Optimization of a Type Three Secretion System-based P. aeruginosa live vector for antigen delivery

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

^* To whom correspondence should be addressed. Email: BPolack{at}chu-grenoble.fr.

	Abstract

During the last years, the use of type III secretion system-based bacterial vectors for immunotherapy purpose has been assessed in various applications. We showed that a type III secretion-based P. aeruginosa vector delivering the 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 effect of the deletion of quorum sensing genes and aroA gene toward toxicity and efficiency of the vector strain. AroA mutation of our strain (making the strain auxotrophic for aromatic aminoacids) conferred a strikingly reduced toxicity, with a bacteria lethal dose more than 100 times higher than with the parental strain. Quorum sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, OVA-delivering aroA-deficient mutant was the most efficient at low dose (10⁵), but dose enhancement was not associated with greater immune response. Quorum-sensing deficient strain was the most efficient at mild dose (10⁶), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at elevated dose (10⁷), in agreement with known negative effects of quorum-sensing molecules upon T cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding to toxicity and efficiency for further developments in both anti-tumour and anti-infectious strategies.