In Silico Design and Characterization of a Multiepitope Vaccine Candidate Against Brucella canis Using a Reverse Vaccinology Approach

Abstract

Brucella canis is a Gram‐negative bacterium that causes canine brucellosis, a zoonotic disease with serious implications for public health and the global economy. Currently, there is no effective preventive vaccine for B. canis. Control measures include diagnostic testing, isolation, and euthanasia of infected animals. However, these measures face significant limitations, such as diagnostic challenges, ethical concerns, and limited success in preventing transmission. Epidemiologically, canine brucellosis exhibits seroprevalence rates ranging from less than 1% to over 15%, with higher rates reported in stray dogs and regions of low socioeconomic development. This study employed a reverse vaccinology approach to design and characterize a multiepitope vaccine candidate against B. canis, aiming to prevent infection caused by this pathogen. A comprehensive in silico analysis of the complete B. canis proteome was conducted to identify proteins with potential as vaccine targets. Predicted epitopes for B and T cells were analyzed, and those with the highest capacity to elicit a robust immune response were selected. These proteins were classified as plasma membrane proteins, outer membrane proteins (OMPs), or proteins with similarity to virulence factors. Selection criteria emphasized their essential roles in bacterial function, lack of homology with proteins from dogs or mice, and presence of fewer than two transmembrane domains. From this process, four candidate proteins were identified. Epitopes for B and T cells within these proteins were predicted and analyzed, selecting the most immunogenic sequences. The overlap between B‐ and T‐cell epitopes narrowed the selection to six final epitopes. These selected epitopes were then assembled into a multiepitope vaccine construct using flexible linkers to ensure structural integrity and molecular adjuvants to enhance immunogenicity. The physicochemical properties, antigenicity, and toxicity of the designed vaccine were evaluated. Additionally, the secondary and tertiary structure of the vaccine was predicted and refined, followed by a molecular interaction analysis with the Toll‐like receptor 4 (TLR4) receptor. The designed vaccine proved to be highly antigenic, nonallergenic, and nontoxic. Validation of its secondary and tertiary structures, along with molecular docking analysis, revealed a high binding affinity to the TLR4 receptor. Molecular dynamics simulations and normal mode analysis further confirmed the vaccine’s structural stability and binding capacity. A multiepitope vaccine candidate against B. canis was successfully designed and characterized using a reverse vaccinology approach. This vaccine construct is expected to induce robust humoral and cellular immune responses, potentially conferring protective immunity against B. canis. The results of this study are promising; however, in vitro and in vivo tests are necessary to validate the vaccine’s protective efficacy. Furthermore, the described method could serve as a framework for developing vaccines against other pathogens.