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

Cin?tica da resposta imune inata induzida por diferentes adjuvantes vacinais.

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.Adjuvantes s?o subst?ncias que atuam na resposta imune inata e quando combinadas a vacinas com ant?genos espec?ficos, s?o capazes de produzir uma resposta mais intensa ao ant?geno alvo. Adjuvantes atuam de v?rias formas para apresentar um ant?geno para o sistema imune. Podem atuar como um sistema de dep?sito para ant?genos, apresentando o ant?geno durante um longo per?odo de tempo. Al?m disso, essas subst?ncias s?o extremamente atrativas para o desenvolvimento de novas formula??es vacinais contra v?rias doen?as como a leishmaniose visceral. Assim, o objetivo desse estudo foi avaliar a resposta imune induzida por uma dose ?nica dos adjuvantes: Hidr?xido de alum?nio (Al(OH) 3), Adjuvante Completo de Freund (ACF), Montanide Pet Gel A (MPGA), glicopiranosil lip?dio A (GLA-SE) e Resiquimod (R-848) inoculados na pele de camundongos. Animais sensibilizados com salina foram utilizados como grupo controle. Os dados obtidos ap?s a sensibiliza??o com os adjuvantes demonstraram que todos adjuvantes foram capazes de promover altera??es hematol?gicas nos leuc?citos totais, bem como nas diferentes subpopula??es. A an?lise fenot?pica do sangue perif?rico mostrou que os animais imunizados com o adjuvante ACF (12h), GLA-SE e R-848 (24h) tiveram uma redu??o na subpopula??o de linf?citos T CD4+, enquanto que os adjuvantes R-848 (12h) e MPGA (24h) apresentaram uma redu??o inicial de linf?citos T CD8+. A popula??o de linf?citos B CD19+ tamb?m foi avaliada, na qual foi poss?vel observar altera??es em todos os grupos, contudo, os animais sensibilizados com os adjuvantes Al(OH)3 e GLA-SE apresentaram um aumento inicial em 24h. Em rela??o ?s c?lulas NK CD49b+, demonstraram distintos perfis nas diferentes imuniza??es com os adjuvantes, onde os animais dois grupos Al(OH)3 e R-848 reduziram nos tempos de 24h e 12h respectivamente. Os animais dos outros grupos apresentaram um aumento tardio: ACF (96h), MPGA (168h) e GLA-SE (168h). A popula??o de mon?citos CD14+ tamb?m foi avaliada, e os resultados mostraram que todos os adjuvantes, com a exce??o do MPGA (96h) apresentaram um aumento recente. A avalia??o dos n?veis s?ricos de ?xido n?trico induzido pelos distintos adjuvantes foi realizada nesse trabalho. Nossos resultados demonstram que os adjuvantes Al(OH)3 e ACF n?o foram capazes de induzir produ??o de NO, em contraste com os outros adjuvantes que mostraram um aumento em 1h e 12h (R-848), 48h (GLA-SE) e 168h e 336h (MPGA). O estudo histomorfol?gico foi realizado no local da imuniza??o para avaliar a habilidade dos adjuvantes promover recrutamento celular. Os animais inoculados com o adjuvante Al(OH)3 n?o apresentaram diferen?as entre os tempos da cin?tica, todavia, difere do grupo controle em toda an?lise. Os outros grupos demonstraram aumento j? nos tempos iniciais. Os animais inoculados com o adjuvante ACF mostraram um aumento no recrutamento entre os tempos de 12h ? 48h. Os animais inoculados com o adjuvante MPGA mostrou aumento na migra??o a partir de 48h, o grupo GLA-SE mostrou aumento entre os temos de 12h e 168h e finalmente os animais inoculados com R-848 mostraram grande recrutamento nos tempos de 1h, 24h e 48h. Assim, nossos dados demonstraram que os adjuvantes vacinais induziram recrutamento celular para o local do in?culo bem como mudan?as nas c?lulas presentes no sangue perif?rico. Contudo, essas altera??es apareceram em tempos distintos, provavelmente devido ao fato de eles terem diferentes constitui??es e, portanto, perfis de respostas imunes distintas que devem ser consideradas na sua sele??o como adjuvantes vacinais. Esses dados nos estimulam a dar prosseguimento nos estudos a fim de compreender melhor esses mecanismos para poder implementar em futuras formula??es vacinais seguras e efetivas.Adjuvants are substances that act in the innate immune response and when combined to vaccines with specific antigens, are capable of producing a more intense response to the target antigen. Adjuvants can act in various ways to present an antigen to the immune system. Can act as a depot system for the antigen, presenting the antigen over a long period of time. Therefore, these substances are extremely attractive for the development of new vaccine formulations against various diseases such as visceral leishmaniasis. Thus, the aim of this study was to evaluate the immune response induced by a single dose of the adjuvants: aluminum hydroxide (Al(OH)3), Freund's Complete Adjuvant (FCA), Montanide Pet Gel A (MPGA), glucopyranosyl lipid A (GLA-SE) and Resiquimod (R-848) applied to the skin of mice. Animals sensitized with saline were used as control group. The data obtained after sensitization with adjuvant demonstrated that all adjuvants were able to promote hematological alterations in total leukocytes levels, as well as in different subpopulations. Phenotypic analysis of peripheral blood showed that the animals immunized with adjuvant CFA (12h), GLA-SE and R-848 (24h) had a reduction in the subpopulation of CD4+ T lymphocytes, whereas the adjuvants R-848 (12h), and MPGA (24h) presented initial reduction of CD8+ T lymphocytes. The population of CD19+ B lymphocytes was also evaluated, in which it was possible to observe changes in all groups, however, the animals sensitized with adjuvant Al(OH)3 and GLA-SE presented an increase in recent time 24h. Regarding CD49b+ NK cells, distinct profiles in different immunizations with adjuvant were shown, where the animals of groups Al(OH)3 and R-848 were reduced in 24h and 12h respectively. The animals of the other groups showed an increase in late times: CFA (96h), MPGA (168h) and GLA-SE (168h). The population of CD14+ monocytes was also evaluated, and the results showed that all adjuvants except MPGA (96h) presented a recent increase. Evaluation of serum levels of nitric oxide induced by different adjuvants was made in this work. Our results showed that the adjuvants Al(OH)3 and CFA were not able to induce NO production, in contrast to the other adjuvants which showed an increase in 1h and 12h (R-848), 48h (GLA-SE) and 168h and 336h (MPGA). A histomorphological study was conducted at the site of immunization to evaluate the ability of adjuvants to induce promoting cell recruitment. The animals inoculated with adjuvant Al(OH)3 showed no difference in recruitment between the times of the kinetics, however, differ in the control group throughout the analysis. The other groups showed increases already in the early times. The animals inoculated with adjuvant CFA showed an increase in recruitment between the times of 12h to 48h. The animals inoculated with adjuvant MPGA showed increased migration from 48h, the GLA-SE group showed an increase between the times of 12h to 168h and finally the animals inoculated with R-848 showed greater recruitment in times of 1h, 24h and 48h. Thus, our data demonstrate that the adjuvant vaccine induced cellular recruitment to the site of inoculation as well as changes in the cells present in peripheral blood. However, these changes appeared at different times, probably due to the fact that they have different constitutions and hence different ways to induce immune responses that should be considered in their selection as vaccine adjuvants. These data encouraged us to continue studies to better understand these mechanisms in order to implement in future safe and effective vaccine formulations

A contribui??o de sistemas de associa??o de adjuvantes no aumento da efic?cia vacinal de dois novos imunobiol?gicos contra a infec??o experimental por Leishmania infantum em camundongos Balb/c.

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 humana (LVH) e a leishmaniose visceral canina (LVC) s?o as mais relevantes doen?as emergentes com alta preval?ncia na Am?rica Latina, sobretudo no Brasil. Apesar disso, ainda s?o limitados os avan?os obtidos tanto no tratamento da doen?a quanto no desenvolvimento de vacinas com intuito de conter sua expans?o. Em se tratando da busca por vacinas mais eficazes, os adjuvantes vacinais surgem como aditivos importantes, capazes de aumentar a imunogenicidade do ant?geno ao induzir uma resposta imune intensa e prolongada. Novas abordagens t?m testado sistemas de duas ou mais associa??es de adjuvantes na busca por uma resposta mais r?pida e efetiva em compara??o ao emprego dos adjuvantes isolados. Dessa forma, o objetivo desta tese foi avaliar a contribui??o de sistemas de associa??o de adjuvantes no aumento da efic?cia vacinal de novos imunobiol?gicos contra a infec??o experimental por L.infantum em camundongos Balb/c. Para tanto, o estudo foi dividido em duas estapas. Primeiro, avaliamos os adjuvantes Saponina (SAP), Resiquimod (R-848) e Monofosforil Lip?dio A (MPL) combinados em sistemas de associa??o duplas e tripla, nas doses de 25%, 33% e 50% referente a dose total de cada adjuvante, a fim de estabelecer o melhor sistema bem como sua dose ideal. Animais inoculados com solu??o salina foram utilizados como grupo controle e todos experimentos foram conduzidos em duplicata. Os animais foram divididos em 16 grupos experimentais (n=6/grupo), Controle Salina (CS), Saponina (SAP), Monofosforil Lip?dio A (MPL), Resquimod (R-848), e as associa??es SAP+MPL (SM), SAP+R-848 (SR), MPL+R-848 (MR) e SAP+MPL+R-848 (SMR), nas doses supracitadas. Os animais receberam uma dose ?nica pela via intrad?rmica e passadas 48 horas foram eutanasiados para obten??o do fragmento de pele do local do in?culo. Metade do fragmento foi destinado para dosagem de quimiocinas (CCL2, CCL3, CCL4, CCL5 e CXCL1) e citocinas (IL-2, IFN-?, TNF-?, IL-4 e IL-10) por Cytometric Bead Array (CBA) e a outra metade para avalia??es histol?gicas. Dentre todos os sistemas avaliados, SM50 levou ao aumento da produ??o de todas as quimiocinas e da citocina TNF-?, enquanto SMR50 aumentou os n?veis das quimiocinas CXCL1, CCL2 e CCL5 e citocinas TNF-? e IFN-? quando comparadas ao grupo controle. Al?m disso, SM50 apresentou aumento do infiltrado inflamat?rio, com aumento do percentual da popula??o de macr?fagos, enquanto que SMR50 tamb?m apresentou aumento no percentual de macr?fagos. Ap?s estes resultados, ambos os sistemas foram escolhidos para serem associados com o ant?geno total de Leishmania braziliensis para compor duas novas vacinas na etapa 2, nas quais foi avaliado o efeito do sistema de associa??o de adjuvantes sobre a imunogenicidade e efic?cia vacinal. Camundongos Balb/c foram divididos em 6 grupos experimentais (n=5/grupo), Controle Salina (CS), grupo imunizado com ant?geno total de L. braziliensis (LB), os sistemas de associa??o dupla e tripla de adjuvantes, SAP+MPL e SAP+MPL+R-848, ambos nas doses de 50%, e outros dois grupos que combinavam o ant?geno total de L. braziliensis com os sistemas de associa??o de adjuvantes (LBSM50 e LBSMR50). Esses foram imunizados com tr?s doses, com intervalo de 15 dias entre as doses, e desafiados com 1 x 107 promastigotas de L. infantum. Ap?s 28 dias, os animais foram necropsiados, o ba?o foi coletado para avaliar a prolifera??o celular, produ??o de IL-2, TNF-? e INF-?, forma??o de c?lulas T de mem?ria efetora e central, al?m da avalia??o da carga parasit?ria tanto no ba?o quanto no f?gado. Observou-se que o sistema LBSMR50 promoveu aumento na produ??o de TNF-? por c?lulas TCD4+, entretanto sua principal a??o foi sobre c?lulas TCD8+, promovendo aumento da prolifera??o celular, produ??o de TNF-? e forma??o de c?lulas de mem?ria efetora. Todavia, ambas as vacinas foram capazes de reduzir a carga parasit?ria no f?gado. Diante do exposto, conclu?mos que o emprego de sistemas de adjuvantes utilizando doses reduzidas induzem uma resposta eficaz, pois uma vez que combinados com o ant?geno promoveram n?o apenas ativa??o do sistema imune no ba?o como tamb?m a redu??o da carga parasit?ria no f?gado, sugerindo que o uso desses sistemas aumentaram a efic?cia vacinal no modelo murino.Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most relevant emerging diseases with high prevalence in Latin America, especially in Brazil. Despite this, the advances obtained both in the treatment of the disease and in the development of vaccines in order to contain its expansion are still limited. In regard to the search for more effective vaccines, vaccine adjuvants appear as important additives, capable of increasing the immunogenicity of the antigen by inducing an intense and prolonged immune response. New approaches have tested systems of two or more adjuvant combinations in the search for a faster and more effective response compared to the use of adjuvants alone. Thus, the aim of this thesis was to evaluate the contribution of adjuvant combination systems in increasing the vaccine efficacy of new immunobiologicals against experimental L.infantum infection in Balb/c mice. Therefore, the study was divided into two stages. First, we evaluated the adjuvants Saponin (SAP), Resiquimod (R-848) and Monophosphoryl Lipid A (MPL) combined in double and triple combination systems at the doses of 25%, 33% and 50% relative to the total dose of each adjuvant , in order to establish the best system as well as its optimal dose. Animals inoculated with saline solution were used as control group and all experiments were conducted in duplicate. The animals were divided into 16 experimental groups (n = 6 / group), Saline Control (CS), Saponin (SAP), Monophosphoryl Lipid A (MPL), Resquimod (R-848) , SAP + R-848 (SR), MPL + R-848 (MR) and SAP + MPL + R-848 (SMR) at the above-mentioned doses. The animals received a single dose via the intradermal route and after 48 hours were euthanized to obtain the skin fragment from the inoculum site. Half of fragment was used to chemokines evaluation (CCL2, CCL3, CCL4, CCL5 e CXCL1) and cytokines (IL-2, IFN-?, TNF-?, IL-4 e IL-10) by Cytometric Bead Array (CBA) and the other half for histological evaluations. Among all evaluated systems, SM50 led to increased production of all chemokines and cytokine TNF-?, while SMR50 increased levels of chemokines CXCL1, CCL2 and CCL5 and cytokines TNF-? e IFN-? when compared to the control group. In addition, SM50 presented an increase in inflammatory infiltrate, with an increase in the percentage of the macrophages population, while SMR50 also showed an increase in the percentage of macrophages. After these results, both systems were chosen to be associated with the total Leishmania braziliensis antigen to compose two new vaccines in step 2, in which the effect of the adjuvant association system on immunogenicity and vaccine efficacy was evaluated. Balb / c mice were divided into 6 experimental groups (n=5 / group), Saline Control (CS), immunized with L. braziliensis total antigen (LB), double and triple combination of adjuvants, SAP + MPL and SAP + MPL + R-848, both at 50% doses, and two other groups that combined total L. braziliensis antigen with adjuvant combination systems (LBSM50 and LBSMR50). These were immunized with three doses, at intervals of 15 days between doses, and challenged with 1 x 107 promastigotes of L. infantum. After 28 days, the animals were necropsied, the spleen was collected to evaluate cell proliferation, production of IL-2, TNF-? e INF-?, effector and central T cell memory formation as well as evaluation of the parasite load both in the spleen and in the liver. t was observed that the LBSMR50 system promoted an increase in the production of TNF-? by TCD4+ cells, but its main action was on TCD8+ cells, T cells, promoting increased cell proliferation, TNF-? production and effector memory cells formation. However, both vaccines were able to reduce the parasitic burden on the liver. In view of the above, we concluded that the use of adjuvant systems using reduced doses induce an effective response, since, when combined with the antigen, they promoted not only the activation of the immune system in the spleen but also the reduction of the parasite load in the liver, suggesting that the use of these systems increased vaccine efficacy in the murine model

Microfluidics and organ-on-a-chip technologies: A systematic review of the methods used to mimic bone marrow.

Bone marrow (BM) is an organ responsible for crucial processes in living organs, e. g., hematopoiesis. In recent years, Organ-on-a-Chip (OoC) devices have been used to satisfy the need for in vitro systems that better mimic the phenomena occurring in the BM microenvironment. Given the growing interest in these systems and the diversity of developed devices, an integrative systematic literature review is required. We have performed this review, following the PRISMA method aiming to identify the main characteristics and assess the effectiveness of the devices that were developed to represent the BM. A search was performed in the Scopus, PubMed, Web of Science and Science Direct databases using the keywords (("bone marrow" OR "hematopoietic stem cells" OR "haematopoietic stem cells") AND ("organ in a" OR "lab on a chip" OR "microfluidic" OR "microfluidic*" OR ("bioreactor" AND "microfluidic*"))). Original research articles published between 2009 and 2020 were included in the review, giving a total of 21 papers. The analysis of these papers showed that their main purpose was to study BM cells biology, mimic BM niches, model pathological BM, and run drug assays. Regarding the fabrication protocols, we have observed that polydimethylsiloxane (PDMS) material and soft lithography method were the most commonly used. To reproduce the microenvironment of BM, most devices used the type I collagen and alginate. Peristaltic and syringe pumps were mostly used for device perfusion. Regarding the advantages compared to conventional methods, there were identified three groups of OoC devices: perfused 3D BM; co-cultured 3D BM; and perfused co-cultured 3D BM. Cellular behavior and mimicking their processes and responses were the mostly commonly studied parameters. The results have demonstrated the effectiveness of OoC devices for research purposes compared to conventional cell cultures. Furthermore, the devices have a wide range of applicability and the potential to be explored

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

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

Cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants : saponin, incomplete Freund's adjuvant, and monophosphoryl Lipid A.

Vaccine adjuvants are substances associated with antigens that are fundamental to the formation of an intense, durable, and fast immune response. In this context, the use of vaccine adjuvants to generate an effective cellular immune response is crucial for the design and development of vaccines against visceral leishmaniasis. The objective of this study was to evaluate innate inflammatory response induced by the vaccine adjuvants saponin (SAP), incomplete Freund’s adjuvant (IFA), and monophosphoryl lipid A (MPL). After a single dose of adjuvant was injected into the skin of mice, we analyzed inflammatory reaction, selective cell migration, and cytokine production at the injection site, and inflammatory cell influx in the peripheral blood. We found that all vaccine adjuvants were able to promote cell recruitment to the site without tissue damage. In addition, they induced selective migration of neutrophils, macrophages, and lymphocytes. The influx of neutrophils was notable at 12 h in all groups, but at other time points it was most evident after inoculation with SAP. With regard to cytokines, the SAP led to production of interleukin (IL)-2, IL-6, and IL-4. IFA promoted production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, IL-17, IL-4, and IL-10. We also observed that MPL induced high production of IL-2, TNF-α, and IFN-γ, in addition to IL-6, IL-17, and IL-10. In peripheral blood, values of certain cell populations in the local response changed after stimulation. Our data demonstrate that the three vaccine adjuvants stimulate the early events of innate immune response at the injection site, suggesting their ability to increase the immunogenicity of co-administered antigens. Moreover, this work provides relevant information about elements of innate and acquired immune response induced by vaccine adjuvants administered alone

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