Human Experimental Challenge With Enterotoxigenic Escherichia coli Elicits Immune Responses to Canonical and Novel Antigens Relevant to Vaccine Development

Abstract

Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in the developing world. Enterotoxigenic E coli vaccinology has been challenged by genetic diversity and heterogeneity of canonical antigens. Examination of the antigenic breadth of immune responses associated with protective immunity could afford new avenues for vaccine development.

Methods: Antibody lymphocyte supernatants (ALS) and sera from 20 naive human volunteers challenged with ETEC strain H10407 and from 10 volunteers rechallenged 4-6 weeks later with the same strain (9 of whom were completely protected on rechallenge) were tested against ETEC proteome microarrays containing 957 antigens.

Results: Enterotoxigenic E coli challenge stimulated robust serum and mucosal (ALS) responses to canonical vaccine antigens (CFA/I, and the B subunit of LT) as well as a small number of antigens not presently targeted in ETEC vaccines. These included pathovar-specific secreted proteins (EtpA, EatA) as well as highly conserved E coli antigens including YghJ, flagellin, and pertactin-like autotransporter proteins, all of which have previously afforded protection against ETEC infection in preclinical studies.

Conclusions: Taken together, studies reported here suggest that immune responses after ETEC infection involve traditional vaccine targets as well as a select number of more recently identified protein antigens that could offer additional avenues for vaccine development for these pathogens.

Figure 1.

Antigen production by challenge strain H10407. Shown are antigens detected after growth of H10407 under conditions that were used to prepare the challenge strain. Values in columns 1, 2, 4, and 5 reflect protein probability of correct tandem mass spectrometry (MS/MS) identification. Relative peptide abundance (*) values are shown in columns 3 and 6. A subset of the antigens detected is listed in the column at left. Columns 1 and 2 represent proteins identified from wild-type (wt) H10407 outer membrane vesicles (omv) or purified outer membrane proteins (omp) fractions, respectively, whereas column 3 compares the relative peptide abundance of each protein in omv and omp preparations. Columns 4 and column 5 depict the identification of supernatant proteins from the wt and the general secretion pathway (gspG) mutant, respectively, whereas column 6 compares the relative abundance of the peptides in the gsp mutant and wt. The top 5 row features correspond to canonical vaccine antigen targets CFA/I with individual protein subunits CfaE (CFA/I tip adhesin), CfaB (CFA/I major fimbrial subunit), and CfaC (CFA/I outer membrane usher protein), and heat-labile toxin subunits (LT-A, and LT-B). Heat-labile enterotoxin and the other type 2 secretion system (T2SS) effector protein, the YghJ metalloprotease, are grouped together. These are followed by noncanonical putative virulence antigens.

Figure 2.

Systemic immunoglobulin (Ig)A and IgG responses to canonical enterotoxigenic Escherichia coli (ETEC) antigens. Serum IgA and IgG endpoint enzyme-linked immunosorbent assay responses to the B subunit of heat-labile enterotoxin (LT-B) and CFA/I fimbriae are shown on day 0 before challenge with ETEC H10407 and on days 7, 10, 28, and 84 after infection. Numbers in the top right corner of each graph indicate the fraction of volunteers with a ≥2-fold increase in titer compared with the day 0 sample. * indicates the peak titer days for volunteer 314 who was the only individual not protected from diarrheal illness on subsequent rechallenge.

Figure 3.

Mucosal immune response to canonical vaccine antigens. Antibody lymphocyte supernatant (immunoglobulin A, antibody lymphocyte supernatants [ALS]) responses of individual subjects (n = 20) to CFA/I (A) and the heat-labile toxin B subunit (LT-B [B]). Subject responses are represented by colored lines (identified by subject number in the key at right) with dashed lines representing those (n = 10) that were challenged once and solid lines representing those (n = 10) who were rechallenged. The x-axis of each graph depicts the days before (day −1) or after (days 7, 10, 28) each challenge, and the y-axis of each graph is the base-2 log of the microarray raw signal intensity. Subject 314 (*) was the only subject not protected on rechallenge.

Figure 4.

(A) Antibody lymphocyte supernatant (ALS) responses to recombinant purified proteins. Heatmap depicts results for individual subjects on day –1 before and day 7 after challenge. Signals shown are base-2 logarithm of raw signal. Subject numbers are shown on the x-axis, and the ordering is the same for both sample days. Proteins are ordered with the pathovar-specific noncanonical secreted antigens EtpA and EatA at top, followed by conserved proteins YghJ and EaeH, and the classic vaccine targets LT, and CFA/I in the last 3 rows. (B) The ALS responses to in vitro transcription—translation proteins. Values correspond to normalized array signals, and antigens are grouped by function with secreted antigens EtpA, EatA, and YghJ; followed by flagellar proteins, FliC (full-length H11 flagellin flagellar structural subunit, H11_1-487, H11 serotype-specific region of, H11_174-399), and FlgE the flagellar hook protein; and membrane-associated proteins, ompW, and DedD; and 3 pertactin-like adhesin-autotransporter (AT) proteins including ^aantigen 43 (ETEC_4662), ^bETEC_2119, and ^cETEC_2366. Is the heat-stable precursor protein identified in this screening?

Figure 5.

H10407 challenge elicits robust mucosal responses to noncanonical antigens. (A) Antibody lymphocyte supernatant (IgA, ALS) responses of individual subjects (n = 20) are represented by colored lines (identified by subject number in the key at right) with dashed lines representing those (n = 10) that were challenged once, and solid lines representing those (n = 10) who were rechallenged. The x-axis of each graph depicts the days before (day –1) or after (days 7, 10, 28) each challenge, and the y-axis of each graph is the base-2 log of the microarray raw signal intensity. Graphs from left to right demonstrate ALS responses, the YghJ metalloprotease, the EatA mucinase, and the EtpA adhesin. Respectively, subject 314 (*) was the only subject not protected against diarrhea on rechallenge. (B) Kinetic enzyme-linked immunosorbent assay data demonstrating ALS (IgA) responses to noncanonical proteins after initial challenge with H10407. P values reflect 2-tailed Mann-Whitney nonparametric comparisons. Dashed lines in each group represent geometric means.