Activation of Human CD11b+ B1 B-Cells by Trypanosoma cruzi-Derived Proteins Is Associated With Protective Immune Response in Human Chagas Disease

B-cells mediate humoral adaptive immune response via the production of antibodies and cytokines, and by inducing T-cell activation. These functions can be attributed to distinct B-cell subpopulations. Infection with Trypanosoma cruzi, the causative agent of Chagas disease, induces a polyclonal B-cell activation and lytic antibody production, critical for controlling parasitemia. Individuals within the chronic phase of Chagas disease may remain in an asymptomatic form (indeterminate), or develop severe cardiomyopathy (cardiac form) that can lead to death. Currently, there is no effective vaccine to prevent Chagas disease, and no treatment to halt the development of the cardiomyopathy once it is installed. The pathology associated with cardiac Chagas disease is a result of an inflammatory reaction. Thus, discovering characteristics of the host's immune response that favor the maintenance of favorable heart function may unveil important immunotherapeutic targets. Given the importance of B cells in antibody production and parasite control, we investigated T. cruzi-derived antigenic fractions responsible for B-cell activation and whether frequencies and functional characteristics of B-cell subpopulations are associated with different clinical outcomes of human Chagas disease. We stimulated cells from indeterminate (I) and cardiac (C) Chagas patients, as well as non-infected individuals (NI), with T. cruzi-derived protein- (PRO), glycolipid- (GCL) and lipid (LIP)-enriched fractions and determined functional characteristics of B-cell subpopulations. Our results showed that the frequency of B-cells was similar amongst groups. PRO, but not GCL nor LIP, led to an increased frequency of B1 B-cells in I, but not C nor NI. Although stimulation with PRO induced higher TNF expression by B1 B-cells from C and I, as compared to NI, it induced expression of IL-10 in cells from I, but not C. Stimulation with PRO induced an increased frequency of the CD11b+ B1 B-cell subpopulation, which was associated with better cardiac function. Chagas patients displayed increased IgM production, and activation of gamma-delta T-cells, which have been associated with B1 B-cell function. Our data showed that PRO activates CD11b+ B1 B-cells, and that this activation is associated with a beneficial clinical status. These findings may have implications in designing new strategies focusing on B-cell activation to prevent Chagas disease cardiomyopathy

Influence of Clinical Status and Parasite Load on Erythropoiesis and Leucopoiesis in Dogs Naturally Infected with Leishmania (Leishmania) chagasi

Background: The bone marrow is considered to be an important storage of parasites in Leishmania-infected dogs, although little is known about cellular genesis in this organ during canine visceral leishmaniasis (CVL). Methodology/Principal Findings: The aim of the present study was to evaluate changes in erythropoiesis and leucopoiesis in bone marrow aspirates from dogs naturally infected with Leishmania chagasi and presenting different clinical statuses and bone marrow parasite densities. The evolution of CVL from asymptomatic to symptomatic status was accompanied by increasing parasite density in the bone marrow. The impact of bone marrow parasite density on cellularity was similar in dogs at different clinical stages, with animals in the high parasite density group. Erythroid and eosinophilic hypoplasia, proliferation of neutrophilic precursor cells and significant increases in lymphocytes and plasma cell numbers were the major alterations observed. Differential bone marrow cell counts revealed increases in the myeloid:erythroid ratio associated to increased numbers of granulopoietic cells in the different clinical groups compared with non-infected dogs. Conclusions: Analysis of the data obtained indicated that the assessment of bone marrow constitutes an additional and useful tool by which to elaborate a prognosis for CVL

Histological study of cell migration in the dermis of hamsters after immunisation with two different vaccines against visceral leishmaniasis.

Vaccine candidates, including live and/or killed parasites, Leishmania -purified fractions, defined recombinant antigens and antigen-encoding DNA-plasmids have been proposed to use as vaccine anti- Leishmania . More recently, the hamsters have been used to pre selection of antigens candidate to apply in further experiments using canine model. In this report we evaluated the kinetics of cell migration in dermal inflammatory infiltrate, circulating leukocytes and the presence of nitric oxide (NO)/induced nitric oxide synthase during the early (1?24 h) and late (48?168 h) periods following inoculation of hamsters with antigenic components of anti-canine visceral leishmaniasis vaccines Leishmune 1 and Leishmania braziliensis antigen (LB) with and without saponin (Sap) adjuvant. Our results show that LB caused an early reduction of lymphocytes in the dermis while Sap and LBSap triggered a late recruitment, suggesting the role of the adjuvant in the traffic of antigen-presenting cells and the induction of lymphocyte migration. In that manner our results suggest that the kinetics of cell migration on hamster model may be of value in the selection of vaccine antigens prior the tests in dogs particularly in respect of the toxicity of the preparations

Immunotherapy and immunochemotherapy in visceral leishmaniasis : promising treatments for this neglected disease.

Leishmaniasis has several clinical forms: self-healing or chronic cutaneous leishmaniasis or post-kala-azar dermal leishmaniasis; mucosal leishmaniasis; visceral leishmaniasis (VL), which is fatal if left untreated.The epidemiology and clinical features of VL vary greatly due to the interaction of multiple factors including parasite strains, vectors, host genetics, and the environment. Human immunodeficiency virus infection augments the severity of VL increasing the risk of developing active disease by 100?2320 times. An effective vaccine for humans is not yet available. Resistance to chemotherapy is a growing problem in many regions, and the costs associated with drug identification and development, make commercial production for leishmaniasis, unattractive.The toxicity of currently drugs, their long treatment course, and limited efficacy are significant concerns. For cutaneous disease, many studies have shown promising results with immunotherapy/immunochemotherapy, aimed to modulate and activate the immune response to obtain a therapeutic cure. Nowadays, the focus of many groups centers on treating canine VL by using vaccines and immunomodulators with or without chemotherapy. In human disease, the use of cytokines like interferon-g associated with pentavalent antimonials demonstrated promising results in patients that did not respond to conventional treatment. In mice, immunomodulation based on monoclonal antibodies to remove endogenous immunosuppressive cytokines (interleukin-10) or block their receptors, antigen-pulsed syngeneic dendritic cells, or biological products like Pam3Cys (TLR ligand) has already been shown as a prospective treatment of the disease. This review addresses VL treatment, particularly immunotherapy and/or immunochemotherapy as an alternative to conventional drug treatment in experimental models, canine VL, and human disease

Higher Expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 Chemokines in the Skin Associated with Parasite Density in Canine Visceral Leishmaniasis

Several previous studies correlated immunopathological aspects of canine visceral leishmaniasis (CVL) with tissue parasite load and/or the clinical status of the disease. Recently, different aspects of the immune response in Leishmania-infected dogs have been studied, particularly the profile of cytokines in distinct compartments. However, the role of chemokines in disease progression or parasite burdens of the visceralising species represents an important approach for understanding immunopathology in CVL. We found an increase in inflammatory infiltrate, which was mainly composed of mononuclear cells, in the skin of animals presenting severe forms of CVL and high parasite density. Our data also demonstrated that enhanced parasite density is positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression. These findings represent an advance in the knowledge of the involvement of skin inflammatory infiltrates in CVL and the systemic consequences and may contribute to developing a rational strategy for the design of new and more efficient prophylactic tools and immunological therapies against CVL

A vaccine therapy for canine visceral leishmaniasis promoted significant improvement of clinical and immune status with reduction in parasite burden.

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-?+ and TCD8+IFN-?+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-? 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

A vaccine therapy for canine visceral leishmaniasis promoted significant improvement of clinical and immune status with reduction in parasite burden.

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-?+ and TCD8+IFN-?+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-? 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

Avalia??o da imunogenicidade de dois novos Imunobiol?gicos candidatos a vacina contra Leishmaniose visceral canina.

Considerando a import?ncia de estrat?gias imunoprofil?ticas para o controle da leishmaniose visceral, a inexist?ncia de drogas capazes de curar c?es infectados e o aumento de casos de resist?ncia aos antimoniais pentavalentes, o presente trabalho buscou avaliar a imunogenicidade de dois novos imunobiol?gicos candidatos a vacina contra leishmaniose visceral canina (LVC). Observa-se uma lacuna em estudos que buscam avaliar a imunogenicidade p?s-vacinal de imunobiol?gicos anti-LVC. Assim, o presente estudo prop?s uma an?lise detalhada da imunogenicidade considerando diversos biomarcadores da resposta imune celular e humoral ap?s as tr?s doses das diferentes estrat?gias vacinais. Trinta e cinco c?es sem ra?a definida foram subdivididos em sete grupos experimentais, entre os quais: (i) grupo controle C (n = 10) que recebeu 1 mL de salina est?ril a 0,9%; (ii) grupo LB (n = 5) que recebeu 600 ?g de prote?na de Leishmania braziliensis em 1 mL de salina est?ril a 0,9%; (iii) grupo Sap (n = 5) que recebeu 1 mg de saponina em 1 mL de salina est?ril a 0,9%; (iv) grupo LBSap (n = 5) que recebeu 600 ?g de prote?na de L. braziliensis associado a 1 mg de saponina em 1 mL de salina est?ril a 0,9%; (v) grupo Sal (n = 5) que recebeu extrato de gl?ndula salivar de Lutzomyia longipalpis (SGE) equivalente ao conte?do de 5 ?cinos da gl?ndula salivar em 1 mL de salina est?ril a 0,9%; (vi) grupo LBSal (n = 5) que recebeu 600 ?g de promastigotas de L. braziliensis associado ao SGE em 1 mL de salina est?ril a 0,9%; (vii) grupo LBSapSal (n = 5) que recebeu 600 ?g de promastigotas de L. braziliensis associado a 1 mg de saponina e ao SGE em 1 mL de salina est?ril a 0,9%. Cada animal recebeu tr?s aplica??es subcut?neas no flanco esquerdo em intervalos de quatro semanas. Os resultados indicam que a saponina quando presente (Sap, LBSap, LBSapSal) induziu em alguns c?es a forma??o de edema ou de pequeno n?dulo local, sem apresentar les?es ulceradas ou outras altera??es adversas. A avalia??o da inocuidade e toxicidade das diferentes vacinas n?o revelou contra-indica??es para utiliza??o. A avalia??o da resposta humoral revelou que os grupos LBSap e LBSapSal apresentaram aumento dos n?veis de IgG total, IgG1 e IgG2 anti-Leishmania, sugerindo um perfil de resposta imune misto Th1/Th2. De forma semelhante, no grupo LBSapSal foi observado aumento dos n?veis de IgG total, IgG1 e IgG2 anti-SGE, que tem sido previamente relacionado ao estabelecimento de uma resposta imune protetora anti- Leishmania. Al?m disto, experimentos utilizando Western blot para avaliar a reatividade de anticorpos caninos anti-prote?nas do SGE evidenciaram o predom?nio de prote?nas com peso molecular de 35, 45 e 75 KDa, particularmente no grupo LBSapSal, sendo relacionado a um padr?o de resist?ncia contra infec??o por Leishmania. A avalia??o da resposta celular contou com o estudo imunofenot?pico de c?lulas mononucleares do sangue perif?rico (PBMC) e revelou aumento do n?mero de linf?citos B CD21+, linf?citos T CD5+ e das subpopula??es T (CD4+ e T CD8+), indicando o estabelecimento de imunidade protetora contra infec??o por Leishmania como previamente relatado na LVC. Al?m disto, foi observada pela an?lise in vitro redu??o do ?ndice de prolifera??o utilizando VSA (vaccine soluble antigen) ou SLcA (soluble Leishmania chagasi antigen) nos grupos LB e Sal, enquanto os grupos LBSap e LBSapSal apresentaram aumento da atividade linfoproliferativa na presen?a de ambos os est?mulos. De forma interessante, foi demonstrado no grupo Sal correla??o negativa entre a atividade linfoproliferativa e linf?citos T CD4+ ou mon?citos CD14+. Estes resultados permitem especular que a imuniza??o realizada no grupo Sal poderia inibir a atividade de mon?citos CD14+ em ativar linf?citos T CD4+ e assim reduzir a atividade linfoproliferativa. Por outro lado, PBMC cultivados in vitro com SLcA do grupo LBSap e com VSA ou SLcA no grupo LBSapSal apresentaram aumento da atividade XI linfoproliferativa acompanhada pela indu??o de linf?citos T CD8+ ant?geno-espec?ficos. Estes resultados sustentam a hip?tese de que a vacina??o com LBSap e LBSapSal estimularia o desenvolvimento de mecanismos imunoprotetores relacionados ao controle do parasitismo por Leishmania considerando a indu??o de linf?citos T CD8+ ant?geno-espec?ficos para prote?nas relacionadas ao agente etiol?gico da LVC. A avalia??o de potenciais c?lulas apresentadoras de ant?genos (APC) revelou aumento do n?mero de mon?citos CD14+ circulantes nos grupos LBSap e LBSapSal. Al?m disto, altas contagens de APC foram associadas ao aumento da express?o de linf?citos MHC-I no grupo LBSap e de linf?citos CD80 e MHC-I no grupo LBSapSal, sugerindo que esta associa??o poderia representar a intera??o entre as respostas imunes inata e adaptativa, refletindo no aumento do perfil de ativa??o durante as imuniza??es. Os resultados obtidos pelas an?lises dos n?veis de ?xido n?trico (NO) pela avalia??o dos n?veis de nitrito no sobrenadante de cultura estimuladas com SLcA no grupo LBSap e no soro do grupo LBSapSal confirmam a hip?tese de que estas vacinas induzem um perfil associado a resist?ncia contra a infec??o por Leishmania. Os resultados obtidos neste trabalho indicam que o potencial imunoprotetor induzido pelas vacinas LBSap e LBSapSal s?o compat?veis com o controle do agente etiol?gico da LVC.Considering the importance of immunoprophylactic strategies for the control of visceral leishmaniasis, and the lack of studies concerning the cellular and humoral events that occur during vaccination in dogs, we have attempted to evaluate the immune response of a promising new vaccines candidate against canine visceral leishmaniasis (CVL). Thirty five mongrel dogs were treated within seven experimental groups as follow: (i) control group C (n = 10) received 1 ml of sterile 0.9% saline; (ii) LB group (n = 5) received 600 ?g of Leishmania braziliensis promastigote protein in 1 ml sterile 0.9% saline; (iii) Sap group (n = 5) received 1 mg of saponin in 1 ml sterile 0.9% saline; (iv) LBSap group (n = 5) received 600 ?g of L. braziliensis promastigote protein and 1 mg of saponin in 1 ml sterile 0.9% saline; (v) Sal group (n = 5) received sand fly gland extract (SGE) prepared from 5 acini of salivary glands of Lutzomyia longipalpis in 1 mL sterile 0.9% saline; (vi) LBSal group (n = 5) received 600 ?g of L. braziliensis promastigote protein plus SGE in 1 mL sterile 0.9% saline; and (vii) the LBSapSal group (n = 5) received 600 ?g of L. braziliensis promastigote protein plus 1 mg of saponin together with SGE in 1 mL sterile 0.9% saline. Each animal received three subcutaneous injections in the right flank at intervals of 4 weeks. The results obtained indicate that some dogs exhibit local swelling or mild local induration reactions, but no ulcerated lesions or other adverse reactions, after receiving saponin as an adjuvant (Sap, LBSap or LBSapSal groups). The overall tolerance of the candidate vaccines in dogs appeared to be adequate. The evaluation of immunogenicity revealed that animals treated with LBSap and LBSapSal presented higher (P < 0.05) amounts of anti- Leishmania total IgG that were associated with increased (P < 0.05) levels of IgG1 and IgG2, suggesting a mixed Th1/Th2 immune response. Likewise, LBSapSal elicited the production of elevated levels of anti-SGE total IgG, IgG1 and IgG2, that was previously associated in establishing an anti-immune L. chagasi response in human. Additionally, anti-SGE Western blot experiments showed predominance in the recognition of seric proteins with molecular weights of 35, 45 and 71 kDa, particularly following LBSapSal vaccination, and were related to the resistance pattern against Leishmania infection. The immunophenotypic evaluation in peripheral blood mononuclear cells (PBMC) reveal in the LBSap and LBSapSal increased (P < 0.05) counts of circulating CD21+ B-cells, CD5+ T-cells and CD4+ and CD8+ T-cell subset, suggesting protective immunity against Leishmania infection as has been suggested previously for CVL. Whilst lower stimulation index by either VSA (vaccine soluble antigen) or SLcA (soluble Leishmania chagasi antigen) were recorded for the LB and Sal groups, higher cell reactivities in the presence of both stumuli were related to LBSap and LBSapSal groups. Interestingly, a negative association was demonstrated between cell reactivity and CD4+ T-lymphocytes or CD14+ monocytes following in vitro stimulation in the Sal group. These data support the hypothesis that Sal treatment inhibits CD14+ monocytes in the promotion of CD4+ T-lymphocyte activation and the induction of cell proliferation. In contrast, when in vitro cultures of peripheral blood mononuclear cells (PBMC) were stimulated with SLcA in the LBSap treatment and VSA or SLcA in the LBSapSal group, increased lymphoproliferation activity was accompanied by a higher frequency of antigen-specific CD8+ T-cells. These results support the hypothesis that induction of CD8+ T-cells may play a protective role in the mechanism of control of parasitism by Leishmania after LBSap and LBSapSal treatment associated with the antigen-specific immune response to antigens from the etiological agent of CVL. The evaluation of potential antigen presenting cells (APC) revealed increased numbers of circulating CD14+ monocytes in the LBSap and LBSapSal groups. Furthermore, the largest APC counts were associated XIII with the highest expression of MHC-I in lymphocytes in the LBSap and CD80 and MHC-I in lymphocytes in the LBSapSal, suggesting that this association could represent interaction between the innate and adaptive immune responses, reflecting an improvement in activation status during immunization. The results obtained from the analysis of nitric oxide (NO) levels (determined as nitrite) in culture supernatants stimulated by SLcA in the LBSap and in the serum of the LBSapSal confirmed the hypothesis that these vaccines induce a potential resistance profile against Leishmania infection. Our data suggested that the potential resistance profile elicited by LBSap and LBSapSal were compatible with effective control of the etiological agent of CVL

Immunity to Leishmania and the rational search for vaccines against canine leishmaniasis.

The control of infection byLeishmania infantum (syn. Leishmania chagasi) in dogs is essential to stop the current spread of zoonotic visceral leishmaniasis. The past few years have seen significant advances in achiev-ing efficient immunization of dogs and, more than ever before, an effective vaccine against canine leishmaniasis can now be considered a feasible goal. This article sum-marizes experimental data gathered from recent dog trials aimed at identifying immunological mechanisms implicated in protection against canine infection to dis-cuss their potential to serve as quantitative surrogate markers of immunization and, more importantly, its usefulness to evaluate whether the immunity induced by the vaccine candidate is strong enough to protect against canine leishmaniasis