Avaliação da Imunogenicidade de Dois NovosImunobiológicos Candidatos a Vacina ContraLeishmaniose Visceral Canina
Considering the importance of immunoprophylactic strategies for the control of visceralleishmaniasis, and the lack of studies concerning the cellular and humoral events thatoccur during vaccination in dogs, we have attempted to evaluate the immune responseof a promising new vaccines candidate against canine visceral leishmaniasis (CVL).Thirty five mongrel dogs were treated within seven experimental groups as follow: (i)control group C (n = 10) received 1 ml of sterile 0.9% saline; (ii) LB group (n = 5)received 600 ?g of Leishmania braziliensis promastigote protein in 1 ml sterile 0.9%saline; (iii) Sap group (n = 5) received 1 mg of saponin in 1 ml sterile 0.9% saline; (iv)LBSap group (n = 5) received 600 ?g of L. braziliensis promastigote protein and 1 mgof saponin in 1 ml sterile 0.9% saline; (v) Sal group (n = 5) received sand fly glandextract (SGE) prepared from 5 acini of salivary glands of Lutzomyia longipalpis in 1mL sterile 0.9% saline; (vi) LBSal group (n = 5) received 600 ?g of L. braziliensispromastigote protein plus SGE in 1 mL sterile 0.9% saline; and (vii) the LBSapSalgroup (n = 5) received 600 ?g of L. braziliensis promastigote protein plus 1 mg ofsaponin together with SGE in 1 mL sterile 0.9% saline. Each animal received threesubcutaneous injections in the right flank at intervals of 4 weeks. The results obtainedindicate that some dogs exhibit local swelling or mild local induration reactions, but noulcerated lesions or other adverse reactions, after receiving saponin as an adjuvant (Sap,LBSap or LBSapSal groups). The overall tolerance of the candidate vaccines in dogsappeared to be adequate. The evaluation of immunogenicity revealed that animalstreated with LBSap and LBSapSal presented higher (P < 0.05) amounts of anti-Leishmania total IgG that were associated with increased (P < 0.05) levels of IgG1 andIgG2, suggesting a mixed Th1/Th2 immune response. Likewise, LBSapSal elicited theproduction of elevated levels of anti-SGE total IgG, IgG1 and IgG2, that was previouslyassociated in establishing an anti-immune L. chagasi response in human. Additionally,anti-SGE Western blot experiments showed predominance in the recognition of sericproteins with molecular weights of 35, 45 and 71 kDa, particularly following LBSapSalvaccination, and were related to the resistance pattern against Leishmania infection. Theimmunophenotypic evaluation in peripheral blood mononuclear cells (PBMC) reveal inthe LBSap and LBSapSal increased (P < 0.05) counts of circulating CD21+ B-cells,CD5+ T-cells and CD4+ and CD8+ T-cell subset, suggesting protective immunity againstLeishmania infection as has been suggested previously for CVL. Whilst lowerstimulation index by either VSA (vaccine soluble antigen) or SLcA (soluble Leishmaniachagasi antigen) were recorded for the LB and Sal groups, higher cell reactivities in thepresence of both stumuli were related to LBSap and LBSapSal groups. Interestingly, anegative association was demonstrated between cell reactivity and CD4+ T-lymphocytesor CD14+ monocytes following in vitro stimulation in the Sal group. These data supportthe hypothesis that Sal treatment inhibits CD14+ monocytes in the promotion of CD4+T-lymphocyte activation and the induction of cell proliferation. In contrast, when invitro cultures of peripheral blood mononuclear cells (PBMC) were stimulated withSLcA in the LBSap treatment and VSA or SLcA in the LBSapSal group, increasedlymphoproliferation activity was accompanied by a higher frequency of antigen-specificCD8+ T-cells. These results support the hypothesis that induction of CD8+ T-cells mayplay a protective role in the mechanism of control of parasitism by Leishmania afterLBSap and LBSapSal treatment associated with the antigen-specific immune responseto antigens from the etiological agent of CVL. The evaluation of potential antigenpresenting cells (APC) revealed increased numbers of circulating CD14+ monocytes inthe LBSap and LBSapSal groups. Furthermore, the largest APC counts were associatedXIIIwith the highest expression of MHC-I in lymphocytes in the LBSap and CD80 andMHC-I in lymphocytes in the LBSapSal, suggesting that this association couldrepresent interaction between the innate and adaptive immune responses, reflecting animprovement in activation status during immunization. The results obtained from theanalysis of nitric oxide (NO) levels (determined as nitrite) in culture supernatantsstimulated by SLcA in the LBSap and in the serum of the LBSapSal confirmed thehypothesis that these vaccines induce a potential resistance profile against Leishmaniainfection. Our data suggested that the potential resistance profile elicited by LBSap andLBSapSal were compatible with effective control of the etiological agent of CVL.
Advisor:Rodrigo Corrêa Oliveira; Luis Carlos Crocco Afonso; Ricardo Toshio Fujiwara; Oscar Bruna Romero; Bartira Rossi Bergman; Alexandre Barbosa Reis
School:Universidade Federal de Ouro Prêto
Source Type:Master's Thesis
Date of Publication:11/30/2007