Completing the pieces of a puzzle: in-depth probing of Toxoplasma gondii rhoptry protein 4 as a promising target for vaccination using an in-silico approach.

The present study aimed to evaluate the key characteristics of Toxoplasma gondii rhoptry protein 4 (TgROP4), including physicochemical parameters, structural features, immunogenic epitopes, and virtual immune simulation, using several bioinformatics-based servers and tools. Based on allergenicity and antigenicity outputs, the TgROP4 protein seemed to have an immunogenic and non-allergenic nature. The quality of the three-dimensional (3D) structure improved after refinement, according to the outcomes of the Ramachandran plot and the ProSA-web servers. ABCpred and SVMTriP web tools were used to predict linear B lymphocyte epitopes and found several promising epitopes. Acceptable antigenicity, hydrophilicity, beta-turn, Bepipred linear epitope 2.0, flexibility, and surface accessibility scores were obtained through the Immune Epitope Database (IEDB). Also, seven discontinuous B-cell epitopes ranging from scores 0.966 to 0.848 were found in the 3D model of TgROP4 via the ElliPro. The IEDB findings showed T-cell epitopes on TgROP4 protein are capable to strongly bind to the major histocompatibility complex classes. In silico immune simulation was performed using C-ImmSim server and showed three injections of TgROP4 protein at 4-week intervals is capable to elicit adequate humoral and cell-mediated immune responses.