Expert Review of Vaccines
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Implications of enterotoxigenic Escherichia coli genomics for vaccine development Åsa Sjöling, Astrid von Mentzer & Ann-Mari Svennerholm To cite this article: Åsa Sjöling, Astrid von Mentzer & Ann-Mari Svennerholm (2015) Implications of enterotoxigenic Escherichia coli genomics for vaccine development, Expert Review of Vaccines, 14:4, 551-560, DOI: 10.1586/14760584.2015.996553 To link to this article: http://dx.doi.org/10.1586/14760584.2015.996553
Published online: 26 Dec 2014.
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Date: 14 December 2016, At: 05:43
Review
Implications of enterotoxigenic Escherichia coli genomics for vaccine development Expert Rev. Vaccines 14(4), 551–560 (2015)
˚ sa Sjo¨ling*1,2, A Astrid von Mentzer1 and Ann-Mari Svennerholm1 1 Department of Microbiology and Immunology, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Box 435, S-40530 Go¨teborg, Sweden 2 Department of Microbiology, Tumor and Cell biology, Karolinska Institutet, Stockholm, Box 280, S-17177, Stockholm, Sweden *Author for correspondence: [email protected]
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Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality caused by diarrhea in children less than 5 years of age in low- and middle-income countries. Despite a wealth of research elucidating the mechanisms of disease, the immunological responses and vaccine development, ETEC is still relatively uncharacterized when it comes to regulation of virulence and detailed immune mechanisms. The recent emergence of next-generation sequencing now offers the possibility to screen genomes of ETEC strains isolated globally to identify novel vaccine targets in addition to those already established. In this review, we discuss how recent findings on ETEC genomics using novel sequencing techniques will aid in finding novel protective antigens that can be used in vaccine approaches. KEYWORDS: colonization factor . enterotoxigenic Escherichia coli . ETEC . ETEC vaccine . genomics . next-generation sequencing
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oral vaccine
Enterotoxigenic Escherichia coli (ETEC) remains to be a major cause of infantile diarrhea in the developing world [1,2]. ETEC infections can range from relatively mild diarrhea to severe symptoms of profuse watery diarrhea that can be fatal. The global mortality in children under 5 years of age due to ETEC is estimated to be close to 300,000 cases yearly [3]. In addition, ETEC infections have been associated with malnutrition and delayed growth [4,5]. Recent studies have shown that ETEC continues to be an important cause of diarrhea-associated morbidity and mortality also in older children and adults in endemic areas [6]. ETEC is also the most common cause of diarrhea in travelers [3], and a cause of food-borne outbreaks of diarrhea in developed countries [7]. The hallmark of ETEC is the presence of either one or both of two enterotoxins, the heat labile toxin (LT) and/or the heat stable toxin (ST). ST forms two variants, STh (originally isolated from a human isolate) and STp (originally isolated from a porcine source), both variants cause disease in humans [8] Globally, LT, ST and LTST
10.1586/14760584.2015.996553
strains generally were detected with frequencies of 27, 40 and 33% in ETEC diarrhea cases isolated between 1961 and 2009 [9]. However, shifts in toxin frequency have been reported and ST producing ETEC strains were found to cause the majority of endemic diarrhea in Bangladesh and Chile during the 90s [10–12], while the LT phenotype seems to have been more common in Argentina and Bolivia [13–16] and recently also in Bangladesh [17]. Furthermore, a clear difference in toxin profile has been observed for ETEC strains isolated from travelers and indigenous children with diarrhea during the same time period in Guatemala [18]. An analysis of the University of Gothenburg ETEC collection comprising over 3500 strains isolated globally since 1980 confirmed the shift in recent years since the prevalence of LT, ST and LTST changed from 32, 41 and 28% in strains isolated 1980–2008 to 40, 30 and, 30% in strains isolated 2009–2011, respectively [19]. Adherence to the epithelium is also a major virulence trait of ETEC and is mediated through different colonization factors (CFs), which usually are fimbriae or fibrillae. So far,
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more than 25 different CFs have been identified and an intensive search is ongoing to identify additional CFs. The most common CFs are CFA/I, CS1-CS7, CS14 and CS17 [2,3,20,21]. ETEC strains typically express 1–3 CFs in specific combinations that are repeatedly isolated from diarrheal feces samples. ETEC strains with toxin–CF combinations such as LTSTh CS1 + CS3, LTSTh CS2 + CS3, LTSTh CFA/I, STp CS6 and LTSTh CS5 + CS6 have repeatedly been isolated from patients with diarrhea globally. Although shifts in prevalence of virulence profiles occur over time, several of the predominant toxin–CF combinations seem to be stable and reoccurring both geographically and over time. However, combinations such as LT CS7 and LT CS17 seem to have increased more recently in Bangladesh [2]. In developing countries, the incidence of ETEC-associated diarrhea decreases during the first 5 years of life since natural ETEC infections seems to protect against re-infection [3,16,22,23], whereas children and adults from industrialized areas who travel to these countries often develop ETEC diarrhea [24,25]. Although ETEC can be isolated from adults in endemic areas, the decrease in prevalence in children exposed to repeated infections indicate that natural immunity develops over time. Protective immunity in ETEC afforded by toxins & CFs
LT and CFs are immunogenic and confer protection against reinfection with ETEC strains expressing similar CFs and/or LT both in humans and experimental animals [23,26]. Numerous allele variants of LT have been identified using sequencing but the most common variants of LT are LT1 and LT2 [27,28]. Most variations are found in the A subunit portion of these toxin variants while the B subunit is largely conserved. In fact, functional immunity against LT is mainly directed against the B-subunits of the LT molecule and protection against LT producing ETEC strains has been afforded by the immunologically cross-reactive cholera toxin B subunit (CTB), shown in both endemic areas and in travelers [29,30]. Hence, an LTB toxoid should target most LT-producing ETEC strains. Current ETEC candidate vaccines express LTB or contain CTB or a hybrid LTB/CTB molecule, LCTBA [31]. The LT holotoxin is frequently used as adjuvant in experimental animal studies, although LT is too toxic to be used in humans. In recent studies, both the common natural variants LT1 and LT2 were shown to have strong adjuvant properties in animal models [32]. However, due to the strong toxicity of the natural LT molecule, in particular when administered orally, research has been ongoing for decades to identify a nontoxic LT molecule with retained adjuvant properties that can be used in combination with oral vaccines in humans. Such a molecule has been developed by Clements and collaborators, that is, a double-mutated LT molecule (dmLT) [33]. The low doses of dmLT used in vaccine studies (10 and 25 mg) have been shown to be safe in humans [34,35] and to provide strong adjuvant properties in experimental animals [36] and also some adjuvant properties to an oral inactivated ETEC vaccine in 552
humans [35]. Thus, an effective ETEC vaccine should most likely contain dmLT or a related adjuvant. Both heat stable toxins, STh and STp, cause disease in humans [8]. Although these ST molecules are small peptides (19 and 18 amino acids, respectively), which do not elicit an immune response unless coupled to a carrier protein [37,38], they have been proposed as putative candidates to be included in an ETEC vaccine. Thus, ST toxoids fused to the dmLT molecule have been shown to be non-toxic and to elicit anti-ST antibody responses in mice [39]. However, ST cross-reacts immunologically with the endogenous GC-C ligands guanylin and uroguanylin [40]. Furthermore, no non-toxic ST toxoid that reproducibly induces strong protective immune responses in humans has been described, although an intensive search is going on to identify such molecules [39,40]. A majority of the CFs are fimbriae or fibrillous structures composed by subunits that protrude from the surface and confer protection against reinfection with strains that express the same CF [23,41]. Most ETEC CFs are plasmid encoded; the operons are usually composed of genes encoding one or two structural subunits, a chaperon and an usher [20,42]. Expression is induced by different physiological factors, for example, pH and temperature and for many CFs bile is an inducer of expression [20,21,43]. Several CFs are positively regulated by members of the AraC family of transcription factors [44]. The CF fimbriae are typically composed of more than 100 major subunit proteins of 15–30 kD each and a tip protein responsible for binding to specific receptors on epithelial cells. Several of the CFs have closely related N-terminal amino acid sequences of their major subunits and/or adherence factors at the tip of the CF, and several CFs cross-react immunologically, for example, the CFA/I-like group, which includes CFA/I, CS1, CS2, CS4, CS14, CS17, CS19 and PCF071, and the CS5-like group, including CS5 and CS7 [20,45]. Type IV-like pili include CS8 (CFA/III) and CS21 (Longus) [20], and additional CFs such as CS12, CS18, CS20, CS26 and newly identified CS27 and CS28 are classified as class 1b fimbria [46]. Additional CFs that share similar sequences are CS13 and the recently described CS23 [47] as well as CS15 (antigen 8786) and CS22 [48]. CFs that do not show any obvious similarity to other known ETEC CFs include CS3 and CS6 [20]. Strong cross-reactive immune responses against different CFs have been demonstrated both in experimental and human studies, for example, for CFs in the CFA/I and CS5 families, respectively [49,50] [SVENNERHOLM A-M, UNPUBLISHED DATA], suggesting that a relatively limited number of CFs would be required in a vaccine targeting ETEC expressing the most common CFs. The considerable heterogeneity in CF profiles of ETEC strains isolated globally and over time has previously been suggested to complicate the relevance for developing a broad-based vaccine based on CFs [9]. Instead, it has been suggested that the tip protein CfaE of the CFA/I family that contains conserved epitopes may be more suitable as a vaccine candidate [44,51]. However, we have recently found that ETEC strains with similar virulence profiles often are remarkably Expert Rev. Vaccines 14(4), (2015)
Implications of ETEC genomics for vaccine development
similar and of clonal descent even if isolated at different continents and several decades, which would argue for a vaccine targeting the most common CFs or other antigens preserved in such successful lineages [52]. Additional vaccine candidates & virulence factors of ETEC that induce protective immunity
In addition to the classical virulence factors of ETEC, several novel CFs and putative toxins have been characterized and described in recent years. Of these, EAST1 is a heat stable enterotoxin ST originally described in enteroaggregative E. coli that is found in some ETEC strains as well as the hemolysin ClyA that is found in almost all ETEC [16]. Additional putative virulence factors that have been described in some ETEC strains include Tia [53], TibAB [54] and CexE [55]. Approaches to find novel molecules exposed on the surface of ETEC, recently led to the identification of several putative virulence loci, including the etpBAC two-partner secretion locus [56,57]. EtpA is situated on the tip of the flagella and mediates initial binding to epithelial cells and has suggested as a putative vaccine antigen [57,58]. Recently, two mucinases, EatA and YghJ, have been proposed as vaccine candidate antigens since they are involved in ETEC colonization and shown to be immunogenic [59–61]. YghJ is secreted through the type II secretion system and degrades the major mucins in the intestine MUC2 and MUC3. This protein is conserved in ETEC and exists in other pathogenic E. coli but is also expressed by non-pathogenic E. coli [61]. A targeted search for autotransporters in ETEC revealed that Ag43 and an autotransporter designated pAT as well as the putative virulence factors CexE and EtpA together with LT were associated with outer membrane vesicles (OMVs) and immunization with OMVs with LT, CexE and EtpA induced antibody responses to each of the antigens in serum and fecal samples. In addition, mice vaccinated with OMVs were significantly protected against subsequent intestinal colonization [58]. Recent progress in ETEC vaccine development
The currently most promising ETEC candidate vaccines are oral live or inactivated E. coli strains expressing some of the most prevalent ETEC CFs, for example, CFA/I, CS3, CS5 and CS6 and an LT toxoid [19,35,36,62]. These candidate vaccines have been estimated to have the potential to protect against up to 80% of all clinical ETEC infections due to the afforded protection against LT and LTST [29] as well as additional protection against the common CFs included in the vaccine. However, their capacity to protect against ETEC that only express ST and lack identified CFs would probably not be effective. Based on analysis of our global ETEC collection, we estimate that ST-only CF-negative strains constitute approximately 11% of all ETEC isolates. Furthermore, protective immunity induced by LT toxoids is short-lasting. A successful vaccination strategy should ideally prevent colonization and delivery of the toxins to the epithelial surface of all types of ETEC bacteria in the small intestine and should preferably generate long-term protection against infection and disease. informahealthcare.com
Review
Hence, a search to identify conserved ETEC surface outer membrane proteins that may protect against a majority of ETEC strains, and preferably also against other pathogenic E. coli, is ongoing but has not yet resulted in identification of strong candidate proteins. The recent emergence of nextgeneration sequencing now offers the possibility to screen the genomes of ETEC strains isolated globally to identify novel vaccine targets in addition to those already established. Reverse vaccinology applied on pathogenic E. coli
The use of high-throughput sequencing of entire genomes of pathogenic bacteria has fostered the ‘reverse vaccinology’ approach. The technique, which was first used to identify meningococcal vaccine candidate proteins from the sequenced genome of a Neisseria meningitides serogroup B strain [63,64], has to date been applied to a number of pathogens including N. meningitides, Streptococcus ssp. and pathogenic E. coli [63,65,66]. The rationale of reverse vaccinology is to use in silico screening of sequenced genomes for genes that encode proteins predicted to be outer membrane proteins with potential to be used as vaccine targets. Proteins predicted to be surface exposed and conserved within the target pathogen are then expressed by recombinant techniques and analyzed for immune responses in mice [63,67]. Reverse vaccinology applied on pathogenic E. coli would hence aim to target conserved surface structures on pathogenic strains that are absent in commensal E. coli. Several of the E. coli pathovars have acquired extrachromosomal genetic information by horizontal gene transfer either as plasmids, by phages or incorporated in the genome in pathogenicity islands that confer their pathogenic traits. Attempts to find conserved antigens in pathogenic E. coli that are not found or conserved in commensal E. coli strains is thus regarded an appealing approach and has been the focus of studies using both whole genome sequencing and subtractive hybridization approaches [66]. To date, most approaches of reverse vaccinology on E. coli pathovars have been applied to extraintestinal E. coli pathogens belonging to uropathogenic E. coli (UPEC) as well as newborn meningitis E. coli. It is important to note that E. coli is a diverse polyphyletic species, roughly divided into the four major phylogroups A, B1, B2 and D/E, and extraintestinal pathogenic E. coli like UPEC and newborn meningitis E. coli mainly cluster in the B2 phylogroup. Comparable analyses of newborn meningitis E. coli strains, an avian pathogenic E. coli and UPEC with the non-pathogenic E. coli K12 strain lead to the identification of 230 candidate genes present in extraintestinal E. coli strains. Of these, nine candidates were further selected based on predictions to encode outer membrane proteins or secreted proteins that conferred protection in a mouse challenge sepsis model [66]. Four candidate proteins were proposed in subsequent studies as candidates for inclusion in a vaccine against ‘all’ E. coli pathogens [66,68]. The most protective antigen in animal models among the four selected ones was a putative lipoprotein ECOK1_3385 with sequence similarities to the Vibrio cholerae accessory colonization factor. 553
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Whole genome sequencing of ETEC strains
Whole genome sequences of ETEC strains now offer the possibility to perform reverse vaccinology approaches specifically targeted toward this pathogen. The ETEC strain E24377A (SThLT, CS1 + CS3, O139) was the first strain sequenced to Lineage 1 completion followed by the model strain CS1 + CS3 24 years (1983–2007) H10407 (LTSThSTp, CFA/I, O78) and a draft sequence of B7A (STLT, CS6, O146) [72,73]. In 2011, five additional Lineage 2 ETEC draft genomes were published [74] CS2 + CS3 27 years (1980–2007) and in 2014 the whole genome sequence of an ETEC strain from Vietnam was Figure 1. Population structure of strains expressing LTSTh CS1 + CS3 (Lineage 1) described [75]. The initial paucity in and LTSTh CS2 + CS3 (Lineage 2) generated as a maximum-likelihood phylogenetic sequencing of ETEC strains has improved tree based on single nucleotide polymorphism differences across the maximum common genome [52]. The close genetic relationships between isolates collected over a substantially and we have recently found, period of nearly three decades from Asia, Africa and the Americas are shown. The two lineby whole genome sequencing of a repreages are also closely related to each other but distant from other major lineages of enterosentative selection of 362 clinical ETEC toxigenic Escherichia coli expressing specific virulence profiles (not shown in figure). isolates collected from Asia, Africa and Latin America [52], that major clonal lineECOK1_3385 was found to be secreted by the type II secre- ages of ETEC expressing the most common toxin–CF combination system and present in strains dispersed over the entire tions have spread globally (FIGURE 1). These lineages have emerged E. coli phylogeny in several pathovars but also in commensals. in modern time and successfully persisted and spread in the last It was, however, argued that the type II secretion pathway is 170–50 years [52]. The description of the new collection of inactivated in most commensals, which might link ETEC genomes is provided by von Mentzer et al. [52]. We found ECOK1_3385 to virulence and thereby justify its use as a vac- that a majority of ETEC clusters in the A and B1 phylogroups cine target [66]. The ECOK1_3385 encoded protein is but we could identify ETEC strains that clustered in B2 and D/E described as secreted and surface-associated lipoprotein from phylogroups as well. These findings implicate that the virulence E. coli (SslE) and is also known as YghJ, the most recent viru- genes defining ETEC might persist in diverse genetic E. coli lence factor described for ETEC [61]. The ECOK1_3385/acces- backgrounds. Extrapolation of these results in our University of sory colonization factor/SslE/YghJ protein will be referred to as Gothenburg ETEC collection of more than 3500 ETEC strains SslE henceforth. Another selected protein, ECOK1_0290, is a isolated globally over more than three decades indicates that a broadly conserved adhesin also described as factor adherence large proportion of all ETEC causing clinical infections in fact E. coli (FdeC) in UPEC. FdeC have some sequence similarity belong to five major lineages expressing the colonization factors to Yersinia pseudotuberculosis invasin and enteropathogenic CFA/I, CS1 + CS3, CS2 + CS3, CS5 + CS6 and CS6 [52]. These E. coli intimin (Eae) and was identified as a main vaccine can- genome sequences, together with those previously described, didate target in a follow-up study [68]. Interestingly, FdeC was seem to cover a representative selection of ETEC isolated worldshown to share 95% identity with EaeH, a putative adhesin wide which may now be used for analyses of prevalence of known identified by subtractive hybridization from the genome vaccine antigens as well as in reverse vaccinology approaches. sequence of the enterotoxigenic E. coli (ETEC) strain H10407 [69]. FdeC was found to be present in all pathogenic Vaccine candidate antigens & their prevalence in ETEC E. coli analyzed and to some extent in commensal E. coli. The Several of the new putative vaccine candidate proteins that have third candidate antigen, ECOK1_3457, was a TonB-dependent been put forward recently are either antigens specific for an siderophore receptor involved in iron acquisition, first identi- ETEC vaccine, for example, EtpA, CexE, EatA, TibAB, Tia and fied in a human E. coli septicemia strain and described as ClyA or antigens suggested for use in broader pathogenic E. coli FitA [70]; the fourth candidate was a hypothetical protein vaccines, for example, SslE, FdeC/EaeH, FitA and c0975. We referred to as c0975 [66]. The prevalence of these suggested can- determined the prevalence of these ‘new’ candidate antigens, as didate proteins remains to be investigated in other pathogenic well as the antigens being components of our inactivated ETEC E. coli. However, since the E. coli pangenome consists of over candidate vaccine, that is, LT, CFA/I, CS3, CS5 and CS6 in our 20,000 genes and only approximately 2000 genes are shared by collection of 362 sequenced ETEC strains by mapping and all E. coli strains [71], identification of conserved pangenome BLASTn. The prevalence of one of the major target molecules E. coli vaccine target antigens must be thoroughly compared for an ETEC vaccine, LT, remains at a level of at least two-thirds with commensal E. coli of all phylogroups. of all ETEC strains isolated. Together with the most prevalent Bangladesh Indonesia Japan Burma/Nepal Egypt Marocco/Tunisia Guatemala Argentina Bolivia Mexico
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Table 1. Prevalence of genes encoding putative enterotoxigenic CFs [19], these antigens cover up to 80% of Escherichia coli-specific vaccine candidate antigens. all ETEC strains isolated from individuals with diarrhea. Hence, a vaccine targeting Putative E. coli Function Prevalence Protection LT, CFA/I, CS3, CS5 and CS6 has the vaccine in ETEC animals/humans potential to provide protection against a (%)† candidate majority of all ETEC strains (TABLE 1) [19]. antigens Among the novel candidates recently put LT Toxin 65† Yes/Yes forward as putative vaccine antigens, EatA † Yes/Yes CFA/I Colonization factor 21 has been suggested to be common in † ETEC [74]. In a recent study in Bolivia, the CS3 Yes/Yes Colonization factor 22 gene encoding EatA was present in 33% of † Yes/Yes CS5 Colonization factor 13 the ETEC strains analyzed [16], and we Yes/Not shown CS6 Colonization factor 31†,‡ could identify EatA in 40% of the isolates in the recent whole genome sequencing EatA Yes/Not shown Serine protease/mucinase 40§ project (TABLE 1) [52]. The gene encoding § Yes/Not shown EtpA Adhesin glycoprotein 23 EatA was mostly found in strains with § common toxin–CF profiles, that is, CexE Yes/Not shown Putative secreted protein 7 LTSTh, CS1 + CS3; LTSTh, CS2 + CS3; § Not shown/Not shown TibAB Adhesin, autotransporter 8 STh, CFA/I, LTSTh, CS5 + CS6, LT § Not shown/Not shown Adhesin glycoprotein 10 CS6 + CS8 and LTSTp CS19 in this latter Tia † Svennerholm and Lundgren 2012 [19]. study while only 9% of the EatA-positive ‡ Combined percentage for CS5 + CS6, CS4 + CS6 and CS6-only strains from Svennerholm and Lundgren [19]. § strains were CF negative. Indeed, 65% of Prevalence in 362 representative enterotoxigenic Escherichia coli strains described by von Mentzer et al. [52]. the EatA-positive strains belonged to one CFA/I: Colonization factor antigen I; CS1: Coli surface antigen 1; CS3: Coli surface antigen 3; CS5: Coli surface antigen 5; CS6: Coli surface antigen 6; EatA: Serine protease autotransporter; ETEC: Enterotoxigenic escherichia of the five major lineages of ETEC coli; EtpA: Two partner secretion adhesin; LT: Heat labile toxin; Tia: Toxigenic invasion loci A; TibAB: Toxigenic invasion loci B. described by von Mentzer et al. [52]. Data taken from ref. [19,23,26,29,58,60]. Another ETEC-specific protein, EtpA has been shown to be located on the tip of the flagella and to be antigenic. The etpABC locus was present in pathogenic E. coli, FdeC is also present in commensals and 23% of the analyzed whole genome sequenced strains. 79% of non-pathogenic strains. these etpABC-positive strains expressed CFs of the CFA/I family, The FitA siderophore receptor that was identified as a putathat is, strains expressing CFA/I, CS1 + CS3, CS2 + CS3 and tive pathogenic E. coli antigen was only present in 7% of the CS14. CexE, has also been proposed as a promising candidate ETEC strains and only in ETEC strains belonging to the protein which induces an immune response [58], however, our B2 and D/E phylogroups, hence representing a minority of in silico analysis of sequenced ETEC isolates revealed that the ETEC (TABLE 2). Extraintestinal E. coli are regarded to mainly gene encoding CexE is exclusively present in CFA/I-positive belong to the B2 phylogroup of E. coli while ETEC strains are strains such as the ETEC type strain H10407 on which the origi- found in all phylogroups with a majority in the A and nal study was performed [55,58]. Genes encoding TibAB and Tia B1 groups [52]. This finding indicates that ETEC and other were found in 8 and 10% of the whole genome sequenced E. coli pathogens that cluster in B2 and D/E have additional strains, respectively. While tibAB was predominantly found in pathways for iron acquisition compared with E. coli pathogens strains expressing CFA/I and CS14, tia was associated with of phylogroups A and B1. Although the association to B2 and strains expressing LTSTh CS5 + CS6 and with CF-negative D/E makes FitA less useful as a vaccine antigen against all strains (29%). Thus, the suggested potential additional vaccine pathogenic E. coli, FitA might provide additional protection antigens may increase the protective coverage of the present LT- capacity against ETEC strains belonging to the D/E group of CF antigen vaccines to a limited extent. But it remains to be seen which some have disseminated globally (i.e., a STp if they may enhance protection. CS6 lineage). In contrast, the SslE protein was found in almost all ETEC strains analyzed and in all E. coli phylogroups. SslE is secreted though the type II secretion system that also exports Putative pangenome E. coli candidate vaccine antigens LT and is involved in mature biofilm formation and has muci& their prevalence in ETEC Certain antigens have been suggested to be included in a pan nase activity [66,77,78]. However, this antigen has also been idenE. coli vaccine (TABLE 2). The putative ETEC adhesion factor tified in commensal E. coli [79] and recently it was shown that EaeH was originally identified in strain H10407 [69] and was non-pathogenic E. coli strains can also secrete SslE [80]. This found to be upregulated at later time-points in a study of tran- finding highlights that novel antigens identified from sequence scriptome responses of adherent ETEC [76]. EaeH, also data and suggested to be incorporated in an ETEC vaccine described as FdeC, was identified in 88% of ETEC, confirming should be thoroughly investigated. The hypothetical protein previous findings (TABLE 2) [68]. Although present in several c0975 was found in 24% of the ETEC isolates [52], of these informahealthcare.com
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Table 2. Prevalence of genes encoding putative pathogenic Escherichia coli vaccine candidate proteins in 362 whole genome sequenced enterotoxigenic E. coli strains. Putative E. coli vaccine candidate antigens
Annotation and putative function
SslE
Secreted and surfaceassociated lipoprotein with putative mucinase function
93
FdeC/EaeH
Bacterial Ig-like domain (group 1) protein putative adhesion and biofilm component
88
FitA
tonB-dependent siderophore receptor
7
c0975
Hypothetical protein
24
Prevalence in ETEC (%)†
†
Prevalence in 362 representative enterotoxigenic Escherichia coli strains described by von Mentzer et al. [52].
strains 39% expressed CS6, either alone or co-expressed with CS5 (TABLE 2). Taken together, these results indicate that SslE and FdeC/ EaeH, although expressed by a majority of ETEC strain, are also expressed and/or secreted by commensal E. coli and hence less suitable as candidate antigens in an ETEC vaccine. The hypothetical protein c0975 as well as FitA, which is specifically linked to ETEC clustering in the B2 D/E group, may be worth further analyses, since these proteins are present in STp CS6 ETEC strains that have disseminated globally [52]. If shown to be specific for pathogenic E. coli, FitA and c0975 might add to the protective capacity of a future ETEC vaccine. CFs revisited: identification of new CFs by a reverse vaccinology approach
Recent vaccine research in our laboratory has focused on the development of a whole cell killed ETEC vaccine based on recombinant overexpression of the most prevalent CFs (CFA/I, CS3, CS5 and CS6) in an ETEC background combined with the LT toxoid, LCTBA, which is administered in combination with the dmLT molecule [19,36]. This vaccine has in recent extensive Phase I studies proved to be very safe and induces strong mucosal immune responses against the different CFs and LT [35]. However, in epidemiological studies worldwide we, and others, have found that between 30 and 50% of the ETEC isolates lack an identified CF [52]. Indeed, in our global analysis of 362 ETEC strains we found that 130 strains lacked a previously described CF. Since additional new CFs might be assumed to provide protective immunity, we have recently launched a search for conserved elements within CFs operons in a targeted reverse vaccinology approach based on conserved sequence motifs in known chaperone–usher CFs [43]. Such new putative CF variants will be further analyzed for prevalence in 556
epidemiological studies – and if prevalent considered for inclusion in a modified ETEC vaccine. This approach might also be further extended to search for presumed surface-exposed structures in CF-negative isolates that could complement a CFbased vaccine approach. Expert commentary
Next-generation sequencing and advanced bioinformatics analysis of genomes will continue to provide clues on novel targets for vaccines that might either target ETEC or all pathogenic E. coli. It has been speculated that all pathogenic E. coli strains could be covered by a vaccine that contains the four antigens: FdeC, SslE, FitA and c0975, but these candidates appear to be less suitable for an ETEC vaccine. In addition, the fact that ETEC strains are found in all E. coli phylogenetic groups, with large intraspecies variation, may suggest that finding one or few targets to provide protection against all ETEC might be difficult. It is also crucial to compare pathogenic E. coli genomes with commensal or non-pathogenic E. coli from all E. coli phylogroups to exclude the whole spectra of genes that might be present also in non-pathogenic strains. Instead, our results imply that a vaccine focused on common ETEC-specific antigens, in particular CFs, is still a highly feasible approach. Nevertheless, such a vaccine can be complemented with novel surface-exposed targets such as conserved antigens and/or new CFs identified by data mining of genome sequences. It is however important to verify surface expression of vaccine target antigens in vivo at the site of infection as well as their capacity to induce protective antibodies. ETEC specific vaccine components should fulfill all these requirements; that is, being expressed at the site of infection, being immunogenic and capable of inducing protection against ETEC infection. Five-year view
The impact of cheaper and faster whole genome sequencing using novel techniques is expected to speed up the pace of bacterial strain sequencing and will aid in the identification of potential new candidate antigens for inclusion in an effective ETEC vaccine with broad protective coverage. However, the finding that successful lineages of ETEC has emerged and spread globally in less than 50 years [52], suggests that novel antigens might emerge and spread rapidly if they confer an evolutionary advantage; hence, surveillance and further analysis of ETEC and other pathogenic E. coli is vital. The expected speed and reduced cost of sequencing in the near future might lead to epidemiological studies and clinical studies based on strains isolated from hospitalized patients using whole genome sequencing as a standard characterization method. Such data will allow surveillance of emerging infections and outbreaks as well as to aid identification of putative virulence factors suitable as vaccine antigen candidates. It is, however, crucial to determine surface expression in vivo during acute infection of such putative vaccine antigens. This may be achieved by RNA-seq of ETEC, in situ in biopsies from infected animals or patients followed by proteomic analyses as well as identification of antibodies toward Expert Rev. Vaccines 14(4), (2015)
Implications of ETEC genomics for vaccine development
the target. The ongoing development of new bioinformatics tools to analyze and predict gene function and protein characteristics in sequenced genomes is likely to allow identification of promising novel vaccine candidates in the future. Acknowledgements
The authors thank Professors G Dougan, N Thomson and T Connor at the Wellcome Trust Sanger Institute UK for financial and technical support of genome sequencing data presented. The authors also would like to thank former and present colleagues at University of Gothenburg who have contributed to sequencing data.
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Financial & competing interests disclosure
This work was funded by the Swedish foundation for Strategic Research, the Swedish Research Council and the PATH EVI program. A-M Svennerholm is a shareholder in Gotovax AB that may receive a small royalty on the sales of ETEC vaccine if it becomes a commercial product. A-M Svennerholm also has a patent in the area. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues .
Enterotoxigenic Escherichia coli (ETEC) remains to be a major cause of infantile diarrhea in the developing world and causes disease by secretion of either one or both of the enterotoxins stable toxin and labile toxin as well as by adhesion to the epithelium by a variety of colonization factors (CFs).
.
Shifts in toxin–CF profiles may occur but several of the predominant toxin–CF combinations seem to be stable and of clonal origin and are reproducibly isolated in Africa, Asia and the Americas over time.
.
The most promising vaccine candidates are still vaccines expressing or containing labile toxin antigen and CFs.
.
The recent advance in whole genome sequencing of ETEC offers the possibility to search genomes for novel prevalent vaccine candidate antigens.
.
Conserved surface-exposed antigens specific for ETEC or for ‘all’ pathogenic E. coli should be identified and might be included in future E. coli candidate vaccines.
.
Identification of conserved pangenome E coli putative vaccine antigens must be thoroughly compared with commensal E. coli of
.
A search for novel colonization factors in genomes that lack described CFs is in progress since ETEC CFs would fulfill requirements for
all phylogroups. an ETEC vaccine with a broad coverage. .
Current vaccines may be improved by additional ETEC-specific conserved antigens identified by reverse vaccinology approaches.
.
Potential novel antigens to be included in future vaccines should be pathogen specific, surface exposed and immunogenic.
and prevention. Clin Microbiol Rev 2005;18:465-83
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Implications of enterotoxigenic Escherichia coli genomics for vaccine development Åsa Sjöling, Astrid von Mentzer & Ann-Mari Svennerholm To cite this article: Åsa Sjöling, Astrid von Mentzer & Ann-Mari Svennerholm (2015) Implications of enterotoxigenic Escherichia coli genomics for vaccine development, Expert Review of Vaccines, 14:4, 551-560, DOI: 10.1586/14760584.2015.996553 To link to this article: http://dx.doi.org/10.1586/14760584.2015.996553
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Date: 14 December 2016, At: 05:43
Review
Implications of enterotoxigenic Escherichia coli genomics for vaccine development Expert Rev. Vaccines 14(4), 551–560 (2015)
˚ sa Sjo¨ling*1,2, A Astrid von Mentzer1 and Ann-Mari Svennerholm1 1 Department of Microbiology and Immunology, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Box 435, S-40530 Go¨teborg, Sweden 2 Department of Microbiology, Tumor and Cell biology, Karolinska Institutet, Stockholm, Box 280, S-17177, Stockholm, Sweden *Author for correspondence: [email protected]
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Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality caused by diarrhea in children less than 5 years of age in low- and middle-income countries. Despite a wealth of research elucidating the mechanisms of disease, the immunological responses and vaccine development, ETEC is still relatively uncharacterized when it comes to regulation of virulence and detailed immune mechanisms. The recent emergence of next-generation sequencing now offers the possibility to screen genomes of ETEC strains isolated globally to identify novel vaccine targets in addition to those already established. In this review, we discuss how recent findings on ETEC genomics using novel sequencing techniques will aid in finding novel protective antigens that can be used in vaccine approaches. KEYWORDS: colonization factor . enterotoxigenic Escherichia coli . ETEC . ETEC vaccine . genomics . next-generation sequencing
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oral vaccine
Enterotoxigenic Escherichia coli (ETEC) remains to be a major cause of infantile diarrhea in the developing world [1,2]. ETEC infections can range from relatively mild diarrhea to severe symptoms of profuse watery diarrhea that can be fatal. The global mortality in children under 5 years of age due to ETEC is estimated to be close to 300,000 cases yearly [3]. In addition, ETEC infections have been associated with malnutrition and delayed growth [4,5]. Recent studies have shown that ETEC continues to be an important cause of diarrhea-associated morbidity and mortality also in older children and adults in endemic areas [6]. ETEC is also the most common cause of diarrhea in travelers [3], and a cause of food-borne outbreaks of diarrhea in developed countries [7]. The hallmark of ETEC is the presence of either one or both of two enterotoxins, the heat labile toxin (LT) and/or the heat stable toxin (ST). ST forms two variants, STh (originally isolated from a human isolate) and STp (originally isolated from a porcine source), both variants cause disease in humans [8] Globally, LT, ST and LTST
10.1586/14760584.2015.996553
strains generally were detected with frequencies of 27, 40 and 33% in ETEC diarrhea cases isolated between 1961 and 2009 [9]. However, shifts in toxin frequency have been reported and ST producing ETEC strains were found to cause the majority of endemic diarrhea in Bangladesh and Chile during the 90s [10–12], while the LT phenotype seems to have been more common in Argentina and Bolivia [13–16] and recently also in Bangladesh [17]. Furthermore, a clear difference in toxin profile has been observed for ETEC strains isolated from travelers and indigenous children with diarrhea during the same time period in Guatemala [18]. An analysis of the University of Gothenburg ETEC collection comprising over 3500 strains isolated globally since 1980 confirmed the shift in recent years since the prevalence of LT, ST and LTST changed from 32, 41 and 28% in strains isolated 1980–2008 to 40, 30 and, 30% in strains isolated 2009–2011, respectively [19]. Adherence to the epithelium is also a major virulence trait of ETEC and is mediated through different colonization factors (CFs), which usually are fimbriae or fibrillae. So far,
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more than 25 different CFs have been identified and an intensive search is ongoing to identify additional CFs. The most common CFs are CFA/I, CS1-CS7, CS14 and CS17 [2,3,20,21]. ETEC strains typically express 1–3 CFs in specific combinations that are repeatedly isolated from diarrheal feces samples. ETEC strains with toxin–CF combinations such as LTSTh CS1 + CS3, LTSTh CS2 + CS3, LTSTh CFA/I, STp CS6 and LTSTh CS5 + CS6 have repeatedly been isolated from patients with diarrhea globally. Although shifts in prevalence of virulence profiles occur over time, several of the predominant toxin–CF combinations seem to be stable and reoccurring both geographically and over time. However, combinations such as LT CS7 and LT CS17 seem to have increased more recently in Bangladesh [2]. In developing countries, the incidence of ETEC-associated diarrhea decreases during the first 5 years of life since natural ETEC infections seems to protect against re-infection [3,16,22,23], whereas children and adults from industrialized areas who travel to these countries often develop ETEC diarrhea [24,25]. Although ETEC can be isolated from adults in endemic areas, the decrease in prevalence in children exposed to repeated infections indicate that natural immunity develops over time. Protective immunity in ETEC afforded by toxins & CFs
LT and CFs are immunogenic and confer protection against reinfection with ETEC strains expressing similar CFs and/or LT both in humans and experimental animals [23,26]. Numerous allele variants of LT have been identified using sequencing but the most common variants of LT are LT1 and LT2 [27,28]. Most variations are found in the A subunit portion of these toxin variants while the B subunit is largely conserved. In fact, functional immunity against LT is mainly directed against the B-subunits of the LT molecule and protection against LT producing ETEC strains has been afforded by the immunologically cross-reactive cholera toxin B subunit (CTB), shown in both endemic areas and in travelers [29,30]. Hence, an LTB toxoid should target most LT-producing ETEC strains. Current ETEC candidate vaccines express LTB or contain CTB or a hybrid LTB/CTB molecule, LCTBA [31]. The LT holotoxin is frequently used as adjuvant in experimental animal studies, although LT is too toxic to be used in humans. In recent studies, both the common natural variants LT1 and LT2 were shown to have strong adjuvant properties in animal models [32]. However, due to the strong toxicity of the natural LT molecule, in particular when administered orally, research has been ongoing for decades to identify a nontoxic LT molecule with retained adjuvant properties that can be used in combination with oral vaccines in humans. Such a molecule has been developed by Clements and collaborators, that is, a double-mutated LT molecule (dmLT) [33]. The low doses of dmLT used in vaccine studies (10 and 25 mg) have been shown to be safe in humans [34,35] and to provide strong adjuvant properties in experimental animals [36] and also some adjuvant properties to an oral inactivated ETEC vaccine in 552
humans [35]. Thus, an effective ETEC vaccine should most likely contain dmLT or a related adjuvant. Both heat stable toxins, STh and STp, cause disease in humans [8]. Although these ST molecules are small peptides (19 and 18 amino acids, respectively), which do not elicit an immune response unless coupled to a carrier protein [37,38], they have been proposed as putative candidates to be included in an ETEC vaccine. Thus, ST toxoids fused to the dmLT molecule have been shown to be non-toxic and to elicit anti-ST antibody responses in mice [39]. However, ST cross-reacts immunologically with the endogenous GC-C ligands guanylin and uroguanylin [40]. Furthermore, no non-toxic ST toxoid that reproducibly induces strong protective immune responses in humans has been described, although an intensive search is going on to identify such molecules [39,40]. A majority of the CFs are fimbriae or fibrillous structures composed by subunits that protrude from the surface and confer protection against reinfection with strains that express the same CF [23,41]. Most ETEC CFs are plasmid encoded; the operons are usually composed of genes encoding one or two structural subunits, a chaperon and an usher [20,42]. Expression is induced by different physiological factors, for example, pH and temperature and for many CFs bile is an inducer of expression [20,21,43]. Several CFs are positively regulated by members of the AraC family of transcription factors [44]. The CF fimbriae are typically composed of more than 100 major subunit proteins of 15–30 kD each and a tip protein responsible for binding to specific receptors on epithelial cells. Several of the CFs have closely related N-terminal amino acid sequences of their major subunits and/or adherence factors at the tip of the CF, and several CFs cross-react immunologically, for example, the CFA/I-like group, which includes CFA/I, CS1, CS2, CS4, CS14, CS17, CS19 and PCF071, and the CS5-like group, including CS5 and CS7 [20,45]. Type IV-like pili include CS8 (CFA/III) and CS21 (Longus) [20], and additional CFs such as CS12, CS18, CS20, CS26 and newly identified CS27 and CS28 are classified as class 1b fimbria [46]. Additional CFs that share similar sequences are CS13 and the recently described CS23 [47] as well as CS15 (antigen 8786) and CS22 [48]. CFs that do not show any obvious similarity to other known ETEC CFs include CS3 and CS6 [20]. Strong cross-reactive immune responses against different CFs have been demonstrated both in experimental and human studies, for example, for CFs in the CFA/I and CS5 families, respectively [49,50] [SVENNERHOLM A-M, UNPUBLISHED DATA], suggesting that a relatively limited number of CFs would be required in a vaccine targeting ETEC expressing the most common CFs. The considerable heterogeneity in CF profiles of ETEC strains isolated globally and over time has previously been suggested to complicate the relevance for developing a broad-based vaccine based on CFs [9]. Instead, it has been suggested that the tip protein CfaE of the CFA/I family that contains conserved epitopes may be more suitable as a vaccine candidate [44,51]. However, we have recently found that ETEC strains with similar virulence profiles often are remarkably Expert Rev. Vaccines 14(4), (2015)
Implications of ETEC genomics for vaccine development
similar and of clonal descent even if isolated at different continents and several decades, which would argue for a vaccine targeting the most common CFs or other antigens preserved in such successful lineages [52]. Additional vaccine candidates & virulence factors of ETEC that induce protective immunity
In addition to the classical virulence factors of ETEC, several novel CFs and putative toxins have been characterized and described in recent years. Of these, EAST1 is a heat stable enterotoxin ST originally described in enteroaggregative E. coli that is found in some ETEC strains as well as the hemolysin ClyA that is found in almost all ETEC [16]. Additional putative virulence factors that have been described in some ETEC strains include Tia [53], TibAB [54] and CexE [55]. Approaches to find novel molecules exposed on the surface of ETEC, recently led to the identification of several putative virulence loci, including the etpBAC two-partner secretion locus [56,57]. EtpA is situated on the tip of the flagella and mediates initial binding to epithelial cells and has suggested as a putative vaccine antigen [57,58]. Recently, two mucinases, EatA and YghJ, have been proposed as vaccine candidate antigens since they are involved in ETEC colonization and shown to be immunogenic [59–61]. YghJ is secreted through the type II secretion system and degrades the major mucins in the intestine MUC2 and MUC3. This protein is conserved in ETEC and exists in other pathogenic E. coli but is also expressed by non-pathogenic E. coli [61]. A targeted search for autotransporters in ETEC revealed that Ag43 and an autotransporter designated pAT as well as the putative virulence factors CexE and EtpA together with LT were associated with outer membrane vesicles (OMVs) and immunization with OMVs with LT, CexE and EtpA induced antibody responses to each of the antigens in serum and fecal samples. In addition, mice vaccinated with OMVs were significantly protected against subsequent intestinal colonization [58]. Recent progress in ETEC vaccine development
The currently most promising ETEC candidate vaccines are oral live or inactivated E. coli strains expressing some of the most prevalent ETEC CFs, for example, CFA/I, CS3, CS5 and CS6 and an LT toxoid [19,35,36,62]. These candidate vaccines have been estimated to have the potential to protect against up to 80% of all clinical ETEC infections due to the afforded protection against LT and LTST [29] as well as additional protection against the common CFs included in the vaccine. However, their capacity to protect against ETEC that only express ST and lack identified CFs would probably not be effective. Based on analysis of our global ETEC collection, we estimate that ST-only CF-negative strains constitute approximately 11% of all ETEC isolates. Furthermore, protective immunity induced by LT toxoids is short-lasting. A successful vaccination strategy should ideally prevent colonization and delivery of the toxins to the epithelial surface of all types of ETEC bacteria in the small intestine and should preferably generate long-term protection against infection and disease. informahealthcare.com
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Hence, a search to identify conserved ETEC surface outer membrane proteins that may protect against a majority of ETEC strains, and preferably also against other pathogenic E. coli, is ongoing but has not yet resulted in identification of strong candidate proteins. The recent emergence of nextgeneration sequencing now offers the possibility to screen the genomes of ETEC strains isolated globally to identify novel vaccine targets in addition to those already established. Reverse vaccinology applied on pathogenic E. coli
The use of high-throughput sequencing of entire genomes of pathogenic bacteria has fostered the ‘reverse vaccinology’ approach. The technique, which was first used to identify meningococcal vaccine candidate proteins from the sequenced genome of a Neisseria meningitides serogroup B strain [63,64], has to date been applied to a number of pathogens including N. meningitides, Streptococcus ssp. and pathogenic E. coli [63,65,66]. The rationale of reverse vaccinology is to use in silico screening of sequenced genomes for genes that encode proteins predicted to be outer membrane proteins with potential to be used as vaccine targets. Proteins predicted to be surface exposed and conserved within the target pathogen are then expressed by recombinant techniques and analyzed for immune responses in mice [63,67]. Reverse vaccinology applied on pathogenic E. coli would hence aim to target conserved surface structures on pathogenic strains that are absent in commensal E. coli. Several of the E. coli pathovars have acquired extrachromosomal genetic information by horizontal gene transfer either as plasmids, by phages or incorporated in the genome in pathogenicity islands that confer their pathogenic traits. Attempts to find conserved antigens in pathogenic E. coli that are not found or conserved in commensal E. coli strains is thus regarded an appealing approach and has been the focus of studies using both whole genome sequencing and subtractive hybridization approaches [66]. To date, most approaches of reverse vaccinology on E. coli pathovars have been applied to extraintestinal E. coli pathogens belonging to uropathogenic E. coli (UPEC) as well as newborn meningitis E. coli. It is important to note that E. coli is a diverse polyphyletic species, roughly divided into the four major phylogroups A, B1, B2 and D/E, and extraintestinal pathogenic E. coli like UPEC and newborn meningitis E. coli mainly cluster in the B2 phylogroup. Comparable analyses of newborn meningitis E. coli strains, an avian pathogenic E. coli and UPEC with the non-pathogenic E. coli K12 strain lead to the identification of 230 candidate genes present in extraintestinal E. coli strains. Of these, nine candidates were further selected based on predictions to encode outer membrane proteins or secreted proteins that conferred protection in a mouse challenge sepsis model [66]. Four candidate proteins were proposed in subsequent studies as candidates for inclusion in a vaccine against ‘all’ E. coli pathogens [66,68]. The most protective antigen in animal models among the four selected ones was a putative lipoprotein ECOK1_3385 with sequence similarities to the Vibrio cholerae accessory colonization factor. 553
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Whole genome sequencing of ETEC strains
Whole genome sequences of ETEC strains now offer the possibility to perform reverse vaccinology approaches specifically targeted toward this pathogen. The ETEC strain E24377A (SThLT, CS1 + CS3, O139) was the first strain sequenced to Lineage 1 completion followed by the model strain CS1 + CS3 24 years (1983–2007) H10407 (LTSThSTp, CFA/I, O78) and a draft sequence of B7A (STLT, CS6, O146) [72,73]. In 2011, five additional Lineage 2 ETEC draft genomes were published [74] CS2 + CS3 27 years (1980–2007) and in 2014 the whole genome sequence of an ETEC strain from Vietnam was Figure 1. Population structure of strains expressing LTSTh CS1 + CS3 (Lineage 1) described [75]. The initial paucity in and LTSTh CS2 + CS3 (Lineage 2) generated as a maximum-likelihood phylogenetic sequencing of ETEC strains has improved tree based on single nucleotide polymorphism differences across the maximum common genome [52]. The close genetic relationships between isolates collected over a substantially and we have recently found, period of nearly three decades from Asia, Africa and the Americas are shown. The two lineby whole genome sequencing of a repreages are also closely related to each other but distant from other major lineages of enterosentative selection of 362 clinical ETEC toxigenic Escherichia coli expressing specific virulence profiles (not shown in figure). isolates collected from Asia, Africa and Latin America [52], that major clonal lineECOK1_3385 was found to be secreted by the type II secre- ages of ETEC expressing the most common toxin–CF combination system and present in strains dispersed over the entire tions have spread globally (FIGURE 1). These lineages have emerged E. coli phylogeny in several pathovars but also in commensals. in modern time and successfully persisted and spread in the last It was, however, argued that the type II secretion pathway is 170–50 years [52]. The description of the new collection of inactivated in most commensals, which might link ETEC genomes is provided by von Mentzer et al. [52]. We found ECOK1_3385 to virulence and thereby justify its use as a vac- that a majority of ETEC clusters in the A and B1 phylogroups cine target [66]. The ECOK1_3385 encoded protein is but we could identify ETEC strains that clustered in B2 and D/E described as secreted and surface-associated lipoprotein from phylogroups as well. These findings implicate that the virulence E. coli (SslE) and is also known as YghJ, the most recent viru- genes defining ETEC might persist in diverse genetic E. coli lence factor described for ETEC [61]. The ECOK1_3385/acces- backgrounds. Extrapolation of these results in our University of sory colonization factor/SslE/YghJ protein will be referred to as Gothenburg ETEC collection of more than 3500 ETEC strains SslE henceforth. Another selected protein, ECOK1_0290, is a isolated globally over more than three decades indicates that a broadly conserved adhesin also described as factor adherence large proportion of all ETEC causing clinical infections in fact E. coli (FdeC) in UPEC. FdeC have some sequence similarity belong to five major lineages expressing the colonization factors to Yersinia pseudotuberculosis invasin and enteropathogenic CFA/I, CS1 + CS3, CS2 + CS3, CS5 + CS6 and CS6 [52]. These E. coli intimin (Eae) and was identified as a main vaccine can- genome sequences, together with those previously described, didate target in a follow-up study [68]. Interestingly, FdeC was seem to cover a representative selection of ETEC isolated worldshown to share 95% identity with EaeH, a putative adhesin wide which may now be used for analyses of prevalence of known identified by subtractive hybridization from the genome vaccine antigens as well as in reverse vaccinology approaches. sequence of the enterotoxigenic E. coli (ETEC) strain H10407 [69]. FdeC was found to be present in all pathogenic Vaccine candidate antigens & their prevalence in ETEC E. coli analyzed and to some extent in commensal E. coli. The Several of the new putative vaccine candidate proteins that have third candidate antigen, ECOK1_3457, was a TonB-dependent been put forward recently are either antigens specific for an siderophore receptor involved in iron acquisition, first identi- ETEC vaccine, for example, EtpA, CexE, EatA, TibAB, Tia and fied in a human E. coli septicemia strain and described as ClyA or antigens suggested for use in broader pathogenic E. coli FitA [70]; the fourth candidate was a hypothetical protein vaccines, for example, SslE, FdeC/EaeH, FitA and c0975. We referred to as c0975 [66]. The prevalence of these suggested can- determined the prevalence of these ‘new’ candidate antigens, as didate proteins remains to be investigated in other pathogenic well as the antigens being components of our inactivated ETEC E. coli. However, since the E. coli pangenome consists of over candidate vaccine, that is, LT, CFA/I, CS3, CS5 and CS6 in our 20,000 genes and only approximately 2000 genes are shared by collection of 362 sequenced ETEC strains by mapping and all E. coli strains [71], identification of conserved pangenome BLASTn. The prevalence of one of the major target molecules E. coli vaccine target antigens must be thoroughly compared for an ETEC vaccine, LT, remains at a level of at least two-thirds with commensal E. coli of all phylogroups. of all ETEC strains isolated. Together with the most prevalent Bangladesh Indonesia Japan Burma/Nepal Egypt Marocco/Tunisia Guatemala Argentina Bolivia Mexico
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Table 1. Prevalence of genes encoding putative enterotoxigenic CFs [19], these antigens cover up to 80% of Escherichia coli-specific vaccine candidate antigens. all ETEC strains isolated from individuals with diarrhea. Hence, a vaccine targeting Putative E. coli Function Prevalence Protection LT, CFA/I, CS3, CS5 and CS6 has the vaccine in ETEC animals/humans potential to provide protection against a (%)† candidate majority of all ETEC strains (TABLE 1) [19]. antigens Among the novel candidates recently put LT Toxin 65† Yes/Yes forward as putative vaccine antigens, EatA † Yes/Yes CFA/I Colonization factor 21 has been suggested to be common in † ETEC [74]. In a recent study in Bolivia, the CS3 Yes/Yes Colonization factor 22 gene encoding EatA was present in 33% of † Yes/Yes CS5 Colonization factor 13 the ETEC strains analyzed [16], and we Yes/Not shown CS6 Colonization factor 31†,‡ could identify EatA in 40% of the isolates in the recent whole genome sequencing EatA Yes/Not shown Serine protease/mucinase 40§ project (TABLE 1) [52]. The gene encoding § Yes/Not shown EtpA Adhesin glycoprotein 23 EatA was mostly found in strains with § common toxin–CF profiles, that is, CexE Yes/Not shown Putative secreted protein 7 LTSTh, CS1 + CS3; LTSTh, CS2 + CS3; § Not shown/Not shown TibAB Adhesin, autotransporter 8 STh, CFA/I, LTSTh, CS5 + CS6, LT § Not shown/Not shown Adhesin glycoprotein 10 CS6 + CS8 and LTSTp CS19 in this latter Tia † Svennerholm and Lundgren 2012 [19]. study while only 9% of the EatA-positive ‡ Combined percentage for CS5 + CS6, CS4 + CS6 and CS6-only strains from Svennerholm and Lundgren [19]. § strains were CF negative. Indeed, 65% of Prevalence in 362 representative enterotoxigenic Escherichia coli strains described by von Mentzer et al. [52]. the EatA-positive strains belonged to one CFA/I: Colonization factor antigen I; CS1: Coli surface antigen 1; CS3: Coli surface antigen 3; CS5: Coli surface antigen 5; CS6: Coli surface antigen 6; EatA: Serine protease autotransporter; ETEC: Enterotoxigenic escherichia of the five major lineages of ETEC coli; EtpA: Two partner secretion adhesin; LT: Heat labile toxin; Tia: Toxigenic invasion loci A; TibAB: Toxigenic invasion loci B. described by von Mentzer et al. [52]. Data taken from ref. [19,23,26,29,58,60]. Another ETEC-specific protein, EtpA has been shown to be located on the tip of the flagella and to be antigenic. The etpABC locus was present in pathogenic E. coli, FdeC is also present in commensals and 23% of the analyzed whole genome sequenced strains. 79% of non-pathogenic strains. these etpABC-positive strains expressed CFs of the CFA/I family, The FitA siderophore receptor that was identified as a putathat is, strains expressing CFA/I, CS1 + CS3, CS2 + CS3 and tive pathogenic E. coli antigen was only present in 7% of the CS14. CexE, has also been proposed as a promising candidate ETEC strains and only in ETEC strains belonging to the protein which induces an immune response [58], however, our B2 and D/E phylogroups, hence representing a minority of in silico analysis of sequenced ETEC isolates revealed that the ETEC (TABLE 2). Extraintestinal E. coli are regarded to mainly gene encoding CexE is exclusively present in CFA/I-positive belong to the B2 phylogroup of E. coli while ETEC strains are strains such as the ETEC type strain H10407 on which the origi- found in all phylogroups with a majority in the A and nal study was performed [55,58]. Genes encoding TibAB and Tia B1 groups [52]. This finding indicates that ETEC and other were found in 8 and 10% of the whole genome sequenced E. coli pathogens that cluster in B2 and D/E have additional strains, respectively. While tibAB was predominantly found in pathways for iron acquisition compared with E. coli pathogens strains expressing CFA/I and CS14, tia was associated with of phylogroups A and B1. Although the association to B2 and strains expressing LTSTh CS5 + CS6 and with CF-negative D/E makes FitA less useful as a vaccine antigen against all strains (29%). Thus, the suggested potential additional vaccine pathogenic E. coli, FitA might provide additional protection antigens may increase the protective coverage of the present LT- capacity against ETEC strains belonging to the D/E group of CF antigen vaccines to a limited extent. But it remains to be seen which some have disseminated globally (i.e., a STp if they may enhance protection. CS6 lineage). In contrast, the SslE protein was found in almost all ETEC strains analyzed and in all E. coli phylogroups. SslE is secreted though the type II secretion system that also exports Putative pangenome E. coli candidate vaccine antigens LT and is involved in mature biofilm formation and has muci& their prevalence in ETEC Certain antigens have been suggested to be included in a pan nase activity [66,77,78]. However, this antigen has also been idenE. coli vaccine (TABLE 2). The putative ETEC adhesion factor tified in commensal E. coli [79] and recently it was shown that EaeH was originally identified in strain H10407 [69] and was non-pathogenic E. coli strains can also secrete SslE [80]. This found to be upregulated at later time-points in a study of tran- finding highlights that novel antigens identified from sequence scriptome responses of adherent ETEC [76]. EaeH, also data and suggested to be incorporated in an ETEC vaccine described as FdeC, was identified in 88% of ETEC, confirming should be thoroughly investigated. The hypothetical protein previous findings (TABLE 2) [68]. Although present in several c0975 was found in 24% of the ETEC isolates [52], of these informahealthcare.com
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Table 2. Prevalence of genes encoding putative pathogenic Escherichia coli vaccine candidate proteins in 362 whole genome sequenced enterotoxigenic E. coli strains. Putative E. coli vaccine candidate antigens
Annotation and putative function
SslE
Secreted and surfaceassociated lipoprotein with putative mucinase function
93
FdeC/EaeH
Bacterial Ig-like domain (group 1) protein putative adhesion and biofilm component
88
FitA
tonB-dependent siderophore receptor
7
c0975
Hypothetical protein
24
Prevalence in ETEC (%)†
†
Prevalence in 362 representative enterotoxigenic Escherichia coli strains described by von Mentzer et al. [52].
strains 39% expressed CS6, either alone or co-expressed with CS5 (TABLE 2). Taken together, these results indicate that SslE and FdeC/ EaeH, although expressed by a majority of ETEC strain, are also expressed and/or secreted by commensal E. coli and hence less suitable as candidate antigens in an ETEC vaccine. The hypothetical protein c0975 as well as FitA, which is specifically linked to ETEC clustering in the B2 D/E group, may be worth further analyses, since these proteins are present in STp CS6 ETEC strains that have disseminated globally [52]. If shown to be specific for pathogenic E. coli, FitA and c0975 might add to the protective capacity of a future ETEC vaccine. CFs revisited: identification of new CFs by a reverse vaccinology approach
Recent vaccine research in our laboratory has focused on the development of a whole cell killed ETEC vaccine based on recombinant overexpression of the most prevalent CFs (CFA/I, CS3, CS5 and CS6) in an ETEC background combined with the LT toxoid, LCTBA, which is administered in combination with the dmLT molecule [19,36]. This vaccine has in recent extensive Phase I studies proved to be very safe and induces strong mucosal immune responses against the different CFs and LT [35]. However, in epidemiological studies worldwide we, and others, have found that between 30 and 50% of the ETEC isolates lack an identified CF [52]. Indeed, in our global analysis of 362 ETEC strains we found that 130 strains lacked a previously described CF. Since additional new CFs might be assumed to provide protective immunity, we have recently launched a search for conserved elements within CFs operons in a targeted reverse vaccinology approach based on conserved sequence motifs in known chaperone–usher CFs [43]. Such new putative CF variants will be further analyzed for prevalence in 556
epidemiological studies – and if prevalent considered for inclusion in a modified ETEC vaccine. This approach might also be further extended to search for presumed surface-exposed structures in CF-negative isolates that could complement a CFbased vaccine approach. Expert commentary
Next-generation sequencing and advanced bioinformatics analysis of genomes will continue to provide clues on novel targets for vaccines that might either target ETEC or all pathogenic E. coli. It has been speculated that all pathogenic E. coli strains could be covered by a vaccine that contains the four antigens: FdeC, SslE, FitA and c0975, but these candidates appear to be less suitable for an ETEC vaccine. In addition, the fact that ETEC strains are found in all E. coli phylogenetic groups, with large intraspecies variation, may suggest that finding one or few targets to provide protection against all ETEC might be difficult. It is also crucial to compare pathogenic E. coli genomes with commensal or non-pathogenic E. coli from all E. coli phylogroups to exclude the whole spectra of genes that might be present also in non-pathogenic strains. Instead, our results imply that a vaccine focused on common ETEC-specific antigens, in particular CFs, is still a highly feasible approach. Nevertheless, such a vaccine can be complemented with novel surface-exposed targets such as conserved antigens and/or new CFs identified by data mining of genome sequences. It is however important to verify surface expression of vaccine target antigens in vivo at the site of infection as well as their capacity to induce protective antibodies. ETEC specific vaccine components should fulfill all these requirements; that is, being expressed at the site of infection, being immunogenic and capable of inducing protection against ETEC infection. Five-year view
The impact of cheaper and faster whole genome sequencing using novel techniques is expected to speed up the pace of bacterial strain sequencing and will aid in the identification of potential new candidate antigens for inclusion in an effective ETEC vaccine with broad protective coverage. However, the finding that successful lineages of ETEC has emerged and spread globally in less than 50 years [52], suggests that novel antigens might emerge and spread rapidly if they confer an evolutionary advantage; hence, surveillance and further analysis of ETEC and other pathogenic E. coli is vital. The expected speed and reduced cost of sequencing in the near future might lead to epidemiological studies and clinical studies based on strains isolated from hospitalized patients using whole genome sequencing as a standard characterization method. Such data will allow surveillance of emerging infections and outbreaks as well as to aid identification of putative virulence factors suitable as vaccine antigen candidates. It is, however, crucial to determine surface expression in vivo during acute infection of such putative vaccine antigens. This may be achieved by RNA-seq of ETEC, in situ in biopsies from infected animals or patients followed by proteomic analyses as well as identification of antibodies toward Expert Rev. Vaccines 14(4), (2015)
Implications of ETEC genomics for vaccine development
the target. The ongoing development of new bioinformatics tools to analyze and predict gene function and protein characteristics in sequenced genomes is likely to allow identification of promising novel vaccine candidates in the future. Acknowledgements
The authors thank Professors G Dougan, N Thomson and T Connor at the Wellcome Trust Sanger Institute UK for financial and technical support of genome sequencing data presented. The authors also would like to thank former and present colleagues at University of Gothenburg who have contributed to sequencing data.
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Financial & competing interests disclosure
This work was funded by the Swedish foundation for Strategic Research, the Swedish Research Council and the PATH EVI program. A-M Svennerholm is a shareholder in Gotovax AB that may receive a small royalty on the sales of ETEC vaccine if it becomes a commercial product. A-M Svennerholm also has a patent in the area. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues .
Enterotoxigenic Escherichia coli (ETEC) remains to be a major cause of infantile diarrhea in the developing world and causes disease by secretion of either one or both of the enterotoxins stable toxin and labile toxin as well as by adhesion to the epithelium by a variety of colonization factors (CFs).
.
Shifts in toxin–CF profiles may occur but several of the predominant toxin–CF combinations seem to be stable and of clonal origin and are reproducibly isolated in Africa, Asia and the Americas over time.
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The most promising vaccine candidates are still vaccines expressing or containing labile toxin antigen and CFs.
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The recent advance in whole genome sequencing of ETEC offers the possibility to search genomes for novel prevalent vaccine candidate antigens.
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Conserved surface-exposed antigens specific for ETEC or for ‘all’ pathogenic E. coli should be identified and might be included in future E. coli candidate vaccines.
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Identification of conserved pangenome E coli putative vaccine antigens must be thoroughly compared with commensal E. coli of
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A search for novel colonization factors in genomes that lack described CFs is in progress since ETEC CFs would fulfill requirements for
all phylogroups. an ETEC vaccine with a broad coverage. .
Current vaccines may be improved by additional ETEC-specific conserved antigens identified by reverse vaccinology approaches.
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Potential novel antigens to be included in future vaccines should be pathogen specific, surface exposed and immunogenic.
and prevention. Clin Microbiol Rev 2005;18:465-83
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