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

Synthetic peptides elicit strong cellular immunity in Visceral Leishmaniasis natural reservoir and contribute to long-lasting polyfunctional T-cells in BALB/c mice.

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

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

Prevalence and Factors Associated with Leishmania infantum Infection of Dogs from an Urban Area of Brazil as Identified by Molecular Methods

Visceral leishmaniasis (VL) is a disease caused by the parasite Leishmania infantum, and dogs are the most important domestic reservoirs of the agent. During recent decades, VL has expanded to large Brazilian urban centers. In the present work, we have demonstrated by using molecular techniques that the rate of canine infection as detected by serology has been considerably underestimated. Two groups of seronegative dogs (infected and non-infected according to molecular methods) were further evaluated from data obtained through interviews with owners of the animals. The factors associated with Leishmania infection in dogs were a family income of less than two minimum salaries, the knowledge of the owner regarding the vector, the dog spending most of its time in the backyard and the dog never having had a previous serological examination. Awareness regarding the factors associated with canine infection will improve health services and the understanding of the disease's expansion in urban areas

Imunogenicidade e eficácia da vacina LBSap em cães após desafio intradérmico com Leishmania chagasi e extrato de glândula salivar de Lutzomyia longipalpis.

Medidas de controle na infecção em cães por L. chagasi/L. infantum são fundamentais para interromper o avanço atual da leishmaniose visceral humana. Recentemente têm sido desenvolvidos inúmeros candidatos a vacinas contra leishmaniose visceral canina (LVC), sendo que, a compreensão dos mecanismos imunes envolvidos na imunogenicidade pós vacinação e pós desafio são cruciais para definir um padrão de resposta associado à proteção contra infecção por Leishmania. Assim, neste trabalho foi investigada a imunogenicidade e o efeito protetor da vacina LBSap em cães após o desafio intradérmico com L. chagasi. Vinte cães sem raça definida foram subdivididos em quatro grupos experimentais: i) controle (n = 5) receberam 1 mL de solução salina estéril 0,9%, ii) Sap (n = 5) receberam 1 mg de saponina em 1 mL de solução salina estéril 0,9%; iii) LB (n = 5) receberam 600 g de proteína de L. braziliensis em 1mL de solução salina estéril 0,9%; iv) LBSap (n = 5) receberam 600 g de proteína de L. braziliensis associado a 1mg de saponina em 1mL de solução salina estéril 0,9%. O protocolo vacinal constou de três imunizações pela via subcutânea em intervalos de 4 semanas. Posteriormente, os cães foram infectados por via intradérmica com 1 × 107 promastigotas de Leishmania (L.) chagasi em fase estacionária de crescimento associado a extrato de glândula salivar de Lutzomyia longipalpis e acompanhados por um período de 435 dias pós desafio (435Dpd). A análise da resposta imune humoral mostrou que antes e durante o acompanhamento longitudinal após o desafio, o grupo LBSap mostrou aumento dos níveis de IgG total, IgG1 e IgG2, sugerindo uma possível resposta imune mista (tipo I e II). A análise inicial da resposta imune celular revelou aumento da freqüência de linfócitos e monócitos no grupo LBSap. A Avaliação do perfil imunofenotípico de células mononucleares do sangue periférico (CMSP) mostrou aumento no número absoluto de linfócitos T CD4+ e linfócitos B CD21+ , bem como contagens elevadas de monócitos CD14+ circulantes no grupo LBSap. Além disso, elevados níveis de linfócitos T CD5+ e LT CD8+ circulantes, foram diretamente correlacionados com a alta expressão de MHC-II em linfócitos no grupo LBSap. Estes resultados mostram que cães imunizados com a vacina LBSap apresentam variações em diversos biomarcadores imunológicos criando um padrão de memória imunológica favorável a uma vacina protetora através de uma maior capacidade na apresentação de antígenos e ativação celular. Além disso, o grupo de cães vacinados com LBSap apresentaram aumento da atividade linfoproliferativa in vitro após estímulo antigênico com VSA (antígeno solúvel vacinal) ou SLcA (antígeno solúvel de L. chagasi), bem como aumento na frequência de linfócitos T CD4+ . O perfil de ativação linfocitária in vitro foi marcado pelo aumento da expressão de linfócitos SLcA-MHC-II específicos ao lado de uma correlação positiva entre as células T CD4+ -SLcA/VSA e a expressão de MHC-II em linfócitos no grupo LBSap. A análise clínica de possíveis alterações sugestivas de LVC revelou que todos os animais permaneceram assintomáticos durante o período de acompanhamento (435Dpd). Finalmente, o estudo da investigação parasitológica realizada através da análise de amostras de medula óssea em cultura (parasitológico em NNN/LIT e análise de esfregaços por microscopia) além de abordagens moleculares (PCR), demonstrou somente PCR+ em um cão do grupo Sap no final do acompanhamento longitudinal (435Dpd). Assim, os resultados obtidos neste estudo indicam que a vacina LBSap induz um perfil de resposta imune compatível com proteção frente ao agente etiológico da LVC. Estes resultados indicam que a via intradérmica de infecção experimental apresenta um curso longo de duração para iniciar as manifestações clínicas e consequentemente a detecção do parasito, necessitando, portanto de um período maior de acompanhamento.The control of L. chagasi/L. infantum infection in dogs is essential to interrupt the current spread of human visceral leishmaniasis. Most recently, various potentials candidates to vaccines against canine visceral leishmaniasis (CVL) have been developed. To obtain a successful vaccine, the understanding of those immunogenicity mechanisms involved after vaccination and challenge is crucial to define the protective responses associated against Leishmania infection. Thus, in this work it was investigated the immunogenicity and protective effect of LBSap vaccination in dogs after challenge. Twenty mongrel dogs were categorized into four groups: i) control (n = 5) received 1 mL of sterile saline 0.9%; ii) Sap (n = 5) received 1 mg of saponin in 1 mL of sterile saline 0.9%; iii) LB (n = 5) received 600 g of L. braziliensis promastigote protein in 1ml sterile 0.9% saline; iv) LBSap (n = 5) received 600 g of L. braziliensis promastigote protein plus 1mg of saponin in 1ml sterile 0.9% saline. The immunization protocol consisted of three subcutaneous injections at intervals of 4 weeks. Afterwards, the dogs were intradermally infected with 1×107 late-log-phase promastigotes of Leishmania (L.) chagasi in the presence of sand fly saliva of Lutzomyia longipalpis and accompanied by a follow-up of 435 days post challenge (435dpc). Analysis of the humoral immune response showed that before and during the longitudinal follow-up after the challenge, the LBSap group showed increased levels of total IgG, IgG1 and IgG2, suggesting a possible mixed immune response (type I and II) after vaccination. Analysis of the cellular immune response revealed increased frequency of lymphocytes and monocytes in the LBSap group. The investigation based on the immunophenotypic profile of peripheral blood mononuclear cells (PBMC) showed an augment in the absolute number of both CD4+ and CD21+ lymphocytes as well as higher counts of circulating CD14+ monocytes in the LBSap group. Moreover, high counts of circulating CD5+ T and CD8+ T lymphocytes were directly correlated with high expression of MHC-II on lymphocytes in the LBSap group. These findings show that LBSap immunized dogs exhibit changes in several immunological biomarkers that may create a pattern of immunological memory favorable to an effective vaccine through a higher ability in antigen presentation and cell activation. In addition, the LBSap group exhibited increased lymphoproliferative activity upon in vitro antigenic stimuli with VSA (vaccine soluble antigen) or SLcA (soluble L. chagasi antigen) as well as increaded frequency of CD4+ -lymphocytes. The in vitro profile of lymphocytic activation was marked by increased SLcA-specific MHC-II-expression alongside a positive correlation between CD4+ T-SLcA/VSA and MHC-II expression in the LBSap group. The clinical evaluation regarding suggestive signs of CVL showed that all animals remained asymptomatic during the all follow-up. Finally, the protection study performed by analysis of bone marrow samples by parasitological (culture in NNN/LIT medium and microscopical investigation) and molecular approaches (PCR) revealed positivity in PCR+ in only one dog of Sap group at the end of the follow-up (435dpc). Thus, this study indicated that the LBSap vaccine presents a profile consistent with immune protection against the etiological agent of CVL. These results showed that the intradermal route of experimental infection has a long course to start the clinical manifestations and therefore the detection of the parasite, and therefore requiring a longer period of follow-up