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This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum.

With the control of vectorial transmission of Chagas disease caused by metacyclic trypomastigotes (MT) in endemic countries, other pathways of infection have become important. The infection caused by blood trypomastigotes (BT) is relevant in places where the blood transfusion and organ transplantation are poorly controlled. This study aimed to evaluate immunopathogenic parameters in the colon during the acute and chronic phases of experimental infection in Swiss mice infected with BT or MT forms of VL-10 strain of Trypanosoma cruzi. We have found that animals infected with MT forms presented lower survival rate, and higher tissue parasitism in the acute phase of the disease, which may be associated with the exacerbated activation of the immune system with the production of pro-inflammatory cytokines even in the chronic phase of infection. Taken together, these results can also be associated to the maintenance of the inflammatory process in chronic phase and an earlier denervation of myenteric plexus in colon. These findings emphasized the importance of the inoculum source and the strain, once different forms of different strains seem to promote distinct diseases.

The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 10

In recent decades, the oral infection of Trypanosoma cruzi has gathered increased attention due to frequent outbreaks that can lead to more severe clinical signs than those usually found in the areas of vector transmission. This study addresses the main routes of infection using metacyclic trypomastigotes (MT) and blood trypomastigotes (BT). Herein, BALB/c mice were infected with the Colombian (TcI) strain via intraperitoneal (IP), oral, intragastric (IG), ocular (OC) and cutaneous (CT) routes with 10

The poor immunogenicity displayed by some antigens has encouraged the development of strategies to improve the immune response and safety of vaccine candidates, resulting in an intense search for substances that potentiate vaccine response. Adjuvants have these properties helping vaccine candidates to induce a strong, durable, and fast immune response. In this study, we evaluated the specific immune response of adjuvants alone, Saponin (SAP), Incomplete Freund’s Adjuvant (IFA) and Monophosphoryl lipid-A SE (MPL-SE®) and in combination with total antigen of L. braziliensis (LB): LBSAP, LBIFA and LBMPL. The specific immune response induced by these compositions demonstrated that they were powerfully immunogenic, increasing cellular infiltration in the skin. Draining lymph nodes cultures showed that LBIFA and LBMPL have higher ability to increase the capacity of APCs to present antigens, with increased frequency of CD11c+CD86+ cells. SAP, MPL, LBSAP, LBIFA and LBMPL could activate lymphocytes increasing expression of CD69 and CD25. LBSAP group was an excellent inducer of pro-inflammatory cytokines at 24 h. At 48 h, higher cytokines production was observed in IFA, LBIFA, MPL and LBMPL groups. Our data demonstrate that LBSAP and LBMPL are potential formulations to be tested in other experimental models. Also, the data obtained could expand the knowledge about immune response after sensitization and also contribute to the development of safe, immunogenic and effective vaccines.

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.

In Brazil, canine visceral leishmaniasis is an important public health problem due to its alarming growth. The high prevalence of infected dogs reinforces the need for a vaccine for use in prophylactic vaccination campaigns. In the present study, we evaluate the immunogenicity and protection of the best dose of Chimera A selected through the screening of cytokines production important in disease. BALB/c mice were vaccinated subcutaneously with three doses and challenged intravenously with 1 × 107 L. infantum promastigotes. Spleen samples were collected to assess the intracellular cytokine profile production, T cell proliferation and parasite load. At first, three different doses of Chimera A (5 μg, 10 μg and 20 μg) were evaluated through the production of IFN-γ and IL-10 cytokines. Since the dose of 20 μg showed the best results, it was chosen to continue the study. Secondarily, Chimera A at dose of 20 μg was formulated with Saponin plus Monophosphoryl lipid A. Vaccination with Chimera A alone and formulated with SM adjuvant system was able to increase the percentage of the proliferation of specific T lymphocytes and stimulated a Th1 response with increased levels of IFN-γ, TNF-α and IL-2, and decreased of IL-4 and IL-10. The vaccine efficacy through real-time PCR demonstrated a reduction in the splenic parasite load in animals that received Chimera A formulated with the SM adjuvant system (92%). Additionally, we observed increased levels of nitric oxide in stimulated-culture supernatants. The Chimera A formulated with the SM adjuvant system was potentially immunogenic, being able to induce immunoprotective mechanisms and reduce parasite load. Therefore, the use of T-cell multi-epitope vaccine is promising against visceral leishmaniasis.

Many vaccine candidates against visceral leishmaniasis (VL) have been proposed, however, to date, none of them have been efficacious for the human or canine disease. On this basis, the design of leishmaniasis vaccines has been constantly changing, and the use of approaches to select specific epitopes seems to be crucial in this scenario. The ability to predict T cell-specific epitopes makes immunoinformatics an even more necessary approach, as in VL an efficient immune response against the parasite is triggered by T lymphocytes in response to Leishmania spp. immunogenic antigens. Moreover, the success of vaccines depends on the capacity to generate long-lasting memory and polyfunctional cells that are able to eliminate the parasite. In this sense, our study used a combination of different approaches to develop potential chimera candidate vaccines against VL. The first point was to identify the most immunogenic epitopes of Leishmania infantum proteins and construct chimeras composed of Major histocompatibility complex (MHC) class I and II epitopes. For this, we used immunoinformatics features. Following this, we validated these chimeras in a murine model in a thorough memory study and multifunctionality of T cells that contribute to a better elucidation of the immunological protective mechanisms of polyepitope vaccines (chimera A and B) using multicolor flow cytometry. Our results showed that in silico-designed chimeras can elicit polyfunctional T cells producing T helper (Th)1 cytokines, a strong immune response against Leishmania antigen, and the generation of central and effector memory T cells in the spleen cells of vaccinated animals that was able to reduce the parasite burden in this organ. These findings contribute two potential candidate vaccines against VL that can be used in further studies, and help in this complex field of vaccine development against this challenging parasite.

The natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitroco-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-γ and TNF-α and decreased levels in IL-4, TGF-β and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products.

Pentavalent antimonial has been the first choice treatment for visceral leishmaniasis; however, it has several side effects that leads to low adherence to treatment. Liposome-encapsulated meglumine antimoniate (MA) arises as an important strategy for chemotherapy enhancement. We evaluated the immunopathological changes using the mixture of conventional and pegylated liposomes with MA. The mice were infected with Leishmania infantum and a single-dose treatment regimen. Comparison was made with groups treated with saline, empty liposomes, free MA, and a liposomal formulation of MA (Lipo MA). Histopathological analyses demonstrated that animals treated with Lipo MA showed a significant decrease in the inflammatory process and the absence of granulomas. The in vitro stimulation of splenocytes showed a significant increase of gamma interferon (IFN-γ) produced by CD8 + T cells and a decrease in interleukin-10 (IL-10) produced by CD4 + and CD8 + T cells in the Lipo MA. Furthermore, the Lipo MA group showed an increase in the IFN-γ/IL-10 ratio in both CD4 + and CD8 + T cell subsets. According to the parasite load evaluation using quantitative PCR, the Lipo MA group showed no L. infantum DNA in the spleen (0.0%) and 41.4% in the liver. In addition, we detected a low positive correlation between parasitism and histopathology findings (inflammatory process and granuloma formation). Thus, our results confirmed that Lipo MA is a promising antileishmanial formulation able to reduce the inflammatory response and induce a type 1 immune response, accompanied by a significant reduction of the parasite burden into hepatic and splenic compartments in treated animals.

Herein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by Leishmania infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n = 6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n = 10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical, and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP, and bilirubin) and hematological (erythrocytes, hemoglobin, hematocrit, and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B lymphocytes, increased NK cells (CD5−CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen- specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-γ+ and TCD8+IFN-γ+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-α and reduced levels of IL-10. Concerning the clinical signs of canine visceral leishmaniasis, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by real-time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment.

Canine visceral leishmaniosis (CVL) is a zoonosis of major public health impact caused by organisms of the genus Leishmania which is transmitted to human and animals by phlebotomine sand flies. The skin is the first point of contact with Leishmania parasites for sandy fly vectors and it is considered an important reservoir compartment in infected dogs. The aim of this study was to determine the main histophatologic alterations in ear skin of dogs naturally infected by Leishmania infantum with different clinical status and different degrees of parasitism. Therefore, thirty-four dogs naturally infected with L. infantum were grouped according to their clinical status in asymptomatic (AD, n = 11), oligosymptomatic (OD, n = 11) and symptomatic dogs (SD, n = 12) as well as their degrees of parasite load in the skin as low (LP, n = 11), median (MP, n = 11) and high (HP, n = 12) parasitism. Additionally, ten dogs were used as control (CD, n = 10). At necropsy, skin samples were collected for further histological and parasitological analysis. The OD and SD groups presented higher parasite burden than AD group. The inflammation was higher in SD group when compared to OD and AD. The LP, MP and HP groups showed an increasing inflammatory process, indicating that a great parasite load is accompanied by a major inflammatory process in the skin. The number of mast cells was higher in the OD and LP groups than CD group, suggesting that these cells may be involved in tissue remodeling, since that an increase of type III collagen fibers and decrease type I collagen fibers were observed in these groups. Taken together, our results enable a better understanding of the alterations in skin of CVL dogs and consequently new insights about the pathogenesis of CVL.

Specific chemotherapy using benznidazole (BNZ) for Chagas disease during the chronic stage is controversial due to its limited efficacy and toxic effects. Although BNZ has been used to treat Chagas disease since the 1970s, few studies about the biodistribution of this drug exist. In this study, BNZ tissue biodistribution in a murine model and its pharmacokinetic profile in plasma were monitored. A bioanalytical high-performance liquid chromatography method with a UV detector (HPLC-UV) was developed and validated according to the European Medicines Agency for quantification of BNZ in organs and plasma samples prepared by liquid-liquid extraction using ethyl acetate. The developed method was linear in the BNZ concentration, which ranged from 0.1 to 100.0 μg/ml for plasma, spleen, brain, colon, heart, lung, and kidney and from 0.2 to 100.0 μg/ml for liver. Validation assays demonstrated good stability for BNZ under all conditions evaluated. Pharmacokinetic parameters confirmed rapid, but low, absorption of BNZ after oral administration. Biodistribution assays demonstrated different maximum concentrations in organs and similar times to maximum concentration and mean residence times, with means of 40 min and 2.5 h, respectively. Therefore, the biodistribution of BNZ is extensive, reaching organs such as the heart and colon, which are the most relevant organs affected by Trypanosoma cruzi infection, and also the spleen, brain, liver, lungs, and kidneys. Simultaneous analyses of tissues and plasma indicated high BNZ metabolism in the liver. Our results suggest that low bioavailability, instead of inadequate biodistribution, could be responsible for therapeutic failure during the chronic phase of Chagas disease.

Reverse vaccinology or immunoinformatics is a computational methodology which integrates data from in silico epitope prediction, associated to other important information as, for example, the predicted subcellular location of the proteins used in the design of the context of vaccine development. This approach has the potential to search for new targets for vaccine development in the predicted proteome of pathogenic organisms. To date, there is no effective vaccine employed in vaccination campaigns against visceral leishmaniasis (VL). For the first time, herein, an in silico, in vitro, and in vivo peptide screening was performed, and immunogenic peptides were selected to constitute VL peptide-based vaccines. Firstly, the screening of in silico potential peptides using dogs naturally infected by L. infantum was conducted and the peptides with the best performance were selected. The mentioned peptides were used to compose Cockt-1 (cocktail 1) and Cockt-2 (cocktail 2) in combination with saponin as the adjuvant. Therefore, tests for immunogenicity, polyfunctional T-cells, and the ability to induce central and effector memory in T-lymphocytes capacity in reducing the parasite load on the spleen for Cockt-1 and Cockt-2 were performed. Among the vaccines under study, Cockt-1 showed the best results, eliciting CD4+ and CD8+ polyfunctional T-cells, with a reduction in spleen parasitism that correlates to the generation of T CD4+ central memory and T CD8+ effector memory cells. In this way, our findings corroborate the use of immunoinformatics as a tool for the development of future vaccines against VL.

Ethnopharmacological relevance Protium spruceanum (Burseraceae) is used in Brazilian traditional medicine as anti-inflammatory, but the factors involved in this activity were not yet characterized. Aims of the study: analyze the aspects involved in the anti-inflammatory activity of polar fractions obtained from extracts of leaves and branches. Materials and methods: Hydromethanolic fraction was obtained by liquid-liquid partition from crude ethanolic extract and its compounds were identified by LC-DAD-MS. Activity tests were performed using LPS + IFN-γ stimulated J774A.1 macrophages. Cytokines were evaluated by CBA kit, NO by Griess method, ROS by DCFH-DA, N-acetylglucosaminidase (NAG) activity by spectrophotometric method, matrix-metalloproteinase (MMP-9) activity by zymography, inducible nitric oxide synthase (iNOS) expression by immunofluorescence and cyclooxygenase (COX-2) expression by Western blot. Results: Fractions induced an increase of IL-6 and IL-10 which leads to the control of pro-inflammatory cytokines levels. The treatment with the fractions also reduced NO production at all concentrations tested in all evaluated periods. ROS production by the macrophages was inhibited by the treatment and the leaves fraction showed the best results with a lower concentration than that observed for the branches. The enzymes assays showed that leaves fraction inhibited NAG and MMP-9 activities, as well as, iNOS and COX-2 expression. These activities can be associated with the presence of procyanidin, catechin, rutin, quercitrin, isoquercitrin and kaempferol-3-O-rhamnoside, major compounds that were identified in the fraction. Conclusions: Anti-inflammatory activity of P. spruceanum is associated to an immunomodulatory effect that leads to inhibition of ROS, NO, NAG, MMP-9, COX-2 and iNOS.

The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-γ, TNF-α, IL-4, IL-13, TGF-β and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-γ and IL-13 expression, and IL-13 and TGF-β expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis.

Visceral leishmaniasis has a great impact on public health, and dogs are considered the main domestic reservoir of Leishmania infantum, the causal parasite. In this study, 159 animals naturally infected by L. infantum from an endemic area of Brazil were evaluated through an analysis of cellular responses, using flow cytometry, and of the hematological parameters. The results confirmed that disease progression is associated with anemia and reductions in eosinophils, monocytes and lymphocytes. The investigation of the immune response, based on the immunophenotypic profile of peripheral blood, showed declines in the absolute numbers of T lymphocytes CD5(+) and their subsets (CD4(+) and CD8(+)) and a drop of B lymphocytes in asymptomatic seropositive (AD-II) and symptomatic seropositive (SD) dogs. Neutrophils, when stimulated with soluble antigen of L. infantum, showed higher synthesis of interferon (IFN)-γ(+) in AD-II and SD groups, with decreased production of interleukin (IL)-4(+) in asymptomatic seronegative dogs positive for L. infantum infection based on polymerase chain reaction testing (AD-I group). In the AD-II and SD groups, subpopulations of stimulated lymphocytes (CD4(+) and CD8(+)) also exhibited greater synthesis of IFN-γ(+) and IL-4(+) in culture. These results suggest that the animals of the AD-II and SD groups exhibited a mixed immune response (Type 1 and 2) and the AD-I group presenting an immune profile very similar to normal control animals.

Background The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8+ T cells, and high NO production. Methods We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge. Results The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5+, CD4+, and CD8+ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load. Conclusions These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector.