Significant differences (experimental challenge In order to evaluate whether the phenotypic profile of PBMCs in vaccinated/challenged dogs was influenced by VSA or SLiA stimulation, as well as to characterize these cells, we conducted an analysis of the phenotypic features of PBMCs of vaccinated/challenged dogs (Determine? 3). in the saliva of the vector. Keywords: LBSapSal vaccine, Canine visceral leishmaniasis, Immunogenicity, Experimental challenge, for humans [3]. Significant efforts are being made by several groups to develop a vaccine against CVL [4-18]. Given their wide spectrum of antigenicity, cost, and safety, the first generation vaccines that composed of crude antigens also represent an excellent tool for immunoprophylaxis [10,11,13-15,19]. In phase I and II clinical trials, Mayrink in dogs that had received ultrasound-disrupted, merthiolated promastigotes of with (BCG). Strong cellular proliferation in response to soluble antigens has also been reported in dogs vaccinated with autoclaved promastigotes plus BCG as the adjuvant [11]. Moreover, in a double-blind randomized efficacy field trial, a single dose of a vaccine composed of alum-precipitated autoclaved vaccine against CVL mixed with BCG was shown to be safe and decreased the incidence of the CVL from 12% to 3.7%, which is equivalent to a 69.3% efficacy rate [20]. In the last few decades, the incorporation of salivary proteins of phlebotomines has been widely used in experimental challenge studies, or in seeking potential targets for vaccine development against infection; such proteins have even been a part of vaccine composition as an adjuvant or co-adjuvant [14,21-29]. Gomes were protected against a challenge with plus salivary gland sonicate [28]. Collin (LJL143 and LJM17) and challenged with uninfected or infected sandflies, observed a cellular immune response at the site of the bite characterized by lymphocytic infiltration and expression of interferon- or interleukin-12 [29]. These results suggest that the use of saliva proteins could be a good strategy in developing an anti-CVL vaccine in dogs. In this context, previous studies in dogs conducted by our group used a first generation vaccine composed of antigens plus saponin as an adjuvant and sand travel salivary gland extract (SGE) (LBSapSal Prasugrel Hydrochloride vaccine). The immunization elicited increases in the anti-saliva and anti-IgG isotypes, higher counts of circulating and specific T CD8+, and high NO production after immunization [14]. The current study included an analysis of the immunogenicity and a parasitological investigation of dogs immunized with LBSapSal vaccine. The dogs were evaluated for up to 885 days after challenge by intradermal inoculation using promastigotes plus SGE. Methods The study protocol was approved by the Ethical Committee for the Use of Experimental Animals at the Universidade Federal de Ouro Preto, Minas Gerais, Brazil. Sand flies and salivary gland extracts Closed colonies of were maintained at 25C and 60%C80% relative humidity according to a published protocol [30]. Sand travel SGE was prepared using the method of Cavalcante for 2?min. The supernatant was collected and stored at -70C prior use. Study animals, vaccination, and experimental challenge In this study, we used the LBSapSal vaccine as previously described by Giunchetti antibodies was confirmed by Bmpr2 indirect fluorescence immunoassay and enzyme-linked immunosorbent assay (ELISA) assessments. Ouro Preto city is considered a non-endemic area for visceral leishmaniasis in Brazil. Besides unfavorable serology, other additional effective approaches were performed aiming to rule out contamination such as Prasugrel Hydrochloride spraying the kennels of UFOP with pyrethroid insecticide and protecting all extension of the kennels with an appropriate and security stainless steel wire mesh to block the access of phlebotomines. At the beginning of the experiments the dogs were approximately Prasugrel Hydrochloride the same age (210??45 days) and had comparable weights (15??5 kilograms) and were randomly chosen from a set with approximately the same number of males and females and divided into four experimental groups: (i) the control group C (in 1?mL of sterile 0.9% saline; (iii) the LBSal group (promastigote protein plus SGE (as above) in 1?mL sterile 0.9% saline; and (iv) the LBSapSal group (promastigote protein plus 1?mg of saponin together with SGE in 1?mL of sterile 0.9% saline. Each group received three subcutaneous injections in the right flank at intervals of 4 weeks. Three.