Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with regulated length of lipopolysaccharide in mice

Non-typhoidal Salmonella enterica (NTS) is a major global foodborne pathogen that poses a major public health concern worldwide, and no vaccines are available for protecting against infection of multiple Salmonella serotypes, therefore, the development of Salmonella vaccines to provide broad protection is valuable. In this work, we aimed to regulate lipopolysaccharide (LPS) synthesis of live Salmonella in vivo for exposing conserved protein antigens on the outer membrane while maintaining smooth LPS patterns in vitro to keep their original ability to invade host cells for inducing cross-protection against infection of multiple Salmonella serotypes. We generated a series of mutants defective in genes to affect the length of LPS. These mutants exhibit in vivo regulated-delayed attenuation and altered length of LPS, and all these mutants were derived from SW067 (ΔpagL7 ΔpagP81::P_lpp lpxE ΔlpxR9 Δfur9) containing ∆pagP81::P_lpp lpxE mutation to reduce their endotoxic activity. Animal experiments demonstrated that all regulated delayed attenuated mutants exhibited reduced ability to colonize the organs of the mice, and SW114 (waaI), SW116 (waaJ), SW118 (waaL), and SW120 (wbaP) induced a significant production of IgG and IgA against OMPs isolated from S. Typhimurium, S. Enteritidis, and S. Choleraesuis. SW114 (waaI), SW116 (waaJ), and SW118 (waaL) were capable of conferring significant protection against infection of wild-type S. Enteritidis and S. Choleraesuis, with SW118 (waaL) triggering significant CD4⁺ T-cell responses as well as the B220^low IgG⁺ B_M cell. In conclusion, regulated delayed attenuated Salmonella vaccines with the whole core oligosaccharides of LPS showed a goo.d ability to expose conserved outer antigens and to trigger strong cross-immune responses against both homologous and heterologous Salmonella infections. These results give new insight into the development of the Salmonella vaccine against multiple serotypes of Salmonella.