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

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.


INTRODUCTION
Enterotoxigenic Escherichia coli (ETEC) cause substantial morbidity due to diarrheal illness in resource-poor areas of the world where young children are disproportionately affected. In children under five years of age, these pathogens are among the leading causes of moderate-to-severe diarrhea and deaths due to acute diarrheal illness. 1,2 ETEC also causes severe illness, clinically indistinguishable from cholera, [3][4][5] and death in older individuals 6 and remains the most common cause of travelers' diarrhea. While oral rehydration therapy and other measures have contributed to a decline in deaths due to diarrheal illness, ETEC have been linked to post-diarrheal sequelae including malnutrition, growth stunting, and impaired cognitive development greatly compounding the impact of these infections. 7 The ETEC pathovar is defined by the production and effective delivery of heat-stable (ST) and/or heat-labile (LT) enterotoxins to epithelial receptors in the small intestine. In the classical ETEC pathogenesis paradigm, plasmid-encoded colonization factor (CF) or coli surface (CS) antigens facilitate small intestinal colonization. 8 Interaction with small intestinal enterocytes leads to toxin-induced alterations in salt and water transport that result in net fluid losses into the intestinal lumen and ensuing watery diarrheal illness ranging from mild to severe and cholera-like. 3 ETEC infections among young children in endemic regions are thought to result in acquired immunity and a decreasing incidence of infection with age. 9 Indeed, controlled human infection studies demonstrate that homologous re-challenge with the ETEC H10407 strain, which encodes CFA/I, results in robust protection against symptomatic ETEC infection. 10 However, precise correlates of protection 11 have not been established, and the majority of immunologic studies have focused on canonical virulence factors, namely the CF/CS antigens and heatlabile toxin. Nevertheless, recent studies indicate that the repertoire of immune responses following infection extends beyond these classical antigens. 12 Because of inherent genetic plasticity of E. coli, no canonical virulence factor is universally conserved in ETEC. Therefore, to achieve broad coverage, most ETEC vaccines under development adopt a polyvalent approach targeting multiple CF/CS antigens and LT. ACE527, was developed as a live-attenuated vaccine combining three strains that collectively express CS1, CS2, CS3, CS5, CS6, CFA/I, and the B subunit of LT. 13 In recent studies vaccination with ACE527 alone failed to protect against severe diarrhea upon challenge with H10407, while ACE527 adjuvanted with double mutant heat-labile toxin (dmLT) 14 afforded significant protection (PE~66%) (Clinical Trials Identifier NCT01739231). 15 Comparatively, H10407 challenge elicits nearly complete protection against homologous rechallenge. 10 To comprehensively assess the adaptive immune response to vaccination we examined the antigen continent of ACE527 and used ETEC protein microarrays to examine antibody responses to the vaccine ± dmLT. These responses were then compared to vaccine placebo controls and to challenge with H10407 to profile potential benchmarks of protection.

RESULTS
Genomic and proteomic characterization of ACE527 vaccine strains Whole genome DNA sequence data was used to verify the genotypes of wild-type parental isolates and the engineering of the ACE527 strains (summarized in supplementary table 1). As anticipated, 13 the three engineered vaccine strains, ACAM2022, ACAM2025, and ACAM2027, collectively encoded the full complement of six CF/CS antigens (CFA/I, CS1,CS2, CS3, CS5, and CS6), and each encoded both the B subunit of heat-labile toxin, as well as the type II secretion system (T2SS) responsible for export of both LT 16 and YghJ 17 (supplementary table 3). Two of the three parental strains, WS1858B and WS3504D were noted to contain the plasmid etpBAC locus which encodes the two-partner secretion system responsible for production and export of the EtpA adhesin, 18 however analysis of the corresponding attenuated vaccine derivatives, ACAM2025 and ACAM2027 revealed that this locus had been lost in the vaccine strain construction (Fig. 1a, b). Similarly, the eatA gene which encodes a serine protease autotransporter protein that degrades MUC2 mucin, 19 was present in each of the parents but absent from ACAM2025 (Fig. 1a, b).
The growth of each parental isolate and corresponding ACAM live-attenuated bacteria was compared to H10407. In general, the growth of the parental wild-type strains paralleled that of H10407, 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 liveattenuated 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 while the growth of ACAM vaccine strains lagged slightly, potentially reflecting the combined effect of OmpC and OmpF mutations on growth 20 (Fig. 1c).
In keeping with the analysis of the individual genomes, mass spectrometry studies verified production of one or more features of the anticipated CF antigens, including outer membrane usher proteins, and major and minor pilin subunits, as well as LT-B in each of the vaccine strains. As anticipated, no EatA protein was detected in the supernatant of ACAM2025 and no EtpA was produced by the vaccine strains (Fig. 1d, supplementary dataset  2). Interestingly, we noted substantially increased expression of several iron-siderophore transport proteins including FepA, CirA, and Fiu in vaccine strains relative to the corresponding parents, possibly due to the absence of OmpC, and OmpF porins, and the complex regulation of the outer membrane architecture by small non-coding rRNAs known to govern the production of CirA and FepA. 21 In contrast to recent studies of the H10407 proteome 12 where the major protein subunit of flagella, flagellin (FliC, serotype H11), and the flagellar hook protein, FlgE were present in abundance, FliC was not universally detected in the ACE527 proteomes, and we were unable to detect the flagellar hook protein, FlgE (Fig. 1d). Despite the identification of the flagellar operons in the genomes of all of the parent and mutant strains, we found that none of the ACE527 or progenitor strains were motile (Fig. 1e).
Collectively, these molecular characterization studies demonstrated that the ACE527 vaccine strains express all of the canonical ETEC antigens targeted in construction of the vaccine. However, they lack some virulence properties previously identified in the H10407 challenge strain including motility, production of key flagellar antigens, and other immunogenic proteins including the secreted EtpA blood group A lectin-adhesin. 12,22 Differential immune responses to ETEC antigens after vaccination and challenge Recent controlled human infection studies with the H10407 strain of ETEC demonstrated that previously naive volunteers mount strong mucosal antibody responses to classical vaccine targets including CFA/I and heat-labile toxin as well as a select number of molecules that have not been traditionally been targeted in vaccines. 12 Here we set out to examine immune response to the classical antigens targeted in ACE527, and to a broadly inclusive ETEC proteome represented on microarrays incorporating more than 4000 proteins.
Mucosal antibody, particularly mucosal IgA, is felt to be an essential determinant of protection against ETEC, that follows natural infection. Antibody secreted by circulating gut-homing lymphocytes can be obtained from peripheral blood mononuclear cells (PBMC) following recent mucosal exposure to antigens from enteric pathogens. 23,24 These antibody lymphocyte supernatants (ALS) serve as a convenient hallmark of recent mucosal antigen exposure. Therefore, we tested ALS samples from individuals who received ACE527 ± dmLT and placebo controls prior to vaccination, after vaccination, and following challenge with ETEC H10407.
Response to classical vaccine antigens Following vaccination with either ACE527 or ACE527 adjuvanted with dmLT, we observed increases in ALS IgA immunoreactivity to most canonical antigens targeted in the vaccine. Seven days after vaccination, we found that each subject mounted robust responses to CFA/I, and the majority (≥75%) responded to B subunit of LT (Fig. 2, Table 1). In addition, we found strong responses in both groups to PCF071, with subjects responding this class 5b fimbrial antigen most closely related to CS1 25 Table 1, supplementary fig. 1, while we observed more modest responses to CS17 another class 5b antigen, or CooD, a minor pilin subunit of CS1. 26 More than half of vaccine recipients also mounted ALS IgA responses to CS2 and/or the CotA CS2 pilin in each of the vaccine groups. Responses to CS3 were strongest in the dmLT adjuvanted cohort where nearly 2/3 of recipients demonstrated increased responses following vaccination (Table 1). Altogether, these data demonstrate that vaccination with ACE527 with or without dmLT results in antibody responses to most classical antigens targeted in the vaccine. CS6 was an exception in this regard. Interestingly, an earlier analysis of samples from individuals immunized with ACE527, prepared from frozen lots of the vaccine, demonstrated a 13-fold increase in peak geometric mean ALS titers from baseline. 27 Whether drying of the lyophilized preparation of the vaccine used in the present studies impacted the stability or presentation of the CS6 antigens to the recipients in the present study is presently unclear.
Response to non-canonical antigens following vaccination and challenge Although ACE527 was directed at classical ETEC CF/CS and LT-B vaccine antigens, recent studies indicate that following ETEC infection, subjects respond to antigens not targeted in canonical vaccine strategies. Following earlier H10407 challenge studies, we found that all subjects mounted robust responses to flagellin (FliC), the major protein subunit of flagella . 12 Interestingly, however, consistent with poor production of flagellin and the lack of motility by the ACE527 vaccine strains, we found that fewer than 1/3 of the recipients of unadjuvanted ACE527 developed significant responses to FliC regardless of serotype. Notably, we found that following administration of the vaccine + dmLT the majority (92%) of recipients mounted responses to at least one flagellin molecule. Interestingly, these enhanced responses were not constrained to serotypes included in the vaccine as 10/13 subjects in the dmLT cohort responded to FliC from serotype H11 (Fig. 3a, Table 2) the serotype for H10407 challenge strain. As this serotype was not included in the vaccine itself, and responses to H-serotype specific regions of the flagellins represented in the vaccine were noticeably absent (supplementary fig. 2), the responses to FliC H11 and other flagellins may reflect recognition of the non-serotype specific highly conserved alpha helical regions comprising the amino and carboxy terminal regions of flagellin molecules (supplementary fig. 3).
We likewise observed differential responses to two secreted non-canonical antigens, the passenger domain of the EatA autotransporter protein, and YghJ a metalloprotease secreted by the type 2 secretion system. Both proteins were recognized following vaccination with ACE527 with 9/12 subjects responding to the EatAp and 6/12 responding to YghJ. These responses were enhanced slightly in the dmLT cohort (Table 1).
Immune responses to non-canonical proteins predominate after challenge In naive subjects and in vaccine recipients challenged with H10407, we noted enhanced responses to a number of proteins that were not recognized following vaccination. Among the most striking differential responses following challenge compared to vaccination were those to flagellar proteins. 9/10 placebo vaccinated subjects responded to both full length and the serotype specific region (AA174-399) of FliC H11 (Table 3). Likewise, 6/10 volunteers responded to the FlgE flagellar hook protein, a response that was absent in vaccinees prior to challenge, consistent with the lack of motility in the ACE527 strains and the absence of FlgE in the ACE527 proteomes.
In addition to flagellin, three secreted antigens YghJ (SslE), 28 EatA, 19 and EtpA, 29 not currently targeted in classical ETEC vaccine approaches have been shown to be immunogenic in humans 12 and associated with protection in animal models. 19,[29][30][31] Although we observed responses to YghJ following vaccination with either ACE527 or ACE527 + dmLT, the response to this protein was significantly increased following challenge with H10407 (Fig. 3b). Similarly, we observed the most robust responses to the EatA passenger domain following challenge with H10407 (Fig. 3c). In contrast, in keeping with the absence of EtpA in the three vaccine strains, vaccinees did not recognize EtpA following immunization, while both the placebo group and the vaccinated subjects mounted robust responses to EtpA upon challenge with H10407 (Fig. 3d, Table 4).

DISCUSSION
A systematic appraisal of immune response to candidate vaccines can aid in vaccine optimization and identification of mechanistic correlates of protection. Here, we combined genomic and proteomic interrogation of a live-attenuated vaccine with immunoproteomic analysis following vaccination and experimental human challenge with ETEC H10407, an extensively characterized strain originally obtained from a patient with severe choleralike diarrheal illness. 32 Previous studies demonstrating that challenge with wild-type ETEC H10407 bacteria affords virtually complete protection against severe diarrhea on subsequent rechallenge 10 provide important benchmarks for comparison of candidate vaccines. Importantly, the studies reported here highlight a number of features that distinguish the vaccine strains from the challenge strain and the corresponding immune responses. First, although the wild-type isolates used to construct the vaccine had previously been serotyped for flagellar (H) antigens, we found that production of flagellin, the major subunit of flagella, by both the parent and the vaccine strains was deficient, and none were motile upon testing. Similarly, contrasting with our earlier analysis of the H10407 challenge strain, 12 we were unable to detect FlgE, the flagellar hook protein, in either the parental or vaccine strains, suggesting that despite the presence of the genes required for assembly, early steps involved in biosynthesis of flagella are deficient 33,34 in the isolates selected for engineering of ACE527. These deficiencies may have negatively impacted the protection afforded by the vaccine in a number of ways including loss of TLR5 mediated stimulation of innate immunity, and the potent adjuvant activity of flagellin. While H serotypes of ETEC vary considerably, 35 motility is a highly conserved virulence characteristic, and the lack of motility of the vaccine strains likely resulted in suboptimal antigen delivery to sampling sites within the intestinal mucosa. 36 In addition, highly conserved regions of flagellin that flank serotype specific regions of the molecule may not only serve as potent stimuli of innate immunity, but contain cross-protective epitopes. 29 Intriguingly, addition of dmLT as a mucosal adjuvant significantly enhanced the response to multiple flagellins independent of whether the specific serotype was present in the vaccine.
The genomes of the three vaccine strains and parents also revealed that the etpBAC locus, which encodes the two-partner secretion system responsible for production and export of the 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 EtpA adhesin, 18 was present in two of the three parental strains but missing from the vaccine altogether, likely the result of engineering the vaccine strains to remove the plasmid-encoded toxins. EtpA is a high molecular weight glycoprotein secreted by ETEC that appears to facilitate bacterial adhesion by serving as a molecular bridge between flagella 37 and the enterocyte surface where it binds to N-acetylgalactosamine (GalNAc) residues 38 particularly when they are presented as the terminal glycan on human A blood group antigen. Interestingly, human challenge studies with H10407 demonstrate that individuals with A blood group are significantly more likely to experience severe diarrhea when challenged with this EtpA-producing strain. 22 These and other recent studies 12 demonstrate that EtpA is highly immunogenic and recognized by the majority of volunteers upon challenge with wild-type H10407, in distinct contrast to those immunized with ACE527 ± dmLT. Although EtpA is required for optimal delivery of both LT and ST enterotoxins, and is protective against ETEC infection in a murine model, further studies are needed to assess its role as a potential protective antigen in humans.
Protective immunity to ETEC is likely complex and may represent the cumulative response to E. coli core proteins, classical vaccine antigens, and more recently discovered proteins. Although the present studies, based on small numbers of human volunteers, do not permit us to establish clear mechanistic correlates of protection, they highlight the utility of combined genomic, proteomic and immunoproteomic platforms in interpreting the response to live-attenuated vaccines.
Comparison of the vaccine antigen content and immunologic responses to those observed with wild-type infection could inform the design, optimization, and engineering of next-generation ETEC vaccines to enhance protective efficacy. Moreover, the platforms used in the present studies could be generalized to interrogation of live-attenuated vaccines for other important pathogens.

Bioinformatics and comparative genomics of ETEC isolates
To select candidate genes for protein expression, we analyzed the previously sequenced genomes of three parental ETEC isolates WS_1858B, WS_2773E, and WS3504D, used in the construction of ACE527, 13 the genome of E. coli H10407 39 the challenge strain used in these studies, and the genomes of a diverse group of clinical isolates. Data describing these strains is presented in supplementary table 1. The genome content of these isolates was compared using Large-Scale BLAST Score Ratio Analysis 40 and encoded products having a signal for potential secretion to the surface were identified using PSORT, 41 TMHMM, 42 and SignalP. 43 The resulting dataset includes 800 antigens identified in H10407, 157 antigens present in one or more of the three isolates of the ACE527 vaccine lacking in the H10407 genome, and an additional 4168 features identified in comparative analysis of 207 clinical ETEC isolates (supplementary data s1). 44,45 The selected 4168 gene features were present in more than 40% of the ETEC isolates and were not present in the genomes of three common E. coli commensal isolates E. coli HS (GenBank Accession number NC_009800), E. coli K-12 (GenBank Accession number NC_007779.1), E. coli ATCC8739 (GenBank Accession number NC_010468) or E. coli IAI1 (GenBank Accession number NC_011741). Gene identifiers, DNA and predicted peptide sequences, and the isolates used as the template for isolation are included in supplementary_data_s1. Informatically selected features encompassed known ETEC antigens including the A and B subunits of heat-labile toxin (LT-A, and LT-B),CFs, the EatA 19 serine protease, the EtpA adhesin, 37 and the metalloprotease YghJ 46 in addition to conserved and serotype specific regions of flagellin molecules represented in the challenge and vaccine strains.
Rapid Annotation using Subsystem Technology (RASTtk v 1.3.0, http:// rast.nmpdr.org) 47 was used to query completed ACE527 genomes for specific virulence factors. Multiplex PCR was used to verify the toxin profiles of parent and ACE527 vaccine strains using primers for estH, estP, and eltB encoding the STh, STp, and the B subunit of LT, respectively. 48 PCR was also used to verify the presence or absence of the eatA and etpA genes. 49 (supplementary table 2). SerotypeFinder v 2.0 (https://cge.cbs.dtu. dk/services/SerotypeFinder/) was used to assign H serotypes from whole genome sequence data. 50
Recombinant antigens including the EtpA adhesin, 52 the passenger domain of EatA, 19 YghJ, 46 antigen 43, 53 EaeH, 54 LT-A and LT-B, and flagellin (FliC) 29 subunits were produced at Washington University in Saint Louis as

Microarray antigen content
The microarrays used in this study were comprised of IVTT expressed proteins selected above and purified proteins representing known ETEC antigens (n = 38) (Supplementary dataset s1). Also included on the array are IVTT control spots (n = 28), positive control spots for IgG secondary antibody (n = 16), positive controls for human IgG (n = 16), positive control spots for IgA secondary antibody (n = 16), and positive controls for human IgA (n = 16).

Vaccination with ACE527 and controlled human infection studies
Samples analyzed in the present study were derived from Phase 1/2b trial of ACE527 conducted in healthy human volunteers at the Centre for Immunization Research at Johns Hopkins University School of Public Health (clinical trial number NCT01739231). 27 In this earlier trial, subjects were randomly assigned to three groups: the placebo group or groups that received three doses of ACE527 (~10 10 cfu/dose) given orally with or without the dmLT adjuvant (25 μg per dose). At 6-7 months following the primary immunization, placebo and vaccine ± dmLT immunization subjects were rescreened for eligibility. Those enrolled were challenged with~2 x 10 7 colony forming units (cfu) of the H10407 ETEC challenge strain after an overnight fast, and assessed in an inpatient setting for development of ETEC-associated diarrhea and other signs and symptoms of enteric illness. , WS-3504D, and WS-1858B used to engineer the vaccine strains were forwarded from the University of Maryland. The toxin profile of each isolate was confirmed by multiplex PCR using primers for eltB, estH, and estP; and etpA and eatA genes fragments were amplified as described previously (supplementary table 2). To verify production of secreted proteins, immunoblotting for EtpA, EatA, and YghJ was performed as previously described. 55 All immunoblots derive from the same experiment and were processed in parallel. Immunoblotting used affinity purified cross absorbed rabbit polyclonal antibodies against each of the respective recombinant proteins. The secondary antibody used for detection was goat anti-rabbit antibody conjugated to IRDye 680RD. (Catalogue number 925-68071).

Production of subcellular fractions and proteome analysis
In characterizing the proteomes of the ACE527 vaccine strains and parents, subcellular fractions including outer membrane proteins (OMPs), outer membrane vesicles (OMV), and concentrated culture supernatants were prepared for tryptic digestion and peptide analysis using high resolution tandem mass spectrometry interfaced to nano-liquid chromatography (LC-MS/MS) as recently described for H10407. 12  Antibody lymphocyte supernatants ALS were prepared from whole blood by isolation of PBMC, followed by in vitro culture of the PBMCs as previously described. 24 The resulting supernatants were then stored at −80°C for future use in these studies.

Statistical analysis
Array comparisons were reported as the proportion of subjects with at least a 50% increase in normalized signal intensity between day 0 and day 7. p values reported in tables represent t-test comparisons of average normalized signals obtained for the two represented groups. In presentation of graphs where multiple comparisons are made, ANOVA was used to Frequency refers to the proportion of subjects with at least a 50% increase in normalized signal intensity between day 0 and day 7 post challenge  Human studies Use of the archived biospecimens, and data in the present study was performed with the approval of the Institution Review Boards of Johns Hopkins University School of Medicine and Washington University School of Medicine.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

DATA AVAILABILITY
Proteomics data have been uploaded to the ProteomeXchange 56,57 database accessible at http://www.proteomexchange.org/ via accession number PXD014724. Protein Microarray data have been uploaded to the GEO database 58,59 https://www. ncbi.nlm.nih.gov/geo/ under accession number GSE134792. Source data for the figures and tables is also provided in the supplementary information.