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

The use of attenuated bacteria as vaccine delivery vehicles for heterologous antigens has been studied extensively in both animals and humans. Attenuated Salmonella is an attractive choice due to its ability to stimulate both cell and humoral-mediated immunity against a heterologous antigen and thus provide protection against pathogen challenge [1–3] when given orally. An optimal live oral Salmonella vaccine would retain its ability to colonize and invade host lymphoid tissues while remaining completely avirulent [4]. Several Salmonella strains with various modifications have been created in an attempt to balance safety and immunogenicity (see reviews [1, 3, 5]), however, there is a need for improved strains. A safe vaccine should carry multiple attenuating mutations. Some target genes for attenuation are those involved in the synthesis of lipopolysaccharide (LPS). LPS is a structural component of the gram-negative outer membrane. Full length (smooth) LPS is comprised of three components – lipid A, also a potent activator of the innate immune response, the core oligosaccharide, and O-antigen, a polymer of repeating sugar units. Four LPS genes that have been shown previously to be useful deletion targets for Salmonella vaccines are galE, pmi, rfaH and rfc. Two of these genes, galE and pmi, are involved in synthesizing sugars required for core and/or O-antigen synthesis. GalE is a UDP-galactose epimerase that inter-converts UDP-glucose and UPD-galactose, an essential part of core sugar and O-antigen. This mutant synthesized core-defective LPS in the absence of galactose but made normal LPS when galactose was available in the growth media [6, 7]. S. Typhimurium galE strains are avirulent and immunogenic [6]. However, this same mutation, transferred to S. Typhi, was not attenuated and was poorly immunogenic in humans [8]. The pmi gene codes for phosphomannose isomerase, which converts fructose-6-P to mannose-6-P [9]. The deletion mutant is unable to synthesize the O-antigen due to unavailability of mannose, an O-antigen sugar. When the mutant is grown in the presence of mannose, the smooth LPS phenotype is exhibited. S. Typhimurium strains with a pmi deletion are attenuated and immunogenic [10]. We constructed and evaluated a Δpmi mutant in our lab, confirming previous results [10–12]. This mutant was attenuated as expected, but also induced antibodies that cross-reacted with outer membrane proteins from other Salmonella serovars and other gram-negative bacteria [12]. A pmi deletion in Typhi has not yet been evaluated in humans. Note that both galE and pmi mutant strains, if grown in the presence of galactose or mannose, respectively, transiently express LPS before colonizing the gut-associated lymphoid tissue (GALT) or organs [11, 13]. Because these mutants are essentially wild-type at the time of immunization and become attenuated in host tissues, we have termed this effect regulated delayed attenuation. RfaH is a transcriptional anti-terminator required for the synthesis of many virulence determinants including O-antigen, core sugar, capsular polysaccharide, and Vi antigen [14, 15]. An rfaH deletion mutant, described as “gently rough”, exhibited some deep-rough characteristics, i.e. lack of O-antigen and outer core, sufficient attenuation, susceptibility to detergents and to some antibiotics, but still proved to be attenuated and immunogenic [14, 16]. Rfc (Wzy) is the O-antigen polymerase that, in conjunction with Wzx (transporter), Wzz (length determinant) and WbaP (O-antigen synthesis initiation), synthesizes, assembles, and transports the O-antigen to the periplasm, where WaaL (ligase) ligates O-antigen to lipid A to form complete LPS [17–19]. rfc mutants exhibit a characteristic “semi-rough” phenotype, making lipid A and core capped with a single O-antigen subunit. An S. Typhimurium rfc mutant administered orally to mice was avirulent and protective against oral challenge with wild-type S. Typhimurium [10]. Unlike pmi and galE mutants, strains with mutations in rfaH or rfc do not transiently express full length O-antigen and therefore may be impaired in their ability to invade and colonize host tissues effectively. A tightly regulated araC PBAD activator-promoter has been used extensively in our lab to regulate gene expression [12, 20]. In a previous study, we replaced the rfaH promoter with araC PBAD and the resulting strain exhibited arabinose-dependent O-antigen synthesis [21]. The promoter substitution mutation was then introduced into attenuated S. Typhimurium strain χ9241 (ΔpabA ΔpabB ΔasdA). The resulting strain was found to be superior to a χ9241 ΔrfaH strain for delivery of the pneumococcal protein PspA, eliciting significantly greater protection against challenge with virulent Streptoccocus pneumoniae [21]. In this study, we evaluated whether this regulated delayed attenuation approach would provide advantages when used with rfc. We replaced the rfc promoter with an araC PBAD promoter to create arabinose inducible production of Rfc and thus regulate rfc expression to mimic transient expression of smooth LPS. We evaluated a series of rfc promoter mutations in vitro and introduced one of them into vaccine strain χ9241. The resulting strain was evaluated for its ability to deliver the heterologous protein PspA to mice.