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

The Salmonella rfc gene encodes the O-antigen polymerase. We constructed three strains in which we replaced the native rfc promoter with the arabinose-dependent araC PBAD 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 Δrfc mutant and the wild-type parent strain. One promoter-replacement mutation, ΔPrfc174, yielded an optimal profile, exhibiting wild-type characteristics when grown with arabinose, and Δrfc characteristics when grown without arabinose. In addition, when administered orally, the ΔPrfc174 strain was completely attenuated in for virulence in mice. The ΔPrfc174 mutation was introduced into attenuated Salmonella vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) followed by introduction of an Asd+ balanced-lethal plasmid to designed for expression of the pneumococcal surface protein PspA. Mice immunized with either χ9241 or its ΔPrfc174 derivative expressing pspA were protected against S. pneumoniae challenge.