Immunization With Skp Delivered on Outer Membrane Vesicles Protects Mice Against Enterotoxigenic Escherichia coli Challenge

Abstract

Outer membrane vesicles (OMVs) are promising vaccine components because they combine antigen and adjuvant in a single formulation. Detoxified Salmonella enterica strains that express penta-acylated lipid A retain OMV immunogenicity but with reduced reactogenicity. We have previously shown that a recombinant form of the enterotoxigenic Escherichia coli (ETEC) 17 kilodalton protein (Skp) protects mice in a pulmonary challenge model, when fused to the glutathione-S-transferase (GST) epitope and combined with cholera toxin. Here we compared directly the efficacy of expressing Skp in detoxified Salmonella OMVs to GST-Skp for their ability to protect mice against ETEC challenge. We observed that the display of Skp on OMVs, in the absence of exogenous adjuvant, protects the mice as well as the recombinant GST-Skp with adjuvant, showing that we can achieve protection when antigen and adjuvant are administered as a single formulation. Collectively, these data demonstrate the utility of using OMVs for the expression and display of antigens for use in vaccine development and validate previously published work demonstrating that immunization with Skp is efficacious in protecting mice against ETEC challenge.

Keywords: enterotoxigenic Escherichia coli; infection; mouse models; outer membrane vesicles; vaccines.

Figure 1

Design and purification of OMV-based Skp vaccine (A) Schematic. This figure was created using Servier Medical Art (https://smart.servier.com/) (B). Coomassie stained SDS-PAGE of the OMV based vaccine stocks used in the study. An amount of 0.5 OD units of OMV material was loaded in each lane. HbpD(Δd1) (110 kDa), HbpD-SkpF1 (129 kDa), HbpD-SkpF2 (131 kDa), and HbpD-SkpF1F2 (131 kDa) are marked by asterisks. The OMV porins (OmpF/C and OmpA) are indicated with arrowheads (C). Coomassie stained SDS-PAGE, showing equal amounts of intact (−tx) and Triton X-100 permeabilized (+tx) OMVs treated with (+pk) or without (−pk) Proteinase K. All Hbp-antigens [as well as HbpD(Δd1) control] were exposed at the OMV surface, based on their sensitivity to externally added Proteinase K.

Figure 2

Impact of vaccination on mouse survival after pulmonary challenge with ETEC (A). Mouse survival is plotted as a function of time (h) after mice were inoculated with ETEC H10407 following intranasal immunization with the indicated antigens, n = 4. Asterisks indicate significantly different (p < 0.05) mouse survival, log-rank test (B). ETEC loads (CFUs/g lung) in mice infected with ETEC H10407 at time of euthanasia or at the end of the study (7 days). Open symbols indicate mice that survived for the duration of the study. Closed symbols indicate mice that were euthanized due to their display of clinical signs of illness, n = 4. Asterisks indicate significantly different (p < 0.05) ETEC loads in mouse lungs, Kruskal-Wallis test (C). Fold change in mouse fecal IgA concentrations after immunization with the indicated antigens. Open symbols indicate mice that survived for the duration of the study. Closed symbols indicate mice that were euthanized due to their display of clinical signs of illness. n = 4. Asterisks indicate significantly different (p < 0.05) fecal IgA concentrations, Kruskal-Wallis test.