Influence of Vector-Encoded Cytokines on Anti-SalmonellaImmunity: Divergent Effects of Interleukin-2 and Tumor Necrosis Factor Alpha

Abstract

Attenuated Salmonella strains are of interest as new vaccine candidates and as vectors of cloned genes of other organisms. Attenuated strains expressing specific cytokines were constructed as a means of manipulating the immune response in various disease settings. In the present study, interleukin-2 (IL-2)-expressing (GIDIL2) or tumor necrosis factor alpha (TNF-␣)-expressing (GIDTNF) strains were compared with the parent strain (BRD509) for the effect of cytokines on anti-Salmonella immunity. Expression of IL-2 resulted in a rapid clearance of the organism soon after vaccination. The reduction in GIDIL2 CFU was 50-to 300-fold higher than that of BRD509 and correlated with a markedly decreased splenomegaly. Furthermore, no evidence for any significant activation, including upregulation of surface markers and production of nitric oxide (NO), was observed in spleens of GIDIL2-injected mice. In contrast, the host response to GIDTNF was marked by an early, strong, splenic cellular influx, but surprisingly, the degree of induced splenomegaly and NO secretion was only 50% of that observed in BRD509-treated mice. Despite this, bacterial colonization of the spleen in GIDTNF-immunized animals was either slightly decreased from or equivalent to that of the BRD509-treated group, suggesting the induction of additional antimicrobial mechanisms by TNF-␣. In vivo protection studies demonstrated that, at limiting doses, GIDIL2 was inferior to GIDTNF and BRD509 in its capacity to protect against virulent challenge. At high doses, however, all three strains exhibited equal protective efficacy. These results demonstrate that the immune response against intracellular bacteria can be manipulated by pathogenexpressed cytokines and open the way for further fine tuning of immune responses not only to Salmonella strains themselves but also to the heterologous gene(s) carried by them.

CD154 Is Essential for Protective Immunity in Experimental Salmonella Infection: Evidence for a Dual Role in Innate and Adaptive Immune Responses

“…The characteristics of the bacterial strains have been previously described (18). BRD509 is an attenuated aroA Ϫ /aroD Ϫ mutant strain of S. enterica serovar Typhimurium, and SL1344 is the parental wild-type strain from which BRD509 was originally derived (19,20).…”

“…To determine the bacterial load in liver and spleen, organs were removed aseptically and homogenized in 3-5 ml of cold sterile water, as previously described (18). A 100-l aliquot of the homogenate or an appropriate dilution was plated on T-soy agar plates, and viable CFU were determined after overnight incubation.…”

“…Accumulation of NO 2 Ϫ was used to determine the production of NO according to the Griess method, as described previously (18). Briefly, 100 l of cell-free culture supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 -15 min.…”

“…90 days previously with a dose of 0.6 ϫ 10 6 BRD509/mouse. Processing of spleen cells for FACS analysis was conducted as described previously (18). Briefly, washed cells were resuspended in staining buffer (PBS/1% FCS/ 0.1% NaN 3 ) to a concentration of 1 ϫ 10 7 /ml.…”

The Organophosphate Paraoxon Has No Demonstrable Effect on the Murine Immune System following Subchronic Low Dose Exposure

“…Erythrocyte-depleted spleen single cell suspensions were prepared, as previously described (13), in supplemented RPMI medium with 5% FCS (GibcoBRL, Paisley, UK). Cells were cultured at a concentration of2-5 x l O" cells/ml in the presence or absence of Con A or LPS at the indicated concentrations.…”