Synthetic Peptides Elicit Strong Cellular Immunity in Visceral Leishmaniasis Natural Reservoir and Contribute to Long-Lasting Polyfunctional T-Cells in BALB/c Mice

Abstract

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.

Chimeric Vaccines Designed by Immunoinformatics-Activated Polyfunctional and Memory T Cells That Trigger Protection against Experimental Visceral Leishmaniasis

“…The chimeric proteins used in this study were designed on the basis of epitopes mapped along Leishmania infantum proteins already described in the literature as vaccine candidates ( Table 1 ). For epitope mapping, an integrative immunoinformatics approach described by [ 9 , 11 ] was used. The epitopes for T cells were selected on the basis of the highest scores provided by the predictive algorithms.…”

“…The proliferation of antigen experienced T cells was assessed by Carboxyfluorescein diacetate succinimidyl ester (CFSE) assay, 30 days after mice challenge, as described by [ 11 ]. Briefly, spleen’s cell suspensions were incubated in 5 μM CFDA-SE and the labeled cells were washed thoroughly before plating in 96-well round-bottom culture plates at a concentration of 5 × 10 5 cells per well.…”

“…Central and effector memory T cells were analyzed 30 days post-challenge, as described by [ 11 ]. Splenocytes from animals were plated at 5 × 10 5 cells per well in duplicate in 96-well round-bottom plates.…”

Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites

“…Regarding the in vitro infectivity of the parasite, we found a higher agreement between the parasite load evaluated by optical microscopy and flow cytometry observing more infective promastigotes in the FCS-supplemented cultures. This result was expected taking into account most articles involving in vitro and in vivo Leishmania infection studies that describe the use of FCS supplementation (de Brito et al 2019;Santarem et al 2014;Siripattanapipong et al 2019). The removal of the FCS leads to the loss of proteins, nucleoside sources, and hemoglobin, directly affecting the growth and infectivity of the promastigotes (Carvalho et al 2009;Krishnamurthy et al 2005).…”

Liposomal Formulation of ChimeraT, a Multiple T-Cell Epitope-Containing Recombinant Protein, Is a Candidate Vaccine for Human Visceral Leishmaniasis

“…Furthermore, adjuvants are necessary to increase the duration of protection and/or reduce the number of vaccine doses [ 34 ]. Previous studies have shown that saponins, which are glycosides capable of stimulating T-cells to produce Th1-type cytokines [ 35 ], have been successful as immuno-adjuvants with recombinant Leishmania proteins [ 36 , 37 , 38 , 39 , 40 , 41 ], chimeras [ 27 , 30 ], and synthetic peptides [ 42 , 43 ]. Although saponins are used in mice and dogs, they are no longer recommended for human use, since they can cause granulomatous reactions, hemolysis, local pain, and other adverse reactions.…”