Affiliation:
1. Center for Infectious Diseases and Vaccinology, The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona 85287
2. Department of Biology, Washington University, St. Louis, Missouri 63130
Abstract
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 arabinose-regulated
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
ribosome-binding site, start codon, and/or codon content to construct RDAS strains χ9095, χ9959, and χ9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain χ9555 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 χ9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain χ9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain χ9241 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.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology
Cited by
59 articles.
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