Tratamento da leishmaniose visceral canina empregando duas abordagens terap?uticas distintas: quimioterapia com antimoniato de meglumina lipossomal e imunoterapia com anticorpo monoclonal bloqueador do receptor de IL-10.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.A leishmaniose visceral (LV) ? um grave problema de sa?de p?blica, em crescente expans?o em v?rias regi?es do mundo. O c?o dom?stico ? o principal reservat?rio urbano do parasito quando a doen?a apresenta perfil epidemiol?gico antropozoon?tico e por esta raz?o, a eutan?sia de c?es soropositivos ? empregada como medida de controle da doen?a. Assim, o tratamento da leishmaniose visceral canina (LVC) tem sido proposto como alternativa ao sacrif?cio de c?es soropositivos. Entretanto, a terap?utica convencional para a LVC tem sido discutida sob v?rios aspectos, entre eles a real compet?ncia de cura dos animais. Dessa forma, nosso estudo buscou avaliar diferentes estrat?gias de tratamento para a doen?a empregando a quimioterapia com antimoniato de meglumina lipossomal e a imunoterapia com anticorpo monoclonal bloqueador do receptor de IL-10. Nossa estrat?gia experimental contou com 20 c?es naturalmente infectados por L. infantum, divididos em dois grupos experimentais: um grupo de c?es que recebeu a quimioterapia composta por seis doses antimoniato de meglumina em lipossomas convencionais e peguilados (6,5mg de Sb5+/kg por dose) por via endovenosa lenta a cada 96h (AMLip - n=9) e o segundo grupo que recebeu a imunoterapia com anticorpo monoclonal bloqueador do receptor de IL-10 em duas doses compostas por 2 mg de cada anticorpo (contra subunidade alfa e beta) totalizando 4 mg de anticorpo por dose, por via intramuscular (Bloq IL-10R ? n=11). Os c?es foram avaliados antes do tratamento (T0) e ap?s 30 (T30), 90 (T90) e 180 (T180) dias das diferentes estrat?gias terap?uticas sob os aspectos hemato-bioqu?micos, imunol?gicos, cl?nicos e parasitol?gicos. Anterior ao tratamento, as formula??es lipossomais compostas por mistura de lipossomas convencionais - DSPC, CHOL e DCP e lipossomas peguilados - DSPC, COL, DCP e DSPE-PEG foram avaliadas e demonstram carater?sticas f?sico-qu?micas satisfat?rias do ponto de vista farmacot?cnico. Da mesma forma, foi realizado uma avalia??o in vitro, da capacidade funcional do anticorpo monoclonal bloqueador do repector de IL-10, demonstrando um aumento da prolifera??o linfocit?ria ant?geno espec?fica tanto para c?lulas T CD4+ quanto para c?lulas T CD8+, uma diminui??o de IL-4 produzido por c?lulas CD4+ al?m de um aumento na produ??o de TNF-? por c?lulas mononucleares do sangue perif?rico (CMSP). Nossos principais resultados revelaram que a quimioterapia com AMLip n?o foi efetiva em restaurar alguns par?metros hematol?gicos, ao contr?rio dos c?es tratados com Bloq Il-10R que foram capazes de restabelecer o n?mero de linf?citos. O quadro bioqu?mico, ? exce??o aos n?veis de fosfatase alcalina, n?o foi reestabelecido nos dois grupos, apesar de manuten??o dos par?metros. A an?lise do perfil imunofenot?pico sangu?neo demonstrou que c?es tratados com AMLip apresentaram aumento de linf?citos T CD3+ circulantes e das subpopula??es de linf?citos TCD4+ e CD8+ al?m de linf?citos B CD21+, c?lulas NK CD5-CD16+ e mon?citos CD14+ nos tempos iniciais p?s tratamento (T30 e T90). J? os c?es tratados com Bloq IL-10R apresentaram aumento apenas de linf?citos TCD3+CD4+ em T30 e T90. Em rela??o ?s avalia??es in vitro, ambos os tratamentos n?o foram capazes de induzir uma capacidade linfoproliferativa ant?geno espec?fica ao fim do acompanhamento experimental (T180), apesar do aumento da prolifera??o de linf?citos T CD4 em T90 ap?s a quimioterapia com AMLip e aumento da linfoprolifera??o de CD4 e CD8 ap?s imunoterapia com Bloq IL-10R em T30 e T90. De forma interessante, foi observado um aumento de linf?citos TCD4+ produtores tanto de IFN-? quanto de IL-4 ap?s estimula??o com ASLi em ambos os grupos apenas em T90 e uma redu??o na produ??o de IL-10 por CMSP em T30 no grupo Bloq IL-10R. Nossos resultados demonstraram que as estrat?gias de tratamento empregadas n?o foram capazes de induzir um perfil de resist?ncia a infec??o por Leishmania ao fim do acompanhamento experimental (T180). Em rela??o a avalia??o cl?nica, os animais submetidos tanto a quimioterapia com AMLip quanto ? imunoterapia com Bloq IL-10R n?o foram capazes de alcan?ar uma melhora de sinais/sintomas cl?nicos sugestivos de LVC al?m de n?o alcan?arem um aumento da massa corporal e redu??o de esplenomegalia ao fim dos respectivos tratamentos (T180), apesar de uma melhora nesses aspectos em T30 e T90. Somando-se a esses fatores, a quimioterapia com AMLip n?o foi eficaz em reduzir a carga parasit?ria no ba?o e pele ap?s ao fim deste regime tratamento. Por outro lado, a imunoterapia com Bloq IL-10R foi eficaz em reduzir a carga parasit?ria no ba?o, apesar da resposta imune ant?geno espec?fica e a avalia??o cl?nica n?o seguir o mesmo padr?o. Nossos resultados sugerem que uma nova interven??o terap?utica ou manuten??o das distintas terapias propostas por um tempo mais prolongado pode ser necess?rio para a ampliar a efic?cia da a??o terap?utica. Al?m disso, nossos resultados enfatizam a necessidade da associa??o entre a quimioterapia e imunoterapia (imunoquimioterapia) como uma poss?vel poderosa ferramenta na terap?utica da LVC.Visceral leishmaniasis (VL) is a serious public health issue, expanding through multiple areas of the worldwide. Domestic dog is the main urban reservoir of the parasite when disease presents an anthropozoonotic profile. Due to this fact, euthanasia of seropositive dogs is widely employed as a disease control mechanism. Thereby, treatment against canine visceral leishmaniasis (CVL) has been suggested as an alternative to sacrificing infected dogs. Morevover, conventional therapy against CVL has been discussed taking multiple variables into account, such as the real healing ability of these drugs. Therefore, our study sought to evaluate different therapeutic strategies against the disease, using chemotherapy with liposomal meglumine antimoniate and immunotherapy with monoclonal antibody anti-IL-10 receptor. Our experimental strategy included 20 naturally infected dogs with L. infantum, divided in two experimental groups: the first group was treated with chemotherapy, including six doses of meglumine antimoniate inside conventional or pegylated liposomes (6,5mg of Sb5+/kg per dose), slowly injected intravenously each 96h (AMLip - n=9) and the second group was treated with immunotherapy including two doses of monoclonal antibody anti-IL-10 receptor (2mg of antibody per dose, against ? and ? subunits) intramuscular (Bloq IL-10R ? n=11). All dogs were evaluated before treatment (T0), 30 (T30), 90 (T90) and 180 (T180) days after treatment. This follow up included haematological, biochemical, immunological, clinical and parasitological approaches. Before treatment, liposomal formulations, including a mixture of conventional - DSPC, COL and DCP and pegylated liposomes - DSPC, COL, DCP and DSPE-PEG were tested and revealed satisfactory physicochemical characteristics. Likewise, in vitro tests were performed to attest functional ability of immunotherapy with monoclonal antibody anti-IL-10 receptor, showing an increase in CD4+ and CD8+ T lymphocytes proliferation, a diminished production of IL-4 by CD4+ lymphocytes and an increase in TNF-? production by peripheral blood mononuclear cells (PBMC). Our most prominent results revealed that chemotherapy with AMLip was not able to restore haematological parameters, unlike the dogs treated with Bloq Il-10R, which were able to reinstate lymphocyte numbers. No group was able to recover biochemical parameters to normal levels, with the exception of alkaline phosphatase. Blood immunophenotypic analysis revealed that dogs treated with AMLip presented an increase of circulating CD3+ T cells lymphocyte levels, as well as higher numbers of CD4+ and CD8+ T cells. Likewise, CD21+ B lymphocytes, CD5-CD16+ NK cells and CD14+ monocytes levels were also increased, at the initial post-treatment time points (T30 and T90). On the other hand, dogs treated with Bloq IL-10R presented only an increase of CD3+CD4+ T cells at the same time points. Regarding in vitro evaluations, no treatment was able to induce CD4+ and CD8+ antigen-specific lymphoproliferative ability after 180 days (T180), despite an increase in CD4+ lymphoproliferation at T90 in dogs submitted to chemotherapy with AMLip and an increase in CD4+ and CD8+ lymphoproliferation at T30 and T90 in dogs submitted to immunotherapy with Bloq IL-10R. Interestingly, an increase of CD4+ cells producing both IFN-? and IL-4 was observed after stimulation with ASLi in both groups at T90. In contrast IL-10 production by PBMC was decreased at T30 in dogs treated with Bloq IL-10R. Our results demonstrated an inability of both treatment strategies to induce a resistance profile to infection at the end of experimental follow-up (T180). Regarding clinical evaluation, neither the group submitted to chemotherapy with AMLip nor the one submitted to immunotherapy with Bloq IL-10R were able to reach an improvement of clinical symptoms suggestive of CVL. Likewise, neither treatments were able to increase body mass and reduce splenomegaly at the end of therapeutic strategies (T180), despite improvements on these aspects at T30 and T90. Adding to that, chemotherapy with AMLip was not able to reduce parasite load at the spleen and skin at the end of the experiment (T180). On the other hand, immunotherapy with Bloq IL-10R was able to reduce splenic parasite load, which is inconsistent with our findings regarding clinical status and antigen-specific immune response. Our results suggest that a new therapeutic approach or an extension of these approaches for a longer period could be necessary to increase therapeutic effectiveness. Moreover, our results emphasize the need to associate chemotherapy and immunotherapy, as a powerful therapeutic tool against CVL

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

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

Implicações da evolução clínica e da carga parasitária em aspectos histopatológicos da pele de cães naturalmente infectados por Leishmania (Leishmania) infantum.

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.A leishmaniose visceral canina (LVC) é uma zoonose de grande impacto na saúde pública sendo a pele o principal ponto de contato de organismos do gênero Leishmania com os hospedeiros invertebrados. O objetivo desse trabalho foi avaliar a carga parasitária, o processo inflamatório e as alterações da matriz extracelular na pele de cães naturalmente infectados por Leishmania (Leishmania) infantum apresentando diferentes formas clínicas da doença. Para isso, foram utilizados 35 cães sem raça definida, provenientes da região de Belo Horizonte, MG. Esses animais foram divididos em três grupos, de acordo com a presença de sinais clínicos, como assintomáticos (n=11, CA), oligossintomáticos (n=12, CO) e sintomáticos (n=12, CS), além de oito animais controle. Posteriormente estes animais foram distribuídos em novos grupos, de acordo com a intensidade do parasitismo cutâneo, sendo classificados em baixo parasitismo (n=12, BP), médio parasitismo (n=11, MP) e alto parasitismo (n=12, AP). Após exame clínico verificou-se que os sinais clínicos mais frequentes, sugestivos de LVC, foram as dermatites (47, 2%), seguidas por linfadenopatia (36, 1%) e emagrecimento (33,3%). Quanto à carga parasitária, verificou-se que essa foi maior nos grupos CS e CO quando comparada a CA. Infiltrado inflamatório foi presente em todos os grupos clínicos, porém esse foi maior nos grupos CA, CO e CS quando comparados ao grupo controle. Ainda, a inflamação foi maior no grupo CS quando comparado aos grupos CA e CO. Ao avaliar esse mesmo parâmetro nos grupos com distintos graus de parasitismo, foi verificado maior inflamação em cães com BP e MP quando comparados aos cães controle. Além disso, cães com MP e AP possuíam maior processo inflamatório que cães com BP. A avaliação da matriz extracelular demonstrou redução de colágeno total nos grupos infectados quando comparados ao grupo controle e nos grupos CO e CS em relação ao grupo CA. Animais com AP e MP apresentaram diminuição da área de colágeno em relação a animais controle. Na quantificação das fibras colágenas do tipo I observou-se redução nos grupos CO e CS quando comparados ao grupo controle e diminuição da mesma nos cães com MP em relação ao grupo controle. Ainda, em relação às fibras colágenas do tipo III foi observado um aumento significativo no grupo CO em relação aos grupos controle e CS e aumento em cães com BP quando comparado a cães controle e com MP. Esses resultados sugerem que a inflamação crônica e o intenso parasitismo dérmico foram diretamente relacionados à gravidade da doença e que esse processo inflamatório está intimamente associado à carga parasitária e as alterações da matriz extracelular desse órgão.Canine visceral leishmaniasis (CVL) is a zoonosis of major public health impact and the skin the main point of contact of organisms of the genus Leishmania with invertebrate hosts. Based on this, the aim of this study was evaluate the parasite load, inflammation and the matrix cellular alterations in the ear skin of dogs naturally infected with Leishmania (Leishmania) infantum with different clinical forms and different intensities of cutaneous parasitism. For that, thirty five dogs naturally infected with Leishmania, mongrels, from the Belo Horizonte, MG were categorized as asymptomatic (n=11), oligosymptomatic (n=12) and symptomatic dogs (n=12) and these were compared to control dogs (n = 8). Later were divided into new groups, according to three different parasites density: low (n=12), medium (n=11) and high parasitism (n=12). The major clinical manifestations of visceral leishmaniasis in dogs were dermatitis (47,2%), lymphadenopathy (36,1%) and weight loss (33,3%). Inflammatory infiltrates were observed in all groups, varying from intense and/or moderate in symptomatic to discrete in asymptomatic and control animals. Moreover, the inflammation was higher in symptomatic dogs when compared to oligosymptomatic and asymptomatic dogs. In assessing this parameter in groups with different degrees of parasitism, greater inflammation was observed in dogs with low, medium and high parasitism when compared to control dogs. In addition, dogs with medium and high parasitism showed higher inflammatory process those dogs with low parasitism. The mast cells’ number was higher in oligosymptomatic dogs compared to control dogs and was higher in dogs with low and medium parasitism when compared to control group. Extracellular matrix assessment demonstrated decrease in the collagen area in all infected groups when compared to control dogs and in symptomatic and oligosymptomatic dogs compared to asymptomatic dogs. Moreover, dogs with high and medium parasitism showed a decrease in collagen area relative to control animals. Regarding collagen type I there was only a significant dicrease in symptomatic and oligosymptomatic dogs compared to the group of uninfected dogs and decrease in dogs with medium parasitism compared to the group of uninfected dogs. Furthermore, it was verified increase in collagen type III in oligosymptomatic dogs compared to control group and symptomatic dogs and increase in dogs with low parasitism in relation the group of uninfected dogs and medium parasitism dogs. The results suggested that chronic dermal inflammation and cutaneous parasitism were directly related to the severity of clinical disease and that this inflammation is closely associated with the parasite load and changes in the extracellular matrix of the skin

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

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

The Use of an Adjuvant System Improves Innate and Adaptive Immune Response When Associated with a <i>Leishmania</i> (<i>Viannia</i>) <i>braziliensis</i> Antigen in a Vaccine Candidate against <i>L.</i> (<i>Leishmania</i>) <i>infantum</i> Infection

Background: The adjuvants’ optimal dose and the administration route can directly influence the epitope recognition patterns and profiles of innate response. We aimed to establish the effect and the optimal dose of adjuvant systems for proposing a vaccine candidate to be employed with Leishmania (Viannia) braziliensis. Methods: We evaluated the adjuvants saponin (SAP), monophosphoryl lipid A (MPL) and resiquimod (R-848) isolated and combined as adjuvant systems in a lower dose corresponding to 25%, 33%, and 50% of each adjuvant total dose. Male outbred BALB/c mice were divided into 13 groups, SAP, MPL, and R-848 isolated, and the adjuvant systems SAP plus MPL (SM), SAP plus R-848 (SR), and MPL plus R-848 (MR). Results: SM50 increased levels of all chemokines analyzed and TNF production, while it presented an increased inflammatory cell infiltrate in the skin with macrophage recruitment. Thus, we proposed a vaccine candidate employing L. (V.) braziliensis antigen associated with the SM adjuvant system against experimental L. (Leishmania) infantum challenge. We observed a significant increase in the frequency of cells expressing the central and effector memory CD4+ T cells phenotype in immunized mice with the LBSM50. In the liver, there was a decreased parasite load when mice received LBSM50. Conclusions: When combined with L. (V.) braziliensis antigen, SM50 increases TNF and IFN-γ, which generates central and effector memory CD4+ T cells. Therefore, using an adjuvant system can promote an effective innate immune response with the potential to compose future vaccines

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-&#947; by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host