Optimization of a Type Three Secretion System-based P. aeruginosa live vector for antigen delivery Running title: optimization of TTSS-based P. aeruginosa vaccine vector Authors:

1 During the last years, the use of type III secretion system-based bacterial vectors for 2 immunotherapy purpose has been assessed in various applications. We showed that a type 3 III secretion-based P. aeruginosa vector delivering the OVA antigen induced an efficient 4 specific CD8+ T lymphocyte immune response against OVA-expressing cells. Because of 5 the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we 6 explored the effect of the deletion of quorum sensing genes and aroA gene toward toxicity 7 and efficiency of the vector strain. AroA mutation of our strain (making the strain 8 auxotrophic for aromatic aminoacids) conferred a strikingly reduced toxicity, with a 9 bacteria lethal dose more than 100 times higher than with the parental strain. Quorum 10 sensing gene mutation alone was associated with a slightly reduced toxicity. In a 11 prophylactic OVA-expressing melanoma mouse model, OVA-delivering aroA-deficient 12 mutant was the most efficient at low dose (10 5), but dose enhancement was not associated 13 with greater immune response. Quorum-sensing deficient strain was the most efficient at 14 mild dose (10 6), but this dose was close to the toxic dose. Combination of both mutations 15 conferred the highest efficiency at elevated dose (10 7), in agreement with known negative 16 effects of quorum-sensing molecules upon T cell activation. In conclusion, we have 17 obtained a promising immunotherapy vector regarding to toxicity and efficiency for further 18 developments in both anti-tumour and anti-infectious strategies. 3 The use of live bacteria and bacterial virulence factors as therapeutic tools in 1 human medicine has been considered for more than a century. The observation that the 2 onset of a bacterial infection could modify the course of a malignant disease (6) was a 3 hallmark in this history, but very few procedures (such as the intra-vesical administration 4 of an attenuated Mycobacterium bovis strain for the cure of non-invasive urothelial 5 carcinoma) had been finally used in routine. In the last 10 years, better characterization of 6 bacterial devices (mainly toxins and secretion systems) and extensive progresses in 7 genomic studies allowed engineering of bacteria (mainly E. coli and Salmonella spp). 8 These domesticated agents can be delivered to mammals organisms for different purposes. 9 Notably, the design of antigen-delivering bacteria triggering antigen-specific cytotoxic 10 CD8+ T lymphocyte response is an emerging investigation field in vaccine development 11 (5). Antigen delivery can …