Figure 3.

CARVE-mediated generation of novel immunotherapeutic STINGPOX. (A) Mammalian expression plasmid screen of bacterial cyclases. HEK293T were transfected with expression plasmids for noted cyclase and then lysed 24 hours post-transfection. Diluted clarified lysates were then used to treat THP- Blue ISG cells, and SEAP reporter assay was performed to measure relative effect on IFN signaling (n=3). (B) CARVE-mediated rescue of OncoVACV +MtbDisA inserted at the B8R locus. Virus underwent traditional CARVE protocol with transfection of HRT encoding DisA (1st round) with or without sgB8R, virus produced with sgB8R in this step underwent a second round of selection where sgB8R was transfected alone, following infection. Resulting virus was used to infect confluent U2OS and plaques were scored for EGFP fluorescence (n = 3). (C, D) Cyclic di-AMP ELISA was used to measure cyclic di-AMP levels in HeLa cell (C) lysates and (D) supernatants - 48 hours post-infection with STINGPOX or OncoVACV (n = 3). (E) 293-Dual hSTING-R232 cells SEAP reporter assay was performed to measure effects of infection with STINGPOX or OncoVACV on IFN signaling (48 hours post-infection; n = 3). (F, G) qRT-PCR analyses of relative (F) IFNB1 and (G) RSAD2 mRNA levels in HT29 cells, 48 hours-post infection with STINGPOX or OncoVACV (MOI = 0.1; n≥2). Data are shown as mean ± SEM (p* < 0.05, p** < 0.01, p**** < 0.001), not significant (ns).