Multiple immunodominant O-epitopes co-expression in live attenuated Salmonella serovars induce cross-protective immune responses against S. Paratyphi A, S. Typhimurium and S. Enteritidis.

Salmonella enterica subsp. enterica (S. enterica) is a significant public health concern and is estimated to cause more than 300,000 deaths annually. Nowadays, the vaccines available for human Salmonellosis prevention are all targeting just one serovar, i.e., S. Typhi, leaving a huge potential risk of Salmonella disease epidemiology change. In this study, we explored the strategy of multiple immunodominant O-epitopes co-expression in S. enterica serovars and evaluated their immunogenicity to induce cross-immune responses and cross-protections against S. Paratyphi A, S. Typhimurium and S. Enteritidis. We found that nucleotide sugar precursors CDP-Abe and CDP-Par (or CDP-Tyv) could be utilized by S. enterica serovars simultaneously, exhibiting O2&O4 (or O4&O9) double immunodominant O-serotypes without obvious growth defects. More importantly, a triple immunodominant O2&O4&O9 O-serotypes could be achieved in S. Typhimurium by improving the substrate pool of CDP-Par, glycosyltransferase WbaV and flippase Wzx via a dual-plasmid overexpressing system. Through immunization in a murine model, we found that double or triple O-serotypes live attenuated vaccine candidates could induce significantly higher heterologous serovar-specific antibodies than their wild-type parent strain. Meanwhile, the bacterial agglutination, serum bactericidal assays and protection efficacy experiments had all shown that these elicited serum antibodies are cross-reactive and cross-protective. Our work highlights the potential of developing a new type of live attenuated Salmonella vaccines against S. Paratyphi A, S. Typhimurium and S. Enteritidis simultaneously.