Contribution of Noncanonical Antigens to Virulence and Adaptive Immunity in Human Infection with Enterotoxigenic E. coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) contributes significantly to the substantial burden of infectious diarrhea among children living in low- and middle-income countries. In the absence of a vaccine for ETEC, children succumb to acute dehydration as well as nondiarrheal sequelae related to these infections, including malnutrition. The considerable diversity of ETEC genomes has complicated canonical vaccine development approaches defined by a subset of ETEC pathovar-specific antigens known as colonization factors (CFs). To identify additional conserved immunogens unique to this pathovar, we employed an "open-aperture" approach to capture all potential conserved ETEC surface antigens, in which we mined the genomic sequences of 89 ETEC isolates, bioinformatically selected potential surface-exposed pathovar-specific antigens conserved in more than 40% of the genomes (n = 118), and assembled the representative proteins onto microarrays, complemented with known or putative colonization factor subunit molecules (n = 52) and toxin subunits. These arrays were then used to interrogate samples from individuals with acute symptomatic ETEC infections. Surprisingly, in this approach, we found that immune responses were largely constrained to a small number of antigens, including individual colonization factor antigens and EtpA, an extracellular adhesin. In a Bangladeshi cohort of naturally infected children <2 years of age, both EtpA and a second antigen, EatA, elicited significant serologic responses that were associated with protection from symptomatic illness. In addition, children infected with ETEC isolates bearing either etpA or eatA genes were significantly more likely to develop symptomatic disease. These studies support a role for antigens not presently targeted by vaccines (noncanonical) in virulence and the development of adaptive immune responses during ETEC infections. These findings may inform vaccine design efforts to complement existing approaches.

Keywords: diarrhea; enteric pathogens; pathogenesis; surface antigens; vaccines.

FIG 1

Serologic response to noncanonical antigens following natural infection. (A) Heat map of log₂-transformed Z-score data indicating ETEC protein microarray responses from days 2 and 30 following presentation to the icddr,b to four noncanonical antigens, EtpA, YghJ, the passenger domain of EatA, and EaeH, and the B subunit of ETEC heat-labile toxin (LT-B). (B) Kinetic ELISA responses to EtpA and EatA following infection. Data are segregated by the presence or absence of the respective antigen in the strain recovered at presentation. Negative-control samples from St. Louis Children’s Hospital (slch) are shown as open circles. *, P < 0.05 by a Wilcoxon matched-pairs signed-rank test.

FIG 2

ALS responses to EtpA or EatA. Shown are microarray data for IgA (top) and IgG responses to EtpA (left) and the passenger domain of EatA (EatA_p) (right) on days 2 and 7 following hospitalization. Data in each graph are segregated according to antigen expression in the infecting strain (negative or positive). P values reflect Kruskal-Wallis values, with post hoc analysis using Dunn’s test adjusted for multiple comparisons for between-group analysis.

FIG 3

Anti-EtpA or anti-EatA IgG responses increase with age. Shown are representative kinetic ELISA data for serum IgG samples obtained from children aged 1 to 24 months enrolled in a birth cohort study. Scatterplots of anti-EtpA and anti-EatA IgG plotted against data for children aged 3 to 24 months with regression lines from linear repeated-measures models overlaid (dotted lines) demonstrate significant increases over time in the responses to the passenger domain of EatA (EatA_p) (top) and EtpA (bottom). See Fig. S4 in the supplemental material for additional plots.

FIG 4

Serum IgG responses preceding asymptomatic ETEC colonization and diarrhea. Shown are peak serum IgG responses for EtpA (left) or the EatA passenger domain (right) preceding either asymptomatic colonization or diarrheal illness with ETEC. Data shown are log₁₀-transformed IgG antibody responses determined by a kinetic ELISA. Bars represent mean values. P values reflect Student’s t testing.