File S1 - Mechanistic Insight into the TH1-Biased Immune Response to Recombinant Subunit Vaccines Delivered by Probiotic Bacteria-Derived Outer Membrane Vesicles

Supporting figures. Figure S1, Isolation of EcN immunostimulatory factors from immunosuppressive ones via OMV formation leads to an induction of a strong immune response. (A) Natural immunosuppressive function of Nissle 1917 E. coli (EcN), focusing on direct effector action on αβ T-lymphocytes. (B) By isolating the targeting and immunostimulatory potential of the EcN membrane from EcN’s secretory immunosuppressive capabilities, and turning it into a bionanoparticle delivery device for the natural immunostimulatory milieu present in bacterial OMVs already, the normal immunosuppressive function of the probiotic bacteria is replaced with a powerful adjuvanting effect. Figure S2, Further comparative analysis of EcN and EcJ OMV vaccine formulations. (A) Dynamic light scattering hydrodynamic z-average particle sizes of EcJ and EcN OMVs (formulations assessed in PBS). (B) OMV zeta potentials assessed in PBS. (C) Dynamic light scattering hydrodynamic z-average particle sizes of EcN and EcN-lpxM OMVs (formulations assessed in PBS). (D) GFP fluorescence-standardized ClyA-GFP(+) vaccine doses of EcN and EcN-lpxM OMVs assayed for total protein content via BCA assay. #No significant difference (P>0.05). All values are given as mean +/− SD. Figure S3, EcN lpxm mutation does not result in detrimental loss of robust humoral immunity stimulation in a mouse model. Terminal titers of antigen-specific IgG from BALB/c mice vaccinated (primed) and boosted once with antigen-normalized doses (n = 5, each group). Experimental groups indicated are as follows: mice injected with recombinant GFP in PBS alone, GFP; with EcN OMVs from ECN containing the lpxM mutation, displaying ClyA-GFP, EcN-LpxM OMV-GFP; with EcN OMVs from non-lpxM mutant EcN, displaying ClyA-GFP, EcN OMV-GFP; with a mixture of recombinant ClyA-GFP and alum, Alum+ClyA-GFP. **P (DOCX)