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Optimization of a Type III Secretion System-Based Pseudomonas aeruginosa Live Vector for Antigen Delivery

TIMC-TheREx (UMR5525 CNRS-UJF), Bâtiment Jean Roget, 8th Floor, UFR de Médecine, Université Joseph Fourier Grenoble 1, 38706 La Tronche Cedex, France

Received 19 June 2007/ Returned for modification 1 November 2007/ Accepted 30 November 2007

During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. We showed that a type III secretion-based Pseudomonas aeruginosa vector delivering the ovalbumin (OVA) antigen induced an efficient specific CD8⁺ T-lymphocyte immune response against OVA-expressing cells. Because of the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we explored the effects of the deletion of quorum-sensing genes and the aroA gene toward toxicity and efficiency of the vector strain. The aroA mutation in our strain (making the strain auxotrophic for aromatic amino acids) conferred a strikingly reduced toxicity, with the bacterial lethal dose being more than 100 times higher than that of the parental strain. The quorum-sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, an OVA-delivering aroA-deficient mutant was the most efficient at a low dose (10⁵), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (10⁶), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (10⁷), in agreement with the known negative effects of quorum-sensing molecules upon T-cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding toxicity and efficiency for further developments in both antitumor and anti-infectious strategies.

Published ahead of print on 19 December 2007.