highlight a HisAK70 heterologous immunization strategy as a promising alternative to evaluate its effectiveness against canine VL.

Diagnostic evaluation of the amastin protein from Leishmania infantum in canine and human visceral leishmaniasis and immunogenicity in human cells derived from patients and healthy controls.

The diagnosis of visceral leishmaniasis (VL) presents problems due to the toxicity and/or high cost of drugs. In addition, no vaccine exists to protect against human disease. In this study, the antigenicity and immunogenicity of amastin protein were evaluated in L. infantum-infected dogs and humans. For the diagnosis, besides the recombinant protein, 1 linear B-cell epitope was synthetized and evaluated in serological assays. Results showed high sensitivity and specificity values to detect the disease when both antigens were employed against a canine and human serological panel. By contrast, when using rA2 and a soluble Leishmania antigenic preparation, sensitivity and specificity values proved to be lower. A preliminary immunogenicity study showed that the amastin protein induced high IFN-γ and low IL-10 production in stimulated PBMC derived from treated VL patients and healthy subjects, thus suggesting a potential use of this protein as an immunogen to protect against human disease.

Alternative strategy for visceral leishmaniosis control: HisAK70-Salmonella Choleraesuis-pulsed dendritic cells.

Here, we describe a novel approach that exploits an attenuated mutant of Salmonella enterica serovar Choleraesuis as carrier to deliver a plasmid encoding protein HisAK70. Subsequently, dendritic cells (DCs) were pulsed with this vaccine vector. The aim of this study was to evaluate the effectiveness of the prepared HisAK70-S. Choleraesuis-pulsed DCs (HisAK70-SAL DCs) against visceral leishmaniosis (VL). In our ex vivo model of infection, the prepared formulations could decrease parasite growth by up to 80% by augmenting the production of IL-12p40 and by reducing arginase activity (ARG). Also, BALB/c mice when immunised with this formulation showed significant reduction in parasite burden in both spleen (20% of reduction) and liver (75% of reduction). The balance of the immune ratios IFN-γ/IL-10, TNF-α/IL-10, and IgG2a/IgG1 reflected the acquisition of an improved resistant phenotype in HisAK70-SAL DCs vaccinated mice compared to control mice. Our results suggest that HisAK70-SAL DCs could be a promising alternative approach for vaccine delivery that has the potential to fight Leishmania infantum (L. infantum) infection.

HisAK70: progress towards a vaccine against different forms of leishmaniosis.

BACKGROUND: Leishmania major and Leishmania infantum are among the main species that are responsible for cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL), respectively. The leishmanioses represent the second-largest parasitic killer in the world after malaria. Recently, we succeeded in generating a plasmid DNA (pCMV-HISA70m2A) and demonstrated that immunized mice were protected against L. major challenge. The efficacy of the DNA-vaccine was further enhanced by the inclusion of KMP-11 antigen into the antibiotic-free plasmid pVAX1-asd. METHODS: Here, we describe the use of a HisAK70 DNA-vaccine encoding seven Leishmania genes (H2A, H2B, H3, H4, A2, KMP11 and HSP70) for vaccination of mice to assess the induction of a resistant phenotype against VL and CL. RESULTS: HisAK70 was successful in vaccinated mice, resulting in a high amount of efficient sterile hepatic granulomas associated with a hepatic parasite burden fully resolved in the VL model; and resulting in 100% inhibition of parasite visceralization in the CL model. CONCLUSIONS: The results suggest that immunization with the HisAK70 DNA-vaccine may provide a rapid, suitable, and efficient vaccination strategy to confer cross-protective immunity against VL and CL.

Characterisation of the ex vivo virulence of Leishmania infantum isolates from Phlebotomus perniciosus from an outbreak of human leishmaniosis in Madrid, Spain.

BACKGROUND: Since mid 2009, an outbreak of human leishmaniosis in Madrid, Spain, has involved more than 560 clinical cases. Many of the cases occurred in people who live in areas around a newly constructed green park (BosqueSur). This periurban park provides a suitable habitat for sand flies (the vectors of Leishmania infantum). Indeed, studies of blood meals from sand flies captured in the area showed a strong association between the insect vector, hares or rabbits, and humans in the area. Interestingly, up to 70% of cases have been found in immunocompetent patients (aged between 46-60 years). This study was designed to evaluate the ex vivo virulence of the L. infantum isolates from Phlebotomus perniciosus captured in this area of Madrid. METHODS: Murine macrophages and dendritic cells were infected ex vivo with L. infantum strain BCN150, isolate BOS1FL1, or isolate POL2FL7. At different times after infection, the infection indices, cytokine production (IL-12p40 and IL-10), NO release and arginase activities were evaluated. RESULTS: Using an ex vivo model of infection in murine bone marrow-derived cells, we found that infection with isolates BOS1FL1 and POL2FL7 undermined host immune defence mechanisms in multiple ways. The main factors identified were changes in both the balance of iNOS versus arginase activities and the equilibrium between the production of IL-12 and IL-10. Infection with isolates BOS1FL1 and POL2FL7 also resulted in higher infection rates compared to the BCN150 strain. Infection index values at 24 h were as follows: BCN150-infected cells, 110 for infected MØ and 115 for infected DC; BOS1FL1-infected cells, 300 for infected MØ and 247 for infected DC; and POL2FL7-infected cells, 275 for infected MØ and 292 for infected DC. CONCLUSIONS: Our data indicate that L. infantum isolates captured from this endemic area exhibited high virulence in terms of infection index, cytokine production and enzymatic activities involved in the pathogenesis of visceral leishmaniosis. Altogether, these data provide a starting point for the study of the virulence behaviour of parasites (BOS1FL1 and POL2FL7) isolated from P. perniciosus during the outbreak of human leishmaniosis in Madrid, Spain, and their involvement in infecting immunocompetent hosts.