Efficacy of intracellular activated promoters for generation of Salmonella-based vaccines - PubMed (original) (raw)
Efficacy of intracellular activated promoters for generation of Salmonella-based vaccines
Xin Xu et al. Infect Immun. 2010 Nov.
Abstract
Salmonella enterica is a versatile vaccine carrier for heterologous antigens. One strategy for vaccine antigen delivery is the use of live attenuated S. enterica strains that translocate heterologous antigens into antigen-presenting cells by means of type III secretion systems (T3SS). The feasibility of this approach has been demonstrated in various experimental vaccination studies. The efficacy of recombinant live vaccines is critically influenced by the optimal level of attenuation and many other factors. For the rational design of approaches involving translocation by T3SS, additional parameters are the level of expression of the heterologous antigens and the selection of carrier proteins for the delivery of antigens to desirable subcellular compartments of the target cell. We deployed the Salmonella pathogenicity island 2 (SPI2)-encoded T3SS for antigen delivery. The SPI2-T3SS and effector proteins are encoded by members of the large SsrAB regulon, including promoters with highly variable strength of expression. We investigated the effect of various in vivo-activated promoters of the SsrAB regulon on the efficacy of recombinant Salmonella vaccines. We observed that the use of promoters with higher strength results in greater synthesis of recombinant antigens and greater stimulation of T-cell responses in cell culture assays for the stimulation of T cells by the model antigen ovalbumin. In contrast, in vaccination experiments, promoters with a low level of expression resulted in the induction of higher amounts of T cells reactive to the model antigen listeriolysin. These results demonstrate that high-level expression of heterologous antigens does not necessarily result in optimal stimulation of immune responses.
Figures
FIG. 1.
Promoter activities of intracellular Salmonella in macrophages and murine DC. Various S. enterica serovar Typhimurium strains harboring chromosomal promoter fusions to genes of the SsrAB virulon were used to infect the macrophagelike murine cell line RAW264.7 (A) or murine BM-DC (B) at an MOI of 10 or 25, respectively. Phagocytosis of the bacteria was allowed for 1 h and subsequently noninternalized bacteria were killed by addition of gentamicin. At 12 h after infection, infected cells were lysed in order to release intracellular bacteria. A fraction of the lysate was used to quantify the amount of viable intracellular bacteria by plating of serial dilutions onto agar plates and determination of CFU (circles). Equal amounts of the lysates were used to determine the luciferase activities of the recovered intracellular bacteria and reporter activities were expressed as relative luciferase units (RLU) per CFU (bars). Means and standard deviations of triplicate assays are shown. Statistical analyses were performed comparing the various promoter fusions to the sseB::luc reporter and are indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
FIG. 2.
Generation of expression cassettes for the expression of heterologous vaccine antigens by promoters of the SsrAB virulon. Expression cassettes consist of hybrid genes for the expression of fusion proteins consisting of SPI2 effector protein SseF, model antigens Llo or OVA, and a C-terminal epitope tag (M45 or HA) for the standardized detection of the amounts of fusion protein. Cassettes also contain sscB encoding a specific chaperone for SseF. The expression is controlled by various _in vivo_-activated promoters of the SsrAB regulon that control the expression of genes within SPI2 (PsseA, PssaG) or genes encoding SPI2 effector proteins outside of SPI2 (PsifA, PsifB, and PsseJ). All plasmids were generated on the basis of the low-copy-number vector pWSK29, and plasmid designations are indicated by p2629, etc.
FIG. 3.
Synthesis of fusion proteins with model antigens. The S. enterica serovar Typhimurium wild-type strain with the empty plasmid vector (vector) or harboring plasmids of the expression of sseF::OVA::M45 (A) or sseF::lisA::HA (B) under the control of various SsrAB-controlled promoters was grown in SPI2-inducing minimal media (PCN-P, pH 5.8). As negative controls, ssrB_-deficient strain (MvP532, Δ_ssrB::FRT) harboring plasmids with _PsseA_-containing expression cassettes was used. Bacteria were harvested after 8 h of culture under inducing conditions, and equal amounts of bacteria (determined based on the optical density at 600 nm) were lysed and subjected to SDS-PAGE and Western blot analyses for the detection of the M45 (A) or HA (B) epitope tag. Blots were probed with fluorescently labeled secondary antibodies, and signal intensities were quantified by using the Odyssey system (Li-Cor). As loading controls, the cytosolic heat shock protein DnaK was detected on the same blot, and signals were quantified. The ratios of the M45 to DnaK or HA to DnaK signals were calculated, and means and standard deviations for three samples are shown. Statistical analyses were performed comparing signals for expression cassettes with various promoters to signals for expression cassettes with PsseA (indicated by plain lines) or by comparing various expression cassettes in a WT background to the PsseA expression cassette in an ssrB background (indicated by lines with arrows). Statistical analysis: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG. 4.
Translocation of fusion proteins by intracellular Salmonella. WT S. enterica serovar Typhimurium harboring plasmids with cassettes for the expression of sseF::lisA::HA under the control of various promoters was used to infect BM-DC at an MOI of 25. The cells were fixed 16 h after infection and processed for immunostaining for DC marker CD11c (blue), intracellular Salmonella (green), and translocated fusion protein SseF-Llo-HA (red). CD11c+ and _Salmonella_-infected cells were identified by confocal laser scanning microscopy and imaged by using the ZEN software package (Zeiss). (A) Representative infected BM-DC with Salmonella WT harboring an expression cassette with PsseJ translocating SseF-Llo-HA. The white line in the SseF-Llo-HA micrograph defines the area used for quantification of the immunofluorescence signals. (B) After infection with Salmonella WT harboring plasmids with various expression cassettes, infected cells with similar amounts of intracellular Salmonella were selected for the various conditions, and the signal intensities of the Cy3 channel for the anti-HA strains were measured with identical exposure times. The mean signal intensities and standard deviations for at least 20 infected cells per strains are shown. Statistical analyses were performed comparing signals for expression cassettes with various promoters to signals for expression cassettes with PsseA (indicated by a plain line) or by comparison to mock-infected cells (indicated by a line with arrows). Statistical analysis: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG. 5.
Stimulation of T-cell responses by recombinant Salmonella vaccines. Murine BM-DC were infected at an MOI of 25 with Salmonella WT, the Δ_purD_ Δ_htrA_ carrier strain, or ssaV or ssrB strains as indicated. Strains harbored the empty plasmid vector or plasmids with expression cassettes for sseF::OVA::M45 under the control of various SsrAB-controlled promoters as indicated. As a positive control, BM-DC were infected with WT Salmonella and stimulated with the SIINFEKL peptide. The infected BM-DC were incubated with B3Z reporter cell line, and after coculture for 24 h, the β-galactosidase substrate chlorophenyl red β-galactopyranoside was added. After additional incubation for 6 h, the reaction was stopped, and the β-galactosidase product was quantified photometrically by measurement of the absorbance at 595 nm. (A) T-cell stimulation was analyzed at various ratios of infected BM-DC to T cells. For further experiments, a DC/T-cell ratio of 1:4 was used. (B) Effect of various SsrAB-controlled promoters on the stimulation of T cell. The means and standard deviations of triplicate samples are shown, and the data sets are representative of six independent experiments. Statistical analyses were performed comparing stimulation by strains harboring expression cassettes with various promoters to strains with expression cassettes with PsseA (indicated by plain lines) or by comparing stimulation by strains with various expression cassettes to the vector control (indicated by lines with arrows). Statistical analysis: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG. 6.
Stimulation of CD4+ and CD8+ T-cell proliferation by recombinant Salmonella vaccines. BM-DC were infected with Salmonella WT (A) or the attenuated Δ_purD_ Δ_htrA_ carrier strain MvP728 (B), each harboring plasmids with various expression cassettes for expression of sseF::OVA:M45. As a control for the release of antigen independent of T3SS translocation, the T3SS-deficient ssaV strain expressing sseF::OVA::M45 under the control of PsseA was used. The infection of BM-DC was performed basically as described for Fig. 5. Infected BM-DC and intracellular bacteria were inactivated after 3 h of infection. Inactivated cells were added to T cells prepared from OT-I or OT-II mice as indicated, and T-cell stimulation was allowed for 24 h. For quantification, the incorporation of [3H]thymidine was determined and is expressed as counts per minute (CPM). The means and standard deviations of triplicate samples are shown for CPM of OT-1 cells (black bars) or OT-II cells (gray bars). The assays are representative of three assays with similar results. Statistical analyses were performed comparing stimulation by strains harboring expression cassettes with various promoters to strains with expression cassettes with PsseA (indicated by plain lines) or by comparing stimulation by strains with various expression cassettes to the vector control (indicated by lines with arrows). Statistical analysis: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG. 7.
Immune response after vaccination with recombinant Salmonella carrier strains. Organ distribution (A) and fecal shedding (B) after oral vaccination of mice with the Δ_purD_ Δ_htrA_ carrier strain MvP728 harboring plasmid p2810. (A) Four mice were sacrificed 15 days after immunization, and serial dilutions of homogenates of liver, spleen, mesenteric lymph nodes (MLN), and Peyer's patches (PP) were plated onto agar plates for determination of the organ burden with Salmonella. For the spleen, PP, and MLN the CFU per organ is indicated, and for the liver the CFU per gram of liver is indicated. (B) The presence of the vaccine strain in fecal pellets was determined over a period of 20 days (days 2, 5, 7, 9, 12, 14, and 20 postvaccination). Approximately 100 mg of fresh fecal pellets was homogenized in 1 ml of PBS, and serial dilutions were platted on LB agar plates containing carbenicillin (50 μg ml−1) for bacterial enumeration of the fecal shedding of individual mice (limit of detection, 0.1 CFU mg of feces−1). The means are indicated by horizontal bars. (C to E) Groups of seven female mice were orally vaccinated with the Δ_purD_ Δ_htrA_ carrier strain MvP728 harboring plasmids for the expression of the sseF::lisA::HA gene fusion under the control of various promoters as indicated or the vector control. Booster immunizations were performed on days 14 and 28 after the primary vaccination. (D) Blood was collected at 10, 21, and 35 days after prime vaccination and pentamer staining for Llo-responsive CD8+ cells in peripheral blood was performed to determine immune responses at various time points after vaccination. The means and standard deviations are shown, and statistical analysis was performed for samples obtained 35 days after vaccination. (E) Mice were sacrificed 35 days after prime vaccination, the spleens were recovered, and suspensions of splenocytes were analyzed for Llo-responsive CD8+ cells as for panel D. Means and standard deviations of analyses of cohorts of immunized mice are given, and statistical analysis of vaccinated cohorts to the vector control was performed. Examples of FACS analyses of splenocytes are shown in panel C, and the percentages of gated cells are indicated. Statistical analysis: n.s., not significant; *, P < 0.05; **, P < 0.01.
References
- Blanchard, N., and N. Shastri. 2010. Cross-presentation of peptides from intracellular pathogens by MHC class I molecules. Ann. N. Y. Acad. Sci. 1183:237-250. - PubMed
- Cheminay, C., and M. Hensel. 2008. Rational design of Salmonella recombinant vaccines. Int. J. Med. Microbiol. 298:87-98. - PubMed
- Cornelis, G. R. 2006. The type III secretion injectisome. Nat. Rev. Microbiol. 4:811-825. - PubMed
- Galan, J. E., and H. Wolf-Watz. 2006. Protein delivery into eukaryotic cells by type III secretion machines. Nature 444:567-573. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous