Systems Biology Analysis of the Radiation-Attenuated Schistosome Vaccine Reveals a Role for Growth Factors in Protection and Hemostasis Inhibition in Parasite Survival.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Epitope Mapping of Exposed Tegument and Alimentary Tract Proteins Identifies Putative Antigenic Targets of the Attenuated Schistosome Vaccine.

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

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 (1-24h) and late (48-168h) periods following inoculation of hamsters with antigenic components of anti-canine visceral leishmaniasis vaccines Leishmune 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.

Effect on cellular recruitment and the innate immune response by combining saponin, monophosphoryl lipid-A and Incomplete Freund's Adjuvant with Leishmania (Viannia) braziliensis antigens for a vaccine formulation.

The poor immunogenicity displayed by some antigens has encouraged the development of strategies to improve the immune response and safety of vaccine candidates, resulting in an intense search for substances that potentiate vaccine response. Adjuvants have these properties helping vaccine candidates to induce a strong, durable, and fast immune response. In this study, we evaluated the specific immune response of adjuvants alone, Saponin (SAP), Incomplete Freund's Adjuvant (IFA) and Monophosphoryl lipid-A SE (MPL-SE®) and in combination with total antigen of L. braziliensis (LB): LBSAP, LBIFA and LBMPL. The specific immune response induced by these compositions demonstrated that they were powerfully immunogenic, increasing cellular infiltration in the skin. Draining lymph nodes cultures showed that LBIFA and LBMPL have higher ability to increase the capacity of APCs to present antigens, with increased frequency of CD11c+CD86+ cells. SAP, MPL, LBSAP, LBIFA and LBMPL could activate lymphocytes increasing expression of CD69 and CD25. LBSAP group was an excellent inducer of pro-inflammatory cytokines at 24 h. At 48 h, higher cytokines production was observed in IFA, LBIFA, MPL and LBMPL groups. Our data demonstrate that LBSAP and LBMPL are potential formulations to be tested in other experimental models. Also, the data obtained could expand the knowledge about immune response after sensitization and also contribute to the development of safe, immunogenic and effective vaccines.

Canine visceral leishmaniasis biomarkers and their employment in vaccines.

The natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitroco-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-γ and TNF-α and decreased levels in IL-4, TGF-ß and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products.

Clinical, hematological and biochemical alterations in hamster (Mesocricetus auratus) experimentally infected with Leishmania infantum through different routes of inoculation.

BACKGROUND: Leishmaniasis remains among the most important parasitic diseases in the developing world and visceral leishmaniasis (VL) is the most fatal. The hamster Mesocricetus auratus is a susceptible model for the characterization of the disease, since infection of hamsters with L. infantum reproduces the clinical and pathological features of human VL. In this context, it provides a unique opportunity to study VL in its active form. The main goal of this study was to evaluate the clinical, biochemical, and hematological changes in male hamsters infected through different routes and strains of L. infantum. METHODS: In the current study, hamsters (Mesocricetus auratus) were infected with the L. infantum strains (WHO/MHOM/BR/74/PP75 and MCAN/BR/2008/OP46) by intradermal, intraperitoneal and intracardiac routes. The animals were monitored for a nine month follow-up period. RESULTS: The hamsters showed clinical signs similar to those observed in classical canine and human symptomatic VL, including splenomegaly, severe weight loss, anemia, and leucopenia. Therefore the OP46 strain was more infective, clinical signs were more frequent and more exacerbated in IC group with 80 to 100 % of the animals showing splenomegaly, in the last month infection. Additionally, desquamation, hair loss and external mucocutaneous lesions and ulcers localized in the snout, accompanied by swelling of the paws in all animals, were observed. Consequently, the animals presented severe weight loss/cachexia, hunched posture, an inability to eat or drink, and non-responsiveness to external stimuli. Furthermore, regardless of strain, route of inoculum and time assessed, the animals showed renal and hepatic alterations, with increased serum levels of urea and creatinine as well as elevated serum levels of aspartate aminotransferase and alanine aminotransferase. CONCLUSIONS: These results strongly suggest that the inoculation through the intracardiac route resulted in a higher severity among infections, especially in the sixth and ninth month after infection via intracardiac, exhibited clinical manifestations and biochemical/hematological findings similar to human visceral leishmaniasis. Therefore, we suggest that this route must be preferentially used in experimental infections for pathogenesis studies of VL in the hamster model.

Systems biology analysis of the radiation- attenuated schistosome vaccine reveals a role for growth factors in protection and hemostasis inhibition in parasite survival

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Epitope mapping of exposed tegument and alimentary tract proteins identifies putative antigenic targets of the attenuated schistosome vaccine

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

The Schistosome Esophagus Is a 'Hotspot' for Microexon and Lysosomal Hydrolase Gene Expression: Implications for Blood Processing.

BACKGROUND: The schistosome esophagus is divided into anterior and posterior compartments, each surrounded by a dense cluster of gland cell bodies, the source of distinct secretory vesicles discharged into the lumen to initiate the processing of ingested blood. Erythrocytes are lysed in the lumen, leucocytes are tethered and killed and platelets are eliminated. We know little about the proteins secreted from the two glands that mediate these biological processes. METHODOLOGY/PRINCIPAL FINDINGS: We have used subtractive RNA-Seq to characterise the complement of genes that are differentially expressed in a head preparation, compared to matched tissues from worm tails. The expression site of representative highlighted genes was then validated using whole munt in situ hybridisation (WISH). Mapping of transcript reads to the S. mansoni genome assembly using Cufflinks identified ~90 genes that were differentially expressed >fourfold in the head preparation; ~50 novel transcripts were also identified by de novo assembly using Trinity. The largest subset (27) of secreted proteins was encoded by microexon genes (MEGs), the most intense focus identified to date. Expression of three (MEGs 12, 16, 17) was confirmed in the anterior gland and five (MEGs 8.1, 9, 11, 15 and 22) in the posterior gland. The other major subset comprised nine lysosomal hydrolases (aspartyl proteases, phospholipases and palmitoyl thioesterase), again localised to the glands. CONCLUSIONS: A proportion of the MEG-encoded secretory proteins can be classified by their primary structure. We have suggested testable hypotheses about how they might function, in conjunction with the lysosomal hydrolases, to mediate the biological processes that occur in the esophagus lumen. Antibodies bind to the esophageal secretions in both permissive and self-curing hosts, suggesting that the proteins represent a novel panel of untested vaccine candidates. A second major task is to identify which of them can serve as immune targets.

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-γ 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.

Dogs immunized with LBSap vaccine displayed high levels of IL-12 and IL-10 cytokines and CCL4, CCL5 and CXCL8 chemokines in the dermis.

The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-γ, TNF-α, IL-4, IL-13, TGF-ß and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-γ and IL-13 expression, and IL-13 and TGF-ß expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis.

Parasite burden in hamsters infected with two different strains of leishmania (Leishmania) infantum: "Leishman Donovan units" versus real-time PCR.

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum's route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates.

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.

Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis.

BACKGROUND: The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL). METHODOLOGY/PRINCIPAL FINDINGS: A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression. CONCLUSIONS/SIGNIFICANCE: These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.

Performance of LBSap vaccine after intradermal challenge with L. infantum and saliva of Lu. longipalpis: immunogenicity and parasitological evaluation.

In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21(+)) and both CD4(+) and CD8(+) T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4(+) T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-ß1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.

Kinetics of cell migration to the dermis and hypodermis in dogs vaccinated with antigenic compounds of Leishmania braziliensis plus saponin.

The search for new immunobiologicals against canine visceral leishmaniasis (CVL) has intensified in the last decade. However, it still remains to be elucidated that mechanisms of the innate immune response in situ after immunization (a.i.). The aim of this study was to investigate the kinetics of cell migration in the skin dogs with distinct antigenic compounds of the LBSap vaccine. Our major findings indicated that saponin adjuvant alone or combined with Leishmania braziliensis antigen induced strong local acute inflammatory reaction. However, these reactions not progressed to ulcerated lesions. Overall, the cell profile found in Sap and LBSap was composed of neutrophils, lymphocytes and eosinophils. There was also increased production of iNOS in Sap and LBSap groups. Thus, we can conclude that dogs immunized by LBSap and the saponin adjuvant elicited a potential innate-immune activations status compatible with effective control of the resistance to infection by Leishmania and contributing to a better understanding of the innate-immunity events induced by the LBSap vaccine.