Regulated delayed expression of rfc enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines

The Salmonella tic gene encodes the O-antigen polymerase. We constructed three strains in which we replaced the native rfc promoter with the arabinose-dependent araC P-BAD promoter so that rfc expression was dependent on exogenously supplied arabinose provided during in vitro growth. The three mutant strains were designed to synthesize different amounts of Rfc by altering the ribosome-binding sequence and start codon. We examined these strains for a number of in vitro characteristics compared to an isogenic A rfc mutant and the wild-type parent strain. One promoter-replacement mutation. Delta P-rfc174, yielded an optimal profile, exhibiting wild-type characteristics when grown with arabinose, and Delta rfc characteristics when grown without arabinose. In addition, when administered orally, the Delta P-rfc174 strain was completely attenuated in for virulence in mice. The Delta P-rfc174 mutation was introduced into attenuated Salmonella vaccine strain chi 9241 (Delta pabA Delta pabB Delta asdA) followed by introduction of an Asd(+) balanced-lethal plasmid to designed for expression of the pneumococcal surface protein PspA. Mice immunized with either chi 9241 or its Delta P-rfc174 derivative expressing pspA were protected against S. pneumoniae challenge. Published by Elsevier Ltd.