Protection Against Salmonella by Vaccination with Toxin–Antitoxin Self-Destructive Bacteria

<b>Background</b>: <i>Salmonella</i> is a major zoonotic foodborne pathogen. Conventional poultry vaccines may present limitations in terms of efficacy, safety, and practicality. <b>Objectives</b>: This study focuses on enhancing the immunogenicity and improving the safety of a novel oral vaccination employing inducible toxin-antitoxin (TA) systems, which lead to self-destruction of virulent <i>Salmonella</i> Enteritidis. <b>Methods</b>: A Hok/Sok (HS) TA system was designed to induce cell death upon absence of arabinose. Point mutations were introduced to the Hok toxin promoter to moderate toxin production. A combination of HS and CeaB/CeiB (CC) TA systems was designed to induce cell death both in low di-cation levels or anaerobic conditions. Survival of <i>Salmonella</i>-carrying TA systems was tested in culture and in the Raw264.7 macrophage cell line. One-day old chicks were inoculated with <i>Salmonella</i> carrying the TA system to evaluate bacterial persistence and induction of a protective immune response. <b>Results</b>: Attenuation of the Hok toxin promoter prolonged bacterial survival in vitro. <i>Salmonella</i> carrying the combined TA systems was eliminated completely both in vitro and in inoculated chickens, eliciting high levels of antibodies and conferring protection against challenge with wild-type <i>Salmonella</i>. <b>Conclusions</b>: These findings highlight the potential of the adaptable TA-based vaccination platform to generate safe and efficacious <i>Salmonella</i> vaccines for poultry, contributing to reduced transmission in the food chain.