Search Results (96)

Canine visceral leishmaniasis (CVL) is a major zoonosis that poses a growing challenge to public health services, as successful disease management requires sensitive, specific, and rapid diagnostic methods capable of identifying infected animals even at a subclinical level. The objective of this study was to evaluate the performance of the recombinant chimeric protein rMELEISH3 as an antigen in ELISA assays for the robust diagnosis of CVL. The protein was expressed in a bacterial system, purified by affinity chromatography, and evaluated through a series of serological assays using serum samples from dogs infected with Leishmania infantum. ROC curve analysis revealed a diagnostic sensitivity of 96.4%, a specificity of 100%, and an area under the curve of 0.996, indicating excellent discriminatory power. Furthermore, rMELEISH3 was recognized by antibodies present in the serum of dogs with low parasite loads, reinforcing the diagnostic potential of the assay in asymptomatic cases. It is concluded that the use of the recombinant antigen rMELEISH3 could significantly contribute to the improvement of CVL surveillance and control programs in endemic areas of Brazil and other countries, by offering a safe, reproducible and effective alternative to the methods currently recommended for the serological diagnosis of the disease. Full article

Human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is a complex parasitic disease marked by dynamic host–parasite interactions and immunomodulation. Extracellular vesicles (EV) derived from immune cells have emerged as key mediators of intercellular communication and potential biomarkers in infectious diseases. In this study, we combined a modified lymphocyte proliferation assay with nano-flow cytometry to quantify and phenotype EV released by CD4⁺, CD8⁺, and CD14⁺ cells in PBMC cultures from CL patients at different clinical stages: before treatment (PBT), during treatment (PDT), and post-treatment (PET) with antimonial. Healthy individuals (HI) were included as physiological controls. Upon stimulation with L. (V.) braziliensis antigens, we observed a distinct modulation of EV subsets. In the PBT group, CD4⁺ and CD14⁺ EV were significantly reduced, while CD8⁺ EV remained elevated. During PDT and PET, EV concentrations were restored across all subsets. These findings suggest that L. (V.) braziliensis selectively modulates the release of immune cell–derived EV, possibly as an immune evasion mechanism. The restoration of EV release following antimonial therapy highlights their potential as sensitive biomarkers for disease activity and treatment monitoring. This study offers novel insights into the immunoregulatory roles of EV in CL and underscores their relevance in host–parasite interactions. Full article

Background/Objectives: Leishmaniasis is a prevailing infectious disease transmitted via infected phlebotomine sandflies. The lack of an efficient vaccine with respect to immunogenic antigens and adjuvanted delivery systems impedes its control. Following the induction of immune responses in mice vaccinated with multi-epitope Leishmania peptides (LeishPts) encapsulated in doubly adjuvanted self-nanoemulsifying drug delivery systems (ST-SNEDDSs), this study aims to assess ST-SNEDDS-based nanoemulsions as vehicles for the delivery of antigenic proteins. Methods: Model antigens (e.g., BSA-FITC, OVA) were encapsulated in ST-SNEDDS after being complexed with the cationic phospholipid dimyristoyl phosphatidylglycerol (DMPG) via hydrophobic ion pairing. The nanoemulsions were characterized with respect to droplet diameter, zeta potential, stability, protein loading, protein release from the nanodroplets in different release media and cell uptake. Results: Both model antigens exhibited high encapsulation efficiency (>95%) and their release from the nanodroplets was shown to be strongly affected by the type of release medium (e.g., PBS, FBS 10% v/v) and the ratio of its volume to that of the oily phase, in agreement with predictions of protein release. Protein-loaded nanoemulsion droplets labeled with Cy-5 were found to be efficiently taken up by macrophages (J774A.1) in vitro. However, no colocalization of the labeled nanodroplets and BSA-FITC could be observed. Conclusions: It was revealed that in contrast with LeishPts, whole protein molecules may not be appropriate antigenic cargo for ST-SNEDDS formulations due to the rapid protein release from the nanodroplets in release media simulating in vitro culture and in vivo conditions such as FBS 10% v/v. Full article

Compounds in sand fly saliva elicit specific immune responses that may play a role in the establishment of canine Leishmania infection. Although canine antibodies to anti-sand fly saliva antigens have been extensively studied, little is known about cellular immune responses against Phlebotomus perniciosus salivary proteins. This study aimed to explore humoral and T-cell-mediated immunity against P. perniciosus salivary proteins in dogs (n = 85) from Mallorca (Spain), a leishmaniosis-endemic area, and find correlations with demographic (age, sex, and breed) and parasite-specific immunological parameters. Anti-sand fly saliva IgG was examined using a P. perniciosus whole salivary gland homogenate (SGH) ELISA and recombinant salivary protein rSP03B ELISA. Interferon gamma (IFN-γ) release whole blood assays with L. infantum soluble antigen (LSA), SGH, and rSP03B were also performed. Positive correlations were found between IgG levels in the SGH and rSP03B tests and between concentrations of SGH IFN-γ and rSP03B IFN-γ. While concentrations of SGH IFN-γ and rSP03B IFN-γ were low and produced only by a minority of dogs (less than 20%), high levels and frequencies of LSA IFN-γ as well as anti-saliva IgG for SGH and rSP03B were detected in a majority of dogs (61% and 75%, respectively). LSA IFN-γ levels were positively correlated with age and Leishmania-specific antibodies. In conclusion, dogs from a leishmaniosis-endemic area presented high humoral immunity against P. perniciosus salivary proteins, but their cellular immunity to these proteins was low and less frequent. Full article

Highly active antiretroviral therapy (HAART) has reduced the incidence of VL/HIV dramatically. However, HAART only partially prevents relapses, with one-year relapse rates ranging from 30 to 60%. Consequently, secondary prophylaxis is recommended for patients with <200 CD4+ cells/μL. In clinical practice, characterizing cellular immune response could help estimate the risk of relapse in VL/HIV coinfected patients. In this study, the lymphoproliferative response after stimulation with soluble Leishmania antigen was assessed in 2022 and 2023 in three cases of VL/HIV coinfection with long-term follow-up (17, 8 and 19 years). PCR and rK-39 results for Leishmania, HIV viral load, CD4 cell count, proliferation index, IFN-γ, IL-2, IP-10, IL-10 and TNF-α were determined. Heterogeneous results were obtained, with only one patient having developed specific cellular immunity against Leishmania. No cases of relapse were observed. The heterogeneity of lymphoproliferative test results in the three cases described highlights the need to identify surrogate markers of cure to guide maintenance or withdrawal of prophylaxis. Full article

Leishmania (Leishmania) infantum chagasi infections range from asymptomatic (AS) to severe visceral leishmaniasis (VL). One of the manifestations is an atypical non-ulcerated cutaneous leishmaniasis (NUCL), which occurs in some locations of Central America with few cases of VL. We conducted a transcriptomic analysis of cell-mediated immunity (CMI) on blood samples from NUCL, AS, VL patients from Amapala, Honduras, and healthy controls. RNA-seq revealed a similar perturbation of gene expression in NUCL and AS. Eight gene signatures of CMI were found in NUCL involved in CD8⁺ T lymphocyte infiltration, reactive oxygen species generation, PD-1 receptor ligand, inflammasome assembly, chemotaxis, complement receptor and suppressor immune cell infiltration. NUCL was distinguished from VL by its up-regulation of differently expressed genes (DEGs) related to T lymphocyte exhaustion, adhesion and transmigration of leukocytes, and down-regulation of oxidative stress genes. In contrast, VL exhibited up-regulated DEGs involved in antigen cross-presentation, and similar to VL from Brazil, down-regulated DEGs involved in innate immunity. Corroborating the transcriptome findings, both the Leishmanin skin test, and the immunopathology of NUCL skin lesion defined NUCL as a proinflammatory condition, intermediate between the AS and VL clinical outcomes. That condition may be the underlying element for the benign nature of the NUCL. Full article

Background/Objectives: The total lysate of Leishmania amazonensis (LaAg) is one of the most extensively studied vaccine formulations against leishmaniasis. Despite demonstrating safety and immunogenicity when administered intramuscularly, LaAg has failed to show efficacy in clinical trials and, in some cases, has even been associated with an enhanced susceptibility to infection. Adjuvants, which are molecules or compounds added to antigens to enhance the immunogenicity or modulate the immune response, are frequently employed in vaccine studies. This study aimed to evaluate different adjuvants to improve the protective efficacy of LaAg in L.amazonensis infection using a BALB/c mouse model. Methods: BALB/c mice were immunized with LaAg in combination with various adjuvants. The delayed-type hypersensitivity (DTH) test was assessed by measuring the infected paw and was used to evaluate the immunogenicity and to determine the most effective adjuvant. The immune response was analyzed through flow cytometry, focusing on cytokine production, immune cell recruitment and lesion size, alongside the control of parasite load at the infection site. The expression levels of iNOS and TGF-β were quantified using RT-qPCR, while IgG1, IgG2a and IgE antibody levels were determined via ELISA. Results: Among the adjuvants tested, only saponin (SAP) elicited a significant DTH response following LaAg challenge. SAP enhanced the immunogenicity of LaAg, as evidenced by increased IFN-γ-producing CD4⁺ and CD8⁺ T cells in the draining lymph nodes at 18 h post-challenge. Additionally, SAP facilitated the recruitment of lymphocytes, macrophages, neutrophils and eosinophils to the infection site. Conclusions: The LaAg + SAP combination conferred partial protection, as demonstrated by a reduction in lesion size and the partial control of parasite load. In conclusion, the addition of SAP as an adjuvant to LaAg effectively modulates the immune response, enhancing the vaccine’s protective efficacy. These findings provide valuable insights into the development of improved vaccines against L.amazonensis infection. Full article

Canine leishmaniosis (CanL) is a zoonotic disease caused by Leishmania infantum, where increased interferon-gamma (IFN-γ) levels are associated with controlling the infection and mild to moderate disease. Therefore, monitoring IFN-γ concentrations is essential for monitoring the immune responses in CanL. This study compared a faster, cost-effective IFN-γ release whole blood assay in tubes (WBA-T) with a standardized version (WBA-S) in 41 dogs at different states of L. infantum infection. WBA-T was performed at 24, 48, and 72 h of incubation with three conditions: blood, blood with L. infantum-soluble antigen (LSA), and blood with mitogen ConA. WBA-S was performed in plates, with blood diluted and incubated for five days using the same conditions. Supernatants (WBA-S) or plasma (WBA-T) were harvested for IFN-γ measurement by ELISA. No significant differences were observed in terms of IFN-γ concentration between WBA-T and WBA-S under LSA conditions. However, the 48 h incubation period during WBA-T showed the highest median of IFN-γ concentration compared to other incubation periods and WBA-S. The IFN-γ concentrations under ConA stimulation in WBA-S were significantly higher than in WBA-T at all incubation times studied. In conclusion, WBA-T stimulated with LSA at 48 h incubation time was shown to be the most appropriate for assessing IFN-γ production. Full article

Background/Objectives: Considering the large number of candidates in vaccine-testing studies against different pathogens and the amount of time spent in the preclinical and clinical trials, there is a pressing need to develop an improved in vivo system to quickly screen vaccine candidates. The model of a polyester–polyurethane sponge implant provides a rapid analysis of the specific stimulus–response, allowing the study of a compartmentalized microenvironment. The sponge implant’s defined measurements were standardized as a compartment to assess the immune response triggered by the vaccinal antigen. The LBSap vaccine (composed of Leishmania braziliensis antigens associated with saponin adjuvant) was used in the sponge model to assess the antigen-specific immunological biomarker, including memory generation after initial contact with the antigen. Methods: Mice strains (Swiss, BALB/c, and C57BL/6) were previously immunized using LBSap vaccine, followed by an antigenic booster performed inside the sponge implant. The sponge implants were assessed after 72 h, and the immune response pattern was analyzed according to leukocyte immunophenotyping and cytokine production. Results: After LBSap vaccination, the innate immune response of the antigenic booster in the sponge implants demonstrated higher levels in the Ly+ neutrophils and CD11c+ dendritic cells with reduced numbers of F4/80+ macrophages. Moreover, the adaptive immune response in Swiss mice demonstrated a high CD3+CD4+ T-cell frequency, consisting of an effector memory component, in addition to a cytoxicity response (CD3+CD8+ T cells), displaying the central memory biomarker. The major cell surface biomarker in the BALB/c mice strain was related to CD3+CD4+ effector memory, while the increased CD3+CD8+ effector memory was highlighted in C57/BL6. The cytokine profile was more inflammatory in Swiss mice, with the highest levels of IL-6, TNF, IFN-g, and IL-17, while the same cytokine was observed in in C57BL/6 yet modulated by enhanced IL-10 levels. Similar to Swiss mice, BALB/c mice triggered an inflammatory environment after the antigenic booster in the sponge implant with the increased levels in the ILL-6, TNF, and IFN-g. Conclusions: The findings emphasized the impact of genetic background on the populations engaged in immune responses, suggesting that this model can be utilized to enhance and track both innate and adaptive immune responses in vaccine candidates. Consequently, these results may inform the selection of the most suitable experimental model for biomolecule testing, taking into account how the unique characteristics of each mouse strain affect the immune response dynamics. Full article

Cutaneous leishmaniasis (CL), caused by Leishmania braziliensis, is closely associated with a severe form of the disease, indicated by a positive Leishmania skin test (LST) that assesses and reflects the presence of immune T cells specific to Leishmania antigens. In this study, we compare the clinical, immunologic, and histopathologic features between Leishmania skin test-positive (LST+) and Leishmania skin test-negative (LST-) in CL. Compared to LST+ patients, LST- patients had larger lesions and had been sicker for longer, presented with more instances of therapeutic failure with meglumine antimonate, (MA) and the healing times were higher than LST+. While granulomas were less frequent and the parasite load was higher in LST-, there were more CD8+ T cells and an enhanced production of Granzyme B in the supernatants of biopsies from LST- subjects. This study shows that in LST-, an impairment in Th1 immune response is associated with a high parasite burden, and the pathology is mediated by CD8+ T cells and the enhanced production of Granzyme B. The abnormalities in the immunologic response in LST- patients lead to a more severe disease with a high rate of failure to therapy. Full article

Significant populations in tropical and sub-tropical locations all over the world are severely impacted by a group of neglected tropical diseases called leishmaniases. This disease is caused by roughly 20 species of the protozoan parasite from the Leishmania genus. Disease prevention strategies that include early detection, vector control, treatment of affected individuals, and vaccination are all essential. The diagnosis is critical for selecting methods of therapy, preventing transmission of the disease, and minimizing symptoms so that the affected individual can have a better quality of life. Nevertheless, the diagnostic methods do eventually have limitations, and there is no established gold standard. Some disadvantages include the existence of cross-reactions with other species, and limited sensitivity and specificity, which are mostly determined by the type of antigen used to perform the tests. A viable alternative for a more precise diagnosis is the application of recombinant antigens, which have been generated using bioinformatics approaches and have shown increased diagnostic accuracy. This approach proves valuable as it spans from epitope selection to predicting the interactions within the antibody–antigen complex through docking analysis. As a result, identifying potential new antigens using bioinformatics resources becomes an effective technique since it may result in an earlier and more accurate diagnosis. Consequently, the primary aim of this review is to conduct a comprehensive overview of the most significant in silico tools developed over time, with a focus on evaluating their efficacy and exploring their potential applications in optimizing the selection of highly specific molecules for a more effective diagnosis of leishmaniasis. Full article

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania. As approved human vaccines are not available, treatment and prevention rely heavily on toxic chemotherapeutic agents, which face increasing resistance problems. The development of effective vaccines against human leishmaniasis is of utmost importance for the control of the disease. Strategies that have been considered for this purpose range from whole-killed and attenuated parasites to recombinant proteins and DNA vaccines. The ideal vaccine must be safe and effective, ensuring lasting immunity through a robust IL-12-driven Th1 adaptive immune response. Despite some success and years of effort, human vaccine trials have encountered difficulties in conferring durable protection against Leishmania, a problem that may be attributed to the parasite’s antigenic diversity and the intricate nature of the host’s immune response. The aim of this review is to provide a thorough overview of recent advances in Leishmania vaccine development, ranging from initial trials to recent achievements, such as the ChAd63-KH DNA vaccine, which underscores the potential for effective control of leishmaniasis through continued research in this field. Full article

Toxoplasmosis represents a significant public health and veterinary concern due to its widespread distribution, zoonotic transmission, and potential for severe health impacts in susceptible individuals and animal populations. The ability to design and produce recombinant proteins with precise antigenic properties is fundamental, as they serve as tools for accurate disease detection and effective immunization strategies, contributing to improved healthcare outcomes and disease control. Most commonly, a prokaryotic expression system is employed for the production of both single antigens and multi-epitope chimeric proteins; however, the cloning strategies, bacterial strain, vector, and expression conditions vary. Moreover, literature reports show the use of alternative microbial systems such as yeast or Leishmania tarentolae. This review provides an overview of the methods and strategies employed for the production of recombinant Toxoplasma gondii antigenic proteins for the serological detection of T. gondii infection and vaccine development. Full article

Th1 and Th2 cytokines determine the outcome of Leishmania major infection and immune protection depends mainly on memory T cells induced during vaccination. This largely hinges on the nature and type of memory T cells produced. In this study, transgenic Leishmania major strains expressing membrane-associated ovalbumin (mOVA) and soluble ovalbumin (sOVA) were used as a model to study whether fully differentiated Th1/Th2 and Th17 cells can recall immune memory and tolerate pathogen manipulation. Naïve OT-II T cells were polarised in vitro into Th1/Th2 cells, and these cells were transferred adoptively into recipient mice. Following the transferral of the memory cells, the recipient mice were challenged with OVA transgenic Leishmania major and a wild-type parasite was used a control. The in vitro-polarised T helper cells continued to produce the same cytokine signatures after being challenged by both forms of OVA-expressing Leishmania major parasites in vivo. This suggests that antigen-experienced cells remain the same or unaltered in the face of OVA-transgenic Leishmania major. Such ability of these antigen-experienced cells to remain resilient to manipulation by the parasite signifies that vaccines might be able to produce immune memory responses and defend against parasitic immune manipulation in order to protect the host from infection. Full article

Leishmaniosis is a vector-borne disease caused by protozoan parasites of the genus Leishmania, which are zoonotic and have an important impact on animal and public health globally. Between 2009 and 2023, blood samples from domestic dogs with clinical suspicion of leishmaniosis were received from 286 veterinary medical centres throughout mainland Portugal. An enzyme-linked immunosorbent assay (ELISA) was utilised to detect antibodies against Leishmania infantum antigens. Additionally, a complete blood count and tests for total proteins, urea, creatinine and alanine aminotransferase, as well as protein electrophoresis, were also performed. No significant relationship between sex and breed was observed. The age distribution was bimodal, with the highest prevalence of disease occurring at 2–5 years of age and a secondary peak occurring at 6 years or over (p < 0.001). No statistical correlation was observed between creatinine and urea across the ELISA serological groups. In contrast, both the gamma globulin levels (r = 0.45; p < 0.001) and the albumin/globulin ratio (r = −0.36; p < 0.001) exhibited moderate correlations with the ELISA. These findings support recent seroprevalence studies in dogs, with some geographical areas in Northern Portugal exhibiting the highest values, which may be the result of geographical shifts in parasite circulation due to climate change. Full article