SalmonellaVaccine Vectors Displaying Delayed Antigen SynthesisInVivoTo Enhance Immunogenicity

Abstract

We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuated Salmonella vaccine (RASV) strains to enhance immune responses by reducing the adverse effects of high-level antigen synthesis. This system includes a chromosomal repressor gene, lacI, expressed from the arabinoseregulated araC P BAD promoter. LacI serves to regulate expression from a plasmid promoter, P trc , that directs antigen synthesis. In the presence of arabinose LacI is produced, which binds to P trc , blocking antigen synthesis. In vivo, an arabinose-poor environment, the concentration of LacI decreases with each cell division, allowing increased antigen synthesis. To optimize the system and for comparison, we altered the lacI ribosomebinding site, start codon, and/or codon content to construct RDAS strains 9095, 9959, and 9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain 9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initial in vitro assessment and the Streptococcus pneumoniae PspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression of lacI did not interfere with the capacity to induce an immune response. Strain 9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain 9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain 9241 also induced significantly greater protective efficacy against challenge with virulent S. pneumoniae. These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.

Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis To Confer Protection against Mycobacterium tuberculosis

“…This system ensures the stability of plasmid vectors producing protective antigens in vivo after immunization of animal hosts and eliminates the use of drug resistance markers. Furthermore, RASV strains have been constructed that are phenotypically similar to wild-type Salmonella at the time of oral vaccination but display (i) regulated delayed attenuation (22), (ii) regulated delayed synthesis of recombinant antigens (71), and (iii) regulated delayed lysis to release protective antigens and confer complete biological containment, after colonizing the host (46).…”

“…Briefly, suicide vectors were used to construct strain 11021 containing the nucleotide sequences designed to delete specific genes or introduce defined deletion-insertion mutations with modified promoter (P), SD, and start codon sequences (Table 1). RASV strain construction and characterization were performed as described previously (22,34,46,49,67,71). Maps of the deletions and deletion-insertion mutations have been described previously for ⌬(gmd-fcl)-26, ⌬pmi-2426, ⌬relA198::araC P BAD lacI TT, and ⌬asd27::TT araC P BAD c2 (49).…”

“…The asdA and murA genes encode enzymes involved in the biosynthesis of the bacterial cell wall (8,13), and the asdA deletion imposes an obligate requirement for diaminopimelic acid (DAP) in noncomplemented mutant strains. Curtiss et al (22), Kong et al (46), and Wang et al (71) have also developed RASVs that, in vivo, display regulated delayed attenuation (22), regulated delayed antigen synthesis (71), and regulated delayed lysis (46), to aid in the induction of high levels of immunogenicity. The genetic mechanisms that make possible these particular phenotypes have been generated by inclusion of a collection of defined deletion or deletion-insertion mutations in specific genes implicated in either the metabolism or virulence of Salmonella that eliminate their gene products or regulate their expression by replacing their original promoters with the tightly arabinose-regulated araC P BAD activator promoter ( Fig.…”

Comparison of a Regulated Delayed Antigen Synthesis System withIn Vivo-Inducible Promoters for Antigen Delivery by Live AttenuatedSalmonellaVaccines

“…In an effort to reduce the metabolic burden imposed by constitutive antigen gene expression and to permit the initiation of antigen synthesis when the bacterium reaches an immunocompetent site in the host, we developed an RDAS system (91). In this system, LacI represses transcription from P trc during in vitro cultivation, with gradual derepression as a consequence of cell divisions occurring during colonization of internal lymphoid tissues following immunization.…”

Live Attenuated Salmonella Vaccines against Mycobacterium tuberculosis with Antigen Delivery via the Type III Secretion System

“…Oral administration of Salmonella allows infection of Peyer's patches via M cells, as well as phagocytosis by dendritic cells sampling the gut mucosa and colonization of the mesenteric lymph nodes, liver, and spleen, generating mucosal, humoral, and cellular immune responses against Salmonella and its heterologous antigens (10,19,24,49,77,81). We have reported the advantages of using new-generation recombinant attenuated Salmonella vaccine (RASV) strains that are phenotypically similar to the wild-type strain at the time of oral vaccination as an alternative for vaccination (23,24,52,79). These RASV strains are able to colonize and persist in the lymphoid tissue without causing disease symptoms when carrying heterologous antigens, thereby inducing higher protective mucosal and systemic immune responses against a number of infectious diseases (27,45,47,48,68,74,83).…”