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

## TL;DR

A new Salmonella vaccine using toxin-antitoxin systems safely eliminates the bacteria and boosts immunity in chickens.

## Contribution

A novel oral Salmonella vaccine using inducible toxin-antitoxin systems for self-destruction of bacteria is developed and tested.

## Key findings

- Modified Hok toxin promoter increased bacterial survival in vitro.
- Combined TA systems eliminated Salmonella in vitro and in chickens, inducing strong antibody responses.
- Vaccinated chickens were protected against wild-type Salmonella challenge.

## Abstract

Background: Salmonella is a major zoonotic foodborne pathogen. Conventional poultry vaccines may present limitations in terms of efficacy, safety, and practicality. Objectives: 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 Salmonella Enteritidis. Methods: 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 Salmonella-carrying TA systems was tested in culture and in the Raw264.7 macrophage cell line. One-day old chicks were inoculated with Salmonella carrying the TA system to evaluate bacterial persistence and induction of a protective immune response. Results: Attenuation of the Hok toxin promoter prolonged bacterial survival in vitro. Salmonella 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 Salmonella. Conclusions: These findings highlight the potential of the adaptable TA-based vaccination platform to generate safe and efficacious Salmonella vaccines for poultry, contributing to reduced transmission in the food chain.