Optimization of a type III secretion system-based Pseudomonas aeruginosa live vector for antigen delivery

Abstract

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 (10(5)), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (10(6)), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (10(7)), 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.

FIG. 1.

Determination of the best time schedule for vaccination using strain CHA-OST. Mice received the same dose of ovalbumin-delivering CHA-OST with different delays from tumor implantation (on day 0): at days −14 and −7, at days −7 and 0, at days 0 and day +7, or at days +7 and +14. A day −14 and day −7 schedule using a CHA-OST strain delivering no antigen was used as a negative control. Mice were sacrificed when the tumor diameter reached 1 cm.

FIG. 2.

In vitro TTSS efficiencies of mutants. (A) Fluorescence intensity in the culture pellet of the wild-type (WT) strain and different mutants transformed by plasmid pS54-GFP_ExsAi under different TTSS-activating conditions. The negative control was CHA-OST without plasmid. Error bars represent 1 standard error. (B) SDS-PAGE analysis of secretion of S54-Ova by the WT strain and different mutants transformed by plasmid pS54-Ova_Exsai. Culture medium was supplemented with both IPTG and EGTA. The negative control was CHA-OST without plasmid.

FIG. 3.

In vivo toxicity (A) and efficiency (B) of TTSS-based mutant vectors. (A) Mouse mortality after subcutaneous injection of 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ mutants. (B) Survival after tumor implantation in mice vaccinated beforehand twice with 10⁵ (thick pale gray line), 10⁶ (thick dark gray line), or 10⁷ (thick black line) mutant vectors delivering S54-Ova: CHA-OST, CHA-ORL, CHA-OA, and CHA-OAL. The negative control was 10⁶ CHA-OST not delivering S54-Ova (thin line).