Immunization with the MipA, Skp, or ETEC_2479 Antigens Confers Protection against Enterotoxigenic E. coli Strains Expressing Different Colonization Factors in a Mouse Pulmonary Challenge Model

Abstract

Achieving cross-protective efficacy against multiple bacterial strains or serotypes is an important goal of vaccine design. Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in underdeveloped nations. We have been interested in identifying and characterizing ETEC antigens that generate protective immune responses independent of ETEC colonization factor (CF) expression. Our previous studies used proteomics to identify the ETEC MipA, Skp, and ETEC_2479 proteins as effective in protecting mice from homologous challenge with ETEC H10407 using a pulmonary inoculation model. This model permits analysis of mouse survival, bacterial clearance, and the production of secretory IgA (sIgA) and has been employed previously for studies of enteric pathogens for which robust oral challenge models do not exist. MipA belongs to a family of proteins involved in remodeling peptidoglycan. Skp rescues misdirected outer membrane proteins. ETEC_2479 is predicted to function as an outer membrane porin. These proteins are conserved in pathogenic ETEC strains as well as in commensal Proteobacteria. Antibodies produced against the ETEC MipA, Skp, and ETEC_2479 proteins also reduced the adherence of multiple ETEC strains differing in CF type to intestinal epithelial cells. Here we characterized the ability of 10 heterologous ETEC strains that differ in CF type to cause clinical signs of illness in mice after pulmonary challenge. ETEC strains C350C1A, E24377A, E7476A, WS2173A, and PE360 caused variable degrees of lethality in this mouse model, while ETEC strains B7A, WS6866B, 2230, ARG-2, and 8786 did not. Subsequent challenge experiments in which mice were first vaccinated intranasally with MipA, Skp, or ETEC_2479, when combined with cholera toxin, showed both that each antigen was protective and that protection was strongly correlated with fecal IgA concentrations. We conclude that the MipA, Skp, or ETEC_2479 antigens generate protection in the mouse pulmonary challenge model against ETEC strains that express different CFs.

Keywords: ETEC; antigens; colonization factor; intranasal immunization; vaccines.

Figure 1

ETEC strains that cause illness in the mouse pulmonary challenge model. (A) Mouse survival is plotted as a function of time (h) after mice were inoculated with the indicated ETEC strains. H10407, n = 29; 350C1A and E24377A, n = 15; E7476A, WS2173A, and PE360 n = 13; B7A, WS6866B, 2230, ARG-2, and 8786, n = 5/group. Asterisks indicate significantly different (p < 0.05) mouse survival, log-rank test. (B) ETEC loads (CFUs/g lung) in mice infected with the indicated ETEC strains at time of euthanasia or at the end of the study (7 d), n = 5/group. Asterisks indicate significantly different (p < 0.05) ETEC loads in mouse lungs, Kruskal-Wallis test.

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 the indicated ETEC strains following intranasal immunization with the indicated antigens, n = 10–15. Asterisks indicate significantly different (p < 0.05) mouse survival, log-rank test. (B) ETEC loads (CFUs/g lung) in mice infected with the indicated ETEC strains at time of euthanasia or at the end of the study (7 d). 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 = 10–15. 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 = 10. Asterisks indicate significantly different (p < 0.05) fecal IgA concentrations, Kruskal-Wallis test.