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

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

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

Ensaio clínico vacinal de fase I e II para avaliação comparativa da toxicidade, imunogenicidade e potência das vacinas Leishmune®, Leish- Tec®, KMP-11 e LBSap contra leishmaniose visceral canina.

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto.O presente estudo avaliou a toxicidade/inocuidade, imunogenicidade e eficácia/potência dos protótipos vacinas KMP-11 e LBSap, de forma comparativa com as vacinas comerciais Leishmune® e Leish-Tec®, em um ensaio clínico vacinal de fase I e II. Para isso, trinta e cinco cães foram classificados em cinco grupos (sete cães por grupo): i) grupo controle receberam 1 mL de solução salina estéril 0,9%; ii) grupo Leish-Tec receberam a vacina comercial Leish-Tec®; iii) Grupo Leishmune receberam a vacina comercial Leishmune®; (iv) grupo LBSap recebeu 600 μg de proteína de promastigotas de L. braziliensis e 1 mg do adjuvante saponina; (v) grupo KMP-11 receberam 100 μg do antígeno recombinante KMP-11 associado a 1 mg do adjuvante saponina. Uma análise detalhada e comparativa da inocuidade/toxicidade vacinal foi realizada através do quadro clínico, bioquímico e hematológico entre as doses de imunização e após o término das três doses vacinais. Embora em alguns cães dos grupos KMP-11, LBSap e Leishmune tenham apresentado alterações no local dos inóculos vacinais, relacionados ao adjuvante saponina, como: nódulos, edema leve, dor local, estes foram transientes e desapareceram após setenta duas horas da vacinação. Nossos resultados de inocuidade indicam que as alterações adversas provocadas pelas imunizações são toleráveis, tendo em vista a importância e funcionalidade que uma vacina canina eficaz promoveria no controle da doença. Nossos resultados da imunogenicidade vacinal demonstram um aumento da população circulante de linfócitos T CD8+ ao final do protocolo vacinal (T1) nos grupos LBSap e Leish-Tec, além de seis meses após o desafio experimental (T2) no grupo LBSap. Também foi observado em T1 aumento de linfócitos B (grupo Leishmune) e de monócitos CD14+ (grupos LBSap, Leishmune e KMP-11), que justificam e reforçam o potencial imunoprofilático destas vacinas. Nas análises in vitro foi observado aumento na atividade linfoproliferativa nos grupos LBSap e Leishmune, sendo no grupo LBSap um aumento antígeno-específico de linfócitos TCD4+ e CD8+. Uma segunda abordagem das análises in vitro buscou avaliar o percentual de células T CD4+ e CD8+ antígeno-específicas produtoras de IFN- e IL-4, onde foi observado no grupo LBSap aumento de ambas as subpopulações produtoras de IFN-, sendo também evidenciado um aumento de T CD8+ produtores de IFN- no grupo Leish-Tec. Nosso resultados de imunogenicidade reforçam a hipótese que o processo vacinal, principalmente, com a vacina LBSap levam a geração de uma resposta imune protetora contra o agente etiológico da LVC compatível com o controle do parasito de L. infantum. Nossos resultados da reatividade sorológicos demonstraram que o TR DPP® foi capaz de distinguir os cães doentes dos vacinados. Após o desafio experimental pela via endovenosa os cães foram acompanhados por 6 meses e foram evidenciadas alterações clínicas sugestivas da infecção por Leishmania, entretanto, na maioria dos cães foi observado uma infecção assintomática. Na avaliação parasitológica da medula óssea foi possível isolar o parasito (mielocultura) bem como quantificar o DNA (qPCR) de Leishmania em todos os grupos, sendo observado redução considerável da carga parasitária da medula óssea em todas as vacinas testadas em relação ao grupo Controle. Entretanto, no grupo de cães imunizados com LBSap esta redução foi mais evidente, chegando a ser 47 vezes menor. Com base no que foi exposto, a vacina LBSap seria a mais indicada para prosseguimento em ensaio clínico vacinal de fase III, em relação a vacina KMP-11.This study evaluated the toxicity/safety, immunogenicity and efficacy/potency of vaccines prototypes KMP-11 and LBSap, in comparison to the commercial vaccines Leishmune® and Leish-Tec®, in a vaccine clinical trial phase I and II. For this, thirty-five dogs were classified into five groups (seven dogs per group): i) control group received 1 mL of sterile 0.9% saline solution; ii) Leish-Tec group received the Leish-Tec® commercial vaccine; iii) Leishmune group received the Leishmune® commercial vaccine; (iv) LBSap groups received 600 μg of L. braziliensis promastigotes protein and 1 mg of saponin adjuvant; (v) KMP-11 group (n=7) received 100 μg recombinant KMP-11 antigen associated to 1 mg saponin adjuvant. A detailed and comparative analysis of safety/toxicity vaccination was performed by clinical, biochemical and hematological parameters between dose and immunization after the end of the three vaccine doses. Although some groups of dogs KMP-11, LBSap and Leishmune have presented changes at the site of vaccination inoculum, related to saponin adjuvant, such as nodules, mild edema, and local pain, these were transient and disappeared seventy two hours after vaccination. Our results indicate that the safety of adverse changes caused by immunizations is tolerable in view of the effective canine vaccine importance and functionality that it promotes in the disease control. Our results of the immunogenicity vaccine demonstrate increase in circulating population of T CD8+ lymphocytes in the end of the immunization protocol (T1) in groups LBSap and Leish-Tec, as long as six months after experimental challenge (T2) in LBSap group. It was also observed in T1, an increase of B lymphocytes (Leishmune group) and monocytes CD14+ (LBSap, Leishmune and KMP-11 groups), that justify and reinforce the potential immunoprophylactic of these vaccines. In the in vitro analyzes an increase in lymphoproliferative activity in groups LBSap and Leishmune was observed, occurring in LBSap group an antigen-specific increase of CD4+ and CD8+ T-lymphocytes. A second approach of in vitro assays aimed to evaluating the percentage of antigen-specific CD4+ and CD8+ lymphocytes producers of IFN- e IL-4, where an increase in both IFN- producing subpopulations in the group LBSap was observed, and it also showed an increase in IFN- producers in CD8+ lymphocytes in the Leish-Tec group. Our immunogenicity results support the hypothesis of the vaccine process, especially with the LBSap vaccine generating a protective immune response against the causative agent of CVL compatible with L. infantum parasite control. Our results of serological reactivity demonstrate that TR DPP® was able to distinguish among diseased and vaccinated dogs. After experimental challenge by intravenous route the dogs were followed for 6 months and clinical changes suggestive of Leishmania infection were observed, however in the majority of dogs asymptomatic infection was observed. In the parasitological evaluation of bone marrow it was possible to isolate the parasite (myeloculture) and quantify the DNA (qPCR) of Leishmania in all groups, significant reduction in parasite burden in the bone marrow was observed in all vaccines tested compared to control group. However, in the group of dogs immunized with LBSap this reduction was more evident, being 47 times smaller. Based on the foregoing, the LBSap vaccine would be the most suitable for further research in phase III clinical trial vaccine in relation to KMP-11 vaccine

Phase I and II Clinical Trial Comparing the LBSap, Leishmune®, and Leish-Tec® Vaccines against Canine Visceral Leishmaniasis

In this study, we performed a phase I and II clinical trial in dogs to evaluate the toxicity and immunogenicity of LBSap-vaccine prototype, in comparison to Leishmune® and Leish-Tec® vaccines. Twenty-eight dogs were classified in four groups: (i) control group received 1 mL of sterile 0.9% saline solution; (ii) LBSap group received 600 μg of Leishmania braziliensis promastigotes protein and 1 mg of saponin adjuvant; (iii) Leishmune®; and (iv) Leish-Tec®. The safety and toxicity of the vaccines were measured before and after three immunizations by clinical, biochemical, and hematological parameters. The clinical examinations revealed that some dogs of LBSap and Leishmune® groups presented changes at the site of vaccination inoculum, such as nodules, mild edema, and local pain, which were transient and disappeared seventy-two hours after vaccination, but these results indicate that adverse changes caused by the immunizations are tolerable. The immunogenicity results demonstrate an increase of B lymphocytes CD21+ regarding the Leishmune® group and monocytes CD14+ concerning LBSap and Leishmune® groups. In the in vitro analyses, an increase in lymphoproliferative activity in LBSap and Leishmune® groups was observed, with an increase of antigen-specific CD4+ and CD8+ T lymphocytes in the LBSap group. A second approach of in vitro assays aimed at evaluating the percentage of antigen-specific CD4+ and CD8+ T lymphocytes producers of IFN-γ and IL-4, where an increase in both IFN-γ producing subpopulations in the LBSap group was observed, also showed an increase in IFN-γ producers in CD8+ lymphocytes in the Leish-Tec® group. Our data regarding immunogenicity indicate that the vaccination process, especially with the LBSap vaccine, generated a protective immune response compatible with L. infantum parasite control. Based on the foregoing, the LBSap vaccine would be suitable for further studies of phase III clinical trial in endemic areas with high prevalence and incidence of canine visceral leishmaniasis (VL) cases