Interrogation of a live-attenuated enterotoxigenic Escherichia coli vaccine highlights features unique to wild-type infection

Abstract

Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of severe diarrheal illness in low- and middle-income countries. The live-attenuated ACE527 ETEC vaccine, adjuvanted with double mutant heat-labile toxin (dmLT), affords clear but partial protection against ETEC challenge in human volunteers. Comparatively, initial wild-type ETEC challenge completely protects against severe diarrhea on homologous re-challenge. To investigate determinants of protection, vaccine antigen content was compared to wild-type ETEC, and proteome microarrays were used to assess immune responses following vaccination and ETEC challenge. Although molecular interrogation of the vaccine confirmed expression of targeted canonical antigens, relative to wild-type ETEC, vaccine strains were deficient in production of flagellar antigens, immotile, and lacked production of the EtpA adhesin. Similarly, vaccination ± dmLT elicited responses to targeted canonical antigens, but relative to wild-type challenge, vaccine responses to some potentially protective non-canonical antigens including EtpA and the YghJ metalloprotease were diminished or absent. These studies highlight important differences in vaccine and wild-type ETEC antigen content and call attention to distinct immunologic signatures that could inform investigation of correlates of protection, and guide vaccine antigen selection for these pathogens of global importance.

Keywords: Bacterial host response; Bacterial infection; Gastroenteritis; Live attenuated vaccines.

Fig. 1

vaccine strain molecular characterization. a PCR confirmation of ACE527 genotypes. Shown are (top) multiplex enterotoxin (eltB, estP, and estH) PCR; (middle) eatA autotransporter gene; (bottom) etpA amplification from wild-type parental ETEC strains, corresponding live-attenuated ACE527 vaccine constructs, and controls. H10407 and MG1655 are shown at right as a positive and negative controls, respectively. b Immunoblots (TCA-precipitated culture supernatants) for secreted antigens EatA passenger domain (EatA_p), the EtpA adhesin, and the YghJ metalloprotease. (Gel and blot images in (a) and (b) respectively were each derived from a single set of experiments). c Growth curves of parental strains (closed symbols) and corresponding vaccine strains (open symbols). H10407 growth curve (blue symbols) is shown for comparison. Summary of proteomic data from interrogation of ACE527 parents and vaccine strains. Parent strain and corresponding mutant are paired on the X axis. d Heat map values reflect the maximum normalized total spectra obtained from analysis of secretome, outer membrane and outer membrane vesicles. Colonization factors are grouped at the top of the map and color-coded on the Y axis by function. e ACE527 parental and vaccine strains are immotile in soft agar assays. H10407 (bottom left) and an isogenic fliC mutant (bottom right) are shown as positive and negative motility controls, respectively

Fig. 2

Immune response to purified colonization factor antigens and secreted ETEC virulence factors following vaccination and challenge. Bar graphs on left depict the difference in mean ALS IgA responses from day-1 to day 7 following vaccination with placebo, ACE527, and ACE527+ dmLT (gray bars), or following challenge with H10407 (blue bars). Antigens listed on the Y axis include all major colonization factors and subunits (top) and secreted virulence proteins LT (A and B subunits), EtpA (carboxy terminal repeat region EtpA_c, and amino terminal secretion domain Etp_n), EatA (passenger domain EatA_p), and YghJ (bottom). The column on the far left indicates antigens present in (+) or absent from (−) the ACE527 vaccine strains. Antigens present in the H10407 challenge strain are depicted in Blue. Heat map at right depicts the proportion of volunteers exhibiting at least a 50% increase in signal intensity at day 7 following vaccination relative to baseline (day 0) values. Challenge values depicted are from day 7 following challenge of the placebo group with H10407

Fig. 3

ALS IgA responses to non-canonical secreted antigens. a Comparison of ALS IgA responses against IVTT-produced flagellins representing the serotypes of the ACE527 strains (H5, H16, H45) and the H10407 challenge strain (H11). Shown are responses to three recombinant protein antigens: b YghJ, c the passenger domain of EatA (EatAp), and d the amino terminal region of EtpA (EtpA_N) secreted by the ETEC H10407 challenge strain and/or the ACE527 vaccine strains. Columns in each group represent data obtained at day 0, day 7 following vaccination, or day 7 following challenge with abbreviated labels of “0”, “7”, and “ch” respectively. Data in each vaccination or control group are represented as corrected for the baseline values obtained at day 0. Each symbol represents the change in normalized ALS IgA signal at baseline (day 0) to day 7 post immunization or after placebo administration. Values on the y-axis for B-D are expressed as the % difference of (signal-background)/background. (*<0.05, **<0.01, ***<0.001, **** ≤ 0.0001 by ANOVA comparison of normalized signal data with corresponding day 0 data without background correction; Kruskal–Wallis test for multiple comparisons). Open bars represent mean values