Lipopolysaccharide structural modification in Salmonella Gallinarum targeting lipid a deacylation and O-antigen reduces virulence and endotoxicity with competitive protection against a wild-type challenge in chickens

Fowl typhoid (FT), caused by Salmonella Gallinarum (SG), inflicts a significant economic burden on the poultry industry. Our study focused on a non-reversible gene deletion to develop a safe and effective SG vaccine strain through lipopolysaccharide (LPS) structural modification. The virulence-attenuated SG JOL914 strain, generated by deleting the global regulator lon gene, was engineered by in-frame deletions targeting the LPS structure. Interruption on LPS deacylation was introduced by pagL deletion, JOL2997, to mitigate endotoxicity induced by the live vaccine. The O-antigen was truncated by rfaL deletion, JOL3004, to compromise virulence and facilitate Differentiating Infected from Vaccinated Animals (DIVA). Both genes were depleted in JOL3016 (ΔlonΔpagLΔrfaL) for their compounding effects. Subcutaneous administration of the engineered strains in chickens showed reduced endotoxicity, particularly demonstrated by JOL3016 with 2.55-fold and 3.79-fold reductions in TNF-α and IL-1β, respectively. Furthermore, discernible effects on body weight gain and the absence of pathological signs demonstrated the safety profiles of the inoculated strains. Administration of mutant strains substantially increased antigen-specific IgY, sIgA, CD4⁺, and CD8⁺ T cells, supporting immune response elicitation. The absence of antibodies against O-antigen in birds immunized with JOL3004 and JOL3016 demonstrated their DIVA capability. The result was corroborated by improved protection with a rational body weight gain and minimal tissue distortion following an SG WT challenge. JOL2997-immunized birds showed promising survival, and negligible invasion of the challenge strain in vital organs, highlighting its protective efficacy. This study underscores the potential of bioengineered vaccine strains to improve poultry health and productivity, reinforcing the necessity for ongoing vaccine development research.