Regulated Antigen Expression in Live RecombinantSalmonella entericaSerovar Typhimurium Strongly Affects Colonization Capabilities and Specific CD4⁺-T-Cell Responses

Abstract

Regulated antigen expression can influence the immunogenicity of live recombinant Salmonella vaccines, but a rational optimization has remained difficult since important aspects of this effect are incompletely understood. Here, attenuated Salmonella enterica serovar Typhimurium SL3261 strains expressing the model antigen GFP_OVA were used to quantify in vivo antigen levels by flow cytometry and to simultaneously follow the crucial early steps of antigen-specific T-cell responses in mice that are transgenic for a T-cell receptor recognizing ovalbumin. Among seven tested promoters, P pagC has the highest activity in murine tissues combined with low in vitro expression, whereas P tac has a comparable in vivo and a very high in vitro activity. Both SL3261 (pP pagC GFP_OVA) and SL3261 (pP tac GFP_OVA) cells can induce potent ovalbumin-specific cellular immune responses following oral administration, but doses almost 1,000-fold lower are sufficient for the in vivo-inducible construct SL3261 (pP pagC GFP_OVA) compared to SL3261 (pP tac GFP_OVA). This efficacy difference is largely explained by impaired early colonization capabilities of SL3261 (pP tac GFP_OVA) cells. Based on the findings of this study, appropriate in vivo expression levels for any given antigen can be rationally selected from the increasing set of promoters with defined properties. This will allow the improvement of recombinant Salmonella vaccines against a wide range of pathogens.

Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen

“…Alternatively, DO11.10 cells were stimulated in vitro with 1 M OVA peptide (amino acids 323-339) for 4 days before adoptive transfer. One day after transfer of unstimulated DO11.10 T cells or 4 days after transfer of prestimulated DO11.10 T cells, mice were orally infected with Ϸ5 ϫ 10 8 colony-forming units (cfu) of Salmonella, as described (18). At various time intervals after infection, mice were killed and Peyer's patches were prepared.…”

Cancer immunotherapy based on recombinant Salmonella enterica serovar Typhimurium aroA strains secreting prostate-specific antigen and cholera toxin subunit B

“…Our western blot data demonstrated that the release of CtxB into the supernatant via its internal sec-dependent signal is, at minimum, very inefficient in Salmonella. Furthermore, although the tac-promoter has a comparable in vitro and in vivo activity, 47 the expression and the stability of CtxB in vivo might be lower than that in vitro. As a consequence, the extracellularly available level of native CtxB (in the form of pentamers) is very low and thus there is only negligible activity of CtxB as an immunostimulant.…”

Enhanced Immunogenicity in the Murine Airway Mucosa with an AttenuatedSalmonellaLive Vaccine Expressing OprF-OprI fromPseudomonas aeruginosa

“…The recombinant vector pTrcHis-F-I, carrying the hybrid gene of Met-Ala-(His) 6 OprF 190-342 -OprI 21-83 from P. aeruginosa was expressed by E. coli XL-1 Blue and purified as described previously (11,24). Subsequently, Met-Ala-(His) 6 OprF 190-342 -OprI 21-83 was adsorbed to aluminum hydroxide [Al(OH) 3 ] (Superfos; Vedbaeck, Denmark). The final concentration of the vaccine was 0.1 mg of OprF-I/ml and 0.3 mg of Al(OH) 3 /ml with 0.05 mg of thimerosal/ml (Caesar & Lorenz, Hilden, Germany) as preservative.…”

“…Escherichia coli Electro10 (Stratagene) was used for cloning. The oprFoprI fusion gene encoding Met-Ala-(His) 6 OprF 190-342 -OprI 21-83 from P. aeruginosa was cloned on pBR322-derived plasmids downstream of two constitutively active promoters (P yz [14] in pDB3 and P trc in pDB4) or the in vivo inducible Salmonella P pagC promoter (7,16) in plasmids pMW151, pMW209, and pMW210. These in vivo inducible constructs differed in the ribosomal binding site (33) upstream of oprF-oprI, resulting in differential in vivo OprF-OprI expression levels.…”

“…Among the many parameters relevant to the efficacy of a Salmonella vaccine are the immunogenicity of the foreign protein, the amount of the induced protein expression, and the induction of the protein expression (6). Thus, experiences from Salmonella constructs with other antigens cannot be easily transferred to the development of a new vaccine.…”