Safety and immunogenicity of Salmonella typhimurium expressing C-terminal truncated human IL-2 in a murine model

Abstract

Salmonella enterica serovar Typhimurium preferentially colonizes tumors in vivo and has proven to be an effective biologic vector. The attenuated S. enterica Typhimurium strain chi4550 was engineered to express truncated human interleukin-2 and renamed SalpIL2. Previously, we observed that a single oral administration of SalpIL2 reduced tumor number and volume, while significantly increasing local and systemic natural killer (NK) cell populations in an experimental metastasis model. Here we report that in nontumor-bearing mice, a single oral dose of SalpIL2 resulted in increased splenic cytotoxic T and NK cell populations that returned to control levels by 4 weeks post oral administration. Though SalpIL2 was detected in mouse tissues for up to 10 weeks, no prolonged alterations in peripheral blood serum chemistry or complete blood cell counts were observed. Similarly, comparative histopathological analysis of tissues revealed no significant increase in pyogranulomas in SalpIL2-treated animals with respect to saline controls. In Rag-1 knockout mice, which have severely impaired B and T cell function, SalpIL2 reduced growth of hepatic metastases. Furthermore, SalpIL2 altered expression of several proinflammatory cytokines and chemokines in the serum of mice with pulmonary osteosarcoma metastases. These data further suggest that SalpIL2 is avirulent and induces a cell-mediated antitumor response.

Keywords: Salmonella Typhimurium; interleukin-2; natural killer cells.

Figure 1

The effect of a single oral administration of SalpIL2 on mouse serum chemistry. Asterisks indicate statistically significant changes in serum chemistry as determined by unpaired Student’s t-test. Results presented are mean values from groups of five SalpIL2-treated and five saline control animals. Notes: Error bars represent one standard deviation. Alkaline phosphatase is denoted as Alk φ. *P < 0.05, **P < 0.01.

Figure 2

The effect of a single oral administration of SalpIL2 on complete blood cell counts. Asterisks indicate differences determined to be statistically significant by unpaired Student’s t-test. White blood cells (WBC), neutrophils (NE), monocytes (MO), eosinophils (EO). Results presented are mean values from groups of five SalpIL2-treated and five saline control animals. Notes: Error bars represent one standard deviation. *P < 0.05.

Figure 3

The effect of a single oral administration of SalpIL2 on blood cell phenotype. Mean corpuscular volume (MCV), red blood cell distribution width (RDW), platelet (PLT) and mean platelet volume (MPV) average values are from groups of five SalpIL2-treated and five control animals per time point. Notes: Error bars represent one standard deviation. Differences determined to be statistically significant by unpaired Student’s t-test are indicated by asterisks. *P < 0.05, ** P < 0.01.

Figure 4

Natural killer (NK) cells and cytotoxic T lymphocytes are significantly increased in the acute systemic immune response to SalpIL2. NK cells (NK 1.1) and cytotoxic T cells (CD8⁺) populations are significantly increased in response to SalpIL2. Helper T cells (CD4⁺) populations are not significantly affected by a single oral administration of 2 × 10⁸ CFU of SalpIL2 or Sal-NG. Notes: Results from triplicate experiments presented as ± standard error of mean, were determined to be statistically significant by unpaired Fisher’s exact test and indicated by asterisks. *P < 0.05.

Figure 5

SalpIL2 antitumor effect is independent of cytotoxic T lymphocytes. SalpIL2 increases NK cell population and reduces the macroscopic volume of hepatic metastases in Rag-1^–/– mice, which have significantly suppressed T lymphocyte populations. Notes: Results from one representative experiment shown, N = 7. *P < 0.05, ***P < 0.001.

Figure 6

Oral inoculation of Salmonella alters serum cytokines during treatment of metastatic osteosarcoma. SalpIL2, but not Sal-NG significantly increases serum G-CSF with respect to saline controls (P < 0.05 and P = 0.276, respectively). Notes: Results presented as SEM from triplicate experiments. *p < 0.05. Abbreviation: G-CSF, granulocyte colony-stimulating factor.

Figure 7

Oral inoculation of Salmonella alters serum cytokines during the prevention of metastatic osteosarcoma. Sal-NG significantly lowers IL-1 β (P < 0.01), KC (P < 0.05) and MCP-1 (P < 0.001) with respect to control animals. SalpIL2 significantly decreases systemic IL-1 β (P < 0.001), IL-3 (P < 0.05), KC (P < 0.01), MCP-1 (P < 0.001), MIP-1 β (P < 0.05), and RANTES (P < 0.05) as compared to saline controls. Notes: *P < 0.05, **P < 0.01, ***P < 0.001.