Estudos de imunobioinformática e imunoprofilático de uma proteína de fusão contra a infecção por Corynebacterium pseudotuberculosis em modelo murino

Abstract

Caseous lymphadenitis (CLA) is a disease that affects small ruminants, and there are no vaccines that provide significant protection against this disease in both goats and sheep. The objective of this work was to evaluate in silico and in vivo the fusion protein MBP:PLD:CP40 as a new immunoprophylactic target for CLA. In silico analysis were performed using immunobioinformatics tools for the construction of the amino acid sequence, assembly and validation of the 3D structure, physicochemical analysis, antigenicity and allergenic potential, prediction of B lymphocytes specific epitopes and those capable of binding to molecules of the complex major histocompatibility (MHC) class I and II, and the prediction of binding to the Toll-Like 2 receptor (TLR2). Expression and purification were performed in vitro in a heterologous system. 40 mice were used for immunization, divided into 4 groups of 10 animals, with 2 immunizations occurring at an interval of 28 days and 4 blood collections occurring at an interval of 14 days. The animals were challenged 42 days after the first immunization and observed for 30 days. Total IgG, IgG1 and IgG2a antibodies were quantified, and a culture of splenocytes from 3 animals from each group was made, with further antigenic stimulation and quantification of the expression of IL-4, IL-12, IL-17, TNF and IFN-γ. The in silico analysis demonstrated that the fusion protein showed antigenicity associated with no prediction of allergenicity, as well as the existence of several B cells epitopes and peptides capable of binding to MHC I and II molecules and anchoring to the TLR2 receptor. Through the cloning and expression of MBP:PLD:CP40, it can be seen that the protein has significant stability and a molecular weight of 115kDa. The fusion protein stimulated a greater production of IgG1 than IgG2a in mice, with significant expression of TNF and IL-17. The fusion protein was able to partially protect mice against C. pseudotuberculosis infection (57.14%), and can be considered as a potential new immunogen for the development of vaccines for CLA.