Regulated Delayed Expression of rfaH in an Attenuated Salmonella enterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen

摘要

RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A ΔrfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >10⁸ CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, ΔPrfaH178, was introduced into the attenuated vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) expressing the pneumococcal surface protein PspA from an Asd⁺ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic ΔrfaH derivative or the isogenic RfaH⁺ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent Streptococcus pneumoniae WU2. Immunization with ΔPrfaH178 mutant strains led to increased levels of protection compared to that of the parent χ9241 and of a ΔrfaH derivative of χ9241.