# Patent

## Title
Live attenuated bacterial vaccine to reduce or inhibit carriage and shedding of enterohemorrhagic Escherichia coli in cattle

## Abstract

The invention provides live, attenuated enterohemorrhagic Escherichia coli (EHEC) bacteria in which the Shiga toxin coding sequences are deleted to abolish Shiga toxin production and one or more of the nucleotide sequences for the bacterial adhesin protein intimin, the locus of enterocyte effacement encoded regulator, and the translocated intimin receptor are mutated to inactivate the activity of the encoded protein(s). This live, attenuated E. coli bacteria is used in a vaccine for reducing or inhibiting carriage and shedding of EHEC in cattle.

## Background
GOVERNMENT LICENSE RIGHTS
The experiments performed in this application were supported in part by the National Institutes of Health, Grant No. NIDDK 1R01DK59012, and USDA, Grant No. CSREES 20053520115345. The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of the above grants.


CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of priority under 35 U.S.C. §119 (e) from provisional U.S. application No. 60/621,872, filed Oct. 26, 2004, the entire content of which is herein incorporated by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to live attenuated bacteria for use in vaccines.
2. Description of the Related Art
Shiga toxin-producing strains of Escherichia coli (STEC), also known as enterohemorrhagic Escherichia coli (EHEC) are important foodborne pathogens associated with foodborne epidemics of bloody diarrhea and hemorrhagic colitis (HC) (Nataro et al., 1998). While HC is often self-limiting, STEC infection can lead to more severe complications including central nervous system (CNS) disturbance and fatal hemolytic uremic syndrome (HUS) (Karmali et al., 1985). HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure and is especially life-threatening for the young and elderly (Carter et al., 1987 and Nataro et al., 1998). Human disease-associated STEC strains are referred as enterohemorrhagic E. coli (EHEC). According to the Center for Disease Control (CDC), there are about 73,000 cases of STEC infections in the U.S. each year. HUS occurs in 5-10% of these cases and leads to as many as 250 deaths (Boyce et al., 1995; Nataro et al., 1998). In the U.S., EHEC are most often serotype O157:H7, but strains in other serogroups including 026, 0111 also cause disease. Although STEC strains are generally susceptible to a variety of antibiotics, there are retrospective studies showing that the use of antibiotics negatively alters the outcome of STEC infections leading to increased incidence of HUS (Nataro et al., 1998). This is likely because lysis of bacteria by some antibiotics leads to increased release of toxin, as well as to increased toxin synthesis during the induction of lysogenic toxin-producing bacteriophage. Second, antibiotic therapy may alter the balance of intestinal flora thereby increasing the systemic absorption of released toxin (Nataro et al., 1998). Infections caused by STEC have became a public health concern since outbreaks of the disease were reported following ingestion of undercooked ground beef in hamburgers distributed by national fast food chains. Outbreaks of infection with STEC are likely to continue because of the capacity for wide distribution of the infecting organism provided by an efficient food distribution system. Currently, there are no proven vaccines or therapeutic agents for infections caused by STEC (EHEC).
Cattle are most frequently identified as the primary source of EHEC infection. EHEC thrives in the ruminant gastrointestinal tract, farm water troughs, raw manure, and other contaminated environmental surfaces (Hovde et al., 1999). The bacteria can survive for more than 2 to 5 months in water containing rumen content (Doyle, 2003 and Killham et al., 2003). The EHEC organisms may be shed at levels up to 106 cfu per gram of feces for several weeks following infection (Naylor et al., 2003 and Omisakin et al., 2003). Although natural symptomatic STEC infections have been reported in young calves (Janke et al., 1989; Person et al., 1989; Pospischil et al., 1987; Schoonderwoerd et al., 1988; and Wray et al., 1989), colonization in adult cattle by EHEC causes no clinical disease. EHEC isolates from cattle produce Shiga toxin (both Stx-1 and/or Stx-2), however, the mechanisms for resistance of adult cattle to STEC disease, despite documented intestinal carriage, is still unknown (Pospischil et al., 1987; and Stordeur et al., 2000). It is important to recognize that the rectum and cecum are principal sites of STEC 0157:H7 colonization during the carrier-shedder state in cattle (Dean-Nystrom, 2003).
The broad outline of the pathogenic mechanisms of STEC infections in humans, due to strain 0157:H7 and other STEC, are well known. After ingestion of contaminated food or water, STEC colonize the intestine (primarily the large bowel), utilizing a mechanism of intimate adherence to intestinal epithelial cells, and elaborate a potent toxin, Shiga toxin (Stx), which is the major virulence factor of this organism (Nataro et al., 1998). Locally produced Stx is then absorbed into the circulation and targets microvascular endothelial cells containing specific receptors for Stx. Low levels of Stx reaching the circulation are able to induce profound vascular lesions in target organs including bowel, central nervous system and kidney (Gyles, 1994).
The hallmark of pathogenicity of EHEC is the production of Stx implicated in the development of HUS (Griffin et al., 1991; Kaper, 1998; Noel et al., 1997; and O'Brien et al., 1992). Stx(s) produced by EHEC belong to a family of bacterial cytotoxins structurally related to those produced by the dysentery bacillus Shigella dysenteriae (Tesh et al., 1991; and O'Brien et al., 1992). Both Stx-1 and Stx-2 are in the class known as AB toxins composed of one A subunit and five identical receptor-binding B subunits (Jackson, 1990,—and Jackson et al., 1990). The B subunit binds to a receptor molecule on the host cell surface (Jackson, 1990; Jackson et al., 1990; and Mobassaleh et al., 1988). The A subunits of both toxins are highly selective N-glycosidases that depurinate a specific adenine residue on the eukaryotic 60S ribosomal subunit thus blocking protein synthesis and leading to the death of the cell (Hovde et al., 1988; and Jackson et al., 1990). Shiga toxins can modulate cytokine secretion and function. For instance, Shiga toxins induce expression and synthesis of cytokines in Caco-2 cells, and their N-glycosidase activity is essential for the induction because proinflammatory cytokine mRNAs, especially IL-8, were induced by Stx1 and Stx2 but not by a non-toxic mutant of Stx1 which lacks N-glycosidase activity (Yamasaki et al., 1999). Microarray analysis demonstrated upregulation of genes belonging to chemokines and cytokines and other genes encoding cell adhesion molecules and transcription factors that are involved in immune response or apoptosis (Matussek et al., 2003).
Attaching and effacing Escherichia coli (AEEC) represent a group of enteric pathogens of humans and animals, including human hEPEC, a major cause of infant diarrhea; EHEC, an important food-borne pathogen; strains causing diarrhea in animals such as rabbit (rEPEC), pig and dog EPEC; and Citrobacter rodentium in mice (Nataro et al., 1998; An et al., 1997; Cantey et al., 1977; and Zhu et al., 1994). Central to the pathogenesis of AEEC infection is the formation of attaching/effacing (A/E) lesions characterized by intimate bacterial attachment to intestinal epithelial cells and effacement of microvilli with disruption of host cell cytoskeleton (Nataro et al., 1998). The genes essential for the A/E phenotype are encoded on the locus of enterocyte effacement (LEE) pathogenicity island (PAI), the complete nucleotide sequence of which has been obtained from hEPEC O127:H6 (strain E2348/69) (Elliott et al., 1998), EHEC O157:H7 (strain EDL933) (Perna et al., 1998), rEPEC strain RDEC-I (015:H-) (Zhu et al., 2001), and C. rodentium associated with colonic hyperplasia in mice (Deng et al., 2001).
LEEs of EPEC, EHEC, rEPEC, and C. rodentium share a 34-kb conserved region containing 40 (RDEC-I) or 41 (hEPEC, EHEC or C. rodentium) open reading frames (ORF) organized into five major polycistronic operons: LEE1, LEE2, LEE3, LEE5 and LEE4, and several minor operons and monocistronic genes (Elliott et al., 1998). The conserved 34-kb core region of LEE PAIs of rEPEC, hEPEC, EHEC, and C. rodentium exhibit nearly identical genetic organization and high homology of LEE-encoded genes (FIG. 1; Elliott et al., 1998; Perna et al., 1998; Zhu et al., 2001; and Deng et al., 2001). The LEE encodes a global regulator Ler (LEE encoded regulator), a type III secretion system (TTSS) (LEE1, LEE2, LEE3), a bacterial adhesin named intimin, a translocated intimin receptor Tir and CesT chaperone for Tir (LEE 5), and several secreted effector proteins (LEE4) including Esp D, B, and F which are delivered into host cells via the TTSS (Elliott et al., 1998; Kaper et al., 2004 and Kenny et al., 1997). TTSSs are critical for the virulence of A/E organisms. While TTSS apparatus deliver LEE-encoded effector molecules, such as Tir, Map, EspF EspG, and EspH, the TTSSs also contribute to delivering virulence factors encoded outside the LEE, such as Cif, and EspF(u) (McNamara et al., 2001; and Marches et al., 2003).
Ler is encoded as the first open reading frame (ORF) in LEE1. It is highly conserved (95-98% amino acid identity) among hEPEC, EHEC and rEPEC 015:H-strain RDEC-I (Elliott et al., 1998; Perna et al., 1998; and Zhu et al., 2001). The deduced amino acid (AA) sequences of Ler from hEPEC share substantial similarities (24% identity and 44% similarity) with H—NS, the histone-like non-structural protein) of Salmonella mainly in the carboxyl terminus (Elliott et al., 2000). It has been shown that Ler plays a central role in the regulation of LEE-encoded gene expression (FIG. 2) and, in EPEC, that Ler positively regulates the LEE operons by acting as an antirepressor protein that overcomes the H—NS-mediated silencing of the LEE2/LEE3, LEE5 and LEE4 (Bustamante et al., 2001; Haack et al., 2003; and Sanchez-Sanmartin et al., 2001) Ler also activates the expression of the genes outside the LEE, such as espC encoded on a second PAI of EPEC (Elliott et al., 2000).
rEPEC constitutes a subset of the AEEC pathotype and strains of different serotypes have been shown to be causative agents of rabbit enteritis (Camguilhem et al., 1989; and Peeters et al., 1988). rEPEC induce A/E lesions in a manner similar to hEPEC and EHEC (Cantey et al., 1977). The extensive phenotypic and genotypic homologies among human and animal A/E strains suggest a common evolutionary origin and perhaps common regulatory mechanisms for LEE-encoded gene expression. A previous study demonstrated in hEPEC that the Ler is essential for in vitro pathogenic effects suggesting that a deletion mutation in the ler gene might attenuate the in vivo virulence of rEPEC (Mellies et al., 1999; and Sperandio et al., 2000).
Bacterial intimate adherence to host epithelial cells is mediated by binding of intimin to the translocated intimin receptor (Tir), which is delivered by A/E organisms to eukaryotic cells (Frankel et al., 1996a, 1996b and 1998; Hartland et al., 1999; Hicks et al., 1998; and Kenny et al., 1997). Intimin is an outer membrane protein (OMP) adhesin that shares homology with the invasin that promotes eukaryotic cell invasion by Yersinia (Jerse et al., 1990). Currently, nearly a dozen genetically and serologically distinct intimin subtypes are reported among A/E organism (Adu-Bobie et al., 1998; Oswald et al., 2000; Ramachandran et al., 2003; and Zhang et al., 2002). All currently known intimin alleles demonstrate more homology in their amino (N)-terminal regions than in their carboxy (c)-terminal regions. Intimins of A/E E. coli (AEEC) including human EPEC O127-.H6 (Intimin-α), EHEC O157:H7 (Intimin-γ), or rEPEC 015-.H-(Intimin-β) show greater than 94% amino acid (aa) identity over the N-terminal two thirds of the molecule while showing only 55% homology over the remaining one third portion at the C-terminus (Zhu et al., 2001). The crystal structure of the C-terminal EPEC intimin fragment (residues 658-939) revealed three adjacent domains: the immunoglobulin-like (Ig) D1 (residues 658-751) and D2 (residues 752-841) and the C-type lectin-like D3 (residues 842-929) (Frankel et al., 1995; and Luo et al., 2000). The immunodominant regions have been demonstrated to be in the domains D1 and D2, as shown by reaction with intimin-specific antiserum (Adu-Bobie et al., 1998). Binding of intimin and Tir is mediated primarily by interactions between the lectin-like D3 domain of intimin and the Tir intimin-binding domain (Luo et al., 2000). Within the Tir-binding region of intimin two conserved cysteine residues (aa 860 and 937 of EPEC intimin) are involved in the formation of a disulfide loop essential for intimin function (Frankel et al., 1995; Hicks et al., 1998; and Luo et al., 2000). This disulfide loop is conserved in Yersinia invasin and all the intimin molecules (Eliott et al., 1998; Ramachandran et al., 2003; and Zhu et al., 2001). Recent studies have shown that other accessory proteins promote bacterial adherence to intestinal epithelial cells, including the Efal from EHEC 0111 (Nicholls et al., 2000) and the Efal homologue LifA from EPEC 0126:H7 (Klapproth et al., 2000), the flegellin of EHEC, and some novel fimbriae (Torres et al., 2003). However, these proteins are not directly involved in the formation of A/E lesions.
Intimin is critical for intimate bacterial adherence. Attenuation of virulence by deletion of intimin has been demonstrated for human EPEC (0127:H6) (Donnenberg et al., 1993a), human EHEC (O157:H7) (Donnenberg et al., 1993b), rEPEC (O103:H2) (Marches et al., 2000), and C. rodentium (Deng et al., 2004).
The role of intimin in in vivo virulence has been tested through isogeneic mutants deficient in expression of functional intimin. Donnenberg and Kaper created an internal 1848-bp deletion in EPEC eae gene and tested its pathogenicity in humans (Donnenberg et al., 1991). While all of the human volunteers received WT EPEC developed diarrhea, the isogeneic eae mutant caused diarrhea in 4 of 11 individuals (Donnenberg et al., 1993a). In a separate study, the isogeneic eae mutant generated by replacing the internal 1.1-kb eae DNA with a 2.9-kb DNA fragment containing a Tet marker of human EHEC 86-24 (0157:H7) was unable to colonize in experimentally inoculated piglets (Donnenberg et al., 1993b). In REPEC O103:H2, an insertion of aphT encoding Kan resistance in the eae gene (between 993 nt and 994 nt) disrupted the expression of intimin and abolished bacterial virulence when tested by experimental inoculation of its natural rabbit host (Marches et al., 2000). Anti-intimin immune responses can modulate the outcome of A/E organism infection. In another study conducted in piglets, passive immunization, achieved by allowing neonatal piglets to suck colostrums from intimin-vaccinated dams for up to 8 h before inoculating with EHEC 0157:H7, protected animals from STEC 0157:H7 colonization and intestinal damage (Dean-Nystrom et al., 2002). Using mutant E. coli heat-labile enterotoxin (LT) lacking the nick site in the A subunit as an adjuvant, intranasally administered 0157:H7 intimin induced an elevation of IgA-specific antibody in the nasal secretion and saliva of calves as well as an elevation of IgG1-specific antibody level against the intimin in the sera and colostrums of cows (Yokomizo et al., 2002). In yet another study, EHEC 0157:H7 intimin C-terminal domain was expressed in transgenic tobacco plant cells and mice immunized with the plants generated an intimin-specific mucosal immune response and exhibited a reduced duration of EHEC 0157:H7 fecal shedding (Judge et al., 2004). Interestingly, vaccination of mice with Int280α induced both type-specific protection to intimin-c organisms and to heterologous intimin types indicating that a highly conserved domain of intimin (Int388-667) including part of C-terminal fragment D1 domain may have potential to induce protection against infections by A/E organism expressing different intimin types (Ghaem-Maghami et al, 2001).
More than 50 serotypes of STEC have been isolated from stool samples of patients with hemorrhagic colitis or HUS. STEC 0157:H7 is the predominant serotype reported as the causative agent world-wide (Nataro et al., 1998). Analysis of HUS samples collected from 1987 to 1991 in the United States indicated that STEC could be implicated in 72% of cases of HUS, and STEC serotype 0157 may be implicated in 80% cases studied (Banatvala et al., 2001). However, infections due to non-0157 STEC are now increasingly recognized (Nataro et al., 1998; and Tarr et al., 1996 and 2002). Of these, STEC 026 and STEC 0111 have been isolated most frequently. In Boston and Virginia, approximately half of all Stx-producing E. coli isolates from patients were of non-0157:H7 serotypes (Park et al., 1996). STEC serogroup 026 and 0111 have been increasingly associated with outbreaks in Europe, Japan, Australia, India, where they account for the majority of HUS cases (Ojeda et al., 1995,—Pierard et al., 1990; and Robins-browne et al., 1998). The prevalence of 0157, 026, and 0111 in humans is in accordance with the findings that these serogroups were most common in fecal samples from animals (Blanco et al., 2004a and 2004b; Borie et al., 1997; and Rey et al., 2003).
Vaccines for animals are aimed at reduction of EHEC secretion in their natural host. A clinical trial of a parenteral STEC vaccine has recently been conducted by the Canadian investigators (Potter et al., 2004). The vaccine formulation containing secreted protein preparations of STEC strain 0157:H7, together with aluminum adjuvant, VSA3 was delivered subcutaneously in the necks of seronegative cattle (Potter et al., 2004). Vaccinated cattle were primed and showed an increase in serum IgG antibody titers. Vaccination was reported to reduce the prevalence of STEC 0157:H7 from 21.3% to 8.8% in feedlot cattle at the day of marketing (Potter et al., 2004). Thus, although an apparent effect was seen, substantial numbers of vaccinated animals still shed STEC in the feces.
In a non-vaccine study, the combinations of several strains of probiotics were shown to inhibit the growth of EHEC 0157 in vitro and reduce the fecal shedding of EHEC 0157:H7 (Doyle, et al, 2003). The fecal shedding and pathogenicity of STEC O26:H11, 0111:NM, 0157:H7 in weaned calves (8 to 10 weeks of age) were compared with and without treatment using a three-strain mixture (Hicks et al., 1998 and Tkalcic et al., 2003). The probiotic E. coli substantially reduced or eliminated fecal shedding of 0157:H7 and 0111:NM. However, STEC were still recovered from one third of calves receiving the probiotic treatment, and the probiotic E. coli did not reduce fecal shedding or gastrointestinal persistence of 026:HIl (Hicks et al., 1998 and Tkalcic et al., 2003). Interestingly, when probiotics were used in calves of less that 1 week of age, reduced fecal shedding of 0111:NM and O26:H11 but not STEC 0157 was observed in most calves (Zhao et al., 2003).
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
SUMMARY OF THE INVENTION
The present invention provides a live, attenuated enterohemorrhagic Escherichia coli (EHEC) of serogroup 0157, 026 or 0111 in which the Shiga toxin (Stx) coding sequences are deleted to abolish Shiga toxin production and one or more of the nucleotide sequences coding for the bacterial adhesin protein intimin (eae), the locus of enterocyte effacement encoded regulator (ler), and the translocated intimin receptor (tir) are mutated to inactivate the activity of the encoded protein (s).
The present invention also provides a vaccine for reducing or inhibiting carriage and shedding of enterohemorrhagic E. coli in cattle which contains an immunogenically effective amount of the live, attenuated enterohemorrhagic E. coli. 
Another aspect of the present invention is directed to a method for reducing or inhibiting carriage and shedding of enterohemorrhagic Escherichia coli in cattle by immunizing cattle with the vaccine of the present invention.
A further aspect of the present invention is directed to a method for producing the isolated live, attenuated enterohemorrhagic E. coli of the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of the genetic organization of RDEC-I LEE pathogenicity island.
FIG. 2 is a schematic illustration of the regulation of LEE operons by Ler.
FIG. 3A is a schematic representation of RDEC-I intimin C-terminal fragment (539-939aa) showing Ig-like (Ig) and Tir-binding domains. Numbers represent aa position of intimin deduced from RDEC-I LEE. The residues of intimin Tir-binding domain (residues 842-939 of SEQ ID NO: 5) are shown. Asterisks indicate two conserved cysteine residues (residues 860 and 937) involved in the formation of a disulfide loop essential for intimin function. The RDEC-l Δeae C-terminal sequence (SEQ ID NO: 6) is also shown, where residues identical with the RDEC-I C-terminus are shown by dots. FIG. 3B is a DNA alignment of a partial sequence of the RDEC-leae gene (SEQ ID NO: 25) and a partial sequence of the RDEC-l Δeae gene (SEQ ID NO: 26) showing a single base deletion (arrow) which introduced a stop codon (underlined) 24-bps immediately after deletion. FIG. 3C is a comparison of OMP profiles on a gel showing native intimin (denoted by arrow) by RDEC-I or truncated intimin (arrow head) by RDEC-l Δeae. Molecular markers are indicated in kDa.
FIGS. 4A and 4B are graphs showing comparisons of cumulative weight change (FIG. 4A) and fecal bacterial shedding (FIG. 4B) between rabbits inoculated with WT RDEC-I or its derivative eae mutant. Averages were derived from six rabbits in each group. Bars designate standard errors.
FIGS. 5A-5D are graphs showing comparisons of fecal bacterial shedding (FIG. 5c), cumulative weight change (FIG. 5B) stool consistency (FIG. 5A), and percent of mucosal surface (cecum) with adherent bacteria (FIG. 5D) between RDEC-l Δeae-immunized (circles) and unimmunized groups (triangles) following challenge with RDEC-H19A. Bars designate standard errors.
FIG. 6 show histological sections from a non-immunized rabbit challenged with RDEC-H19A showing intimate bacterial adherence and effacement of microvilli. Bacteria are adhering to the cecal enterocytes lining the villi (solid arrow). The intestinal villi became irregular and a cluster of cells has been desquamated from the villi (arrow). The lumina propria was infiltrated with polymorphonuclear leukocytes (semisolid arrow heads). H&E staining, magnification, ×1,000.
FIGS. 7A-7D are graphs showing comparisons between un-immunized/challenged (vertical line), immunized/challenged (semi-solid), and immunized/un-challenged (hatched columns) rabbits of histopathological findings for edema (FIG. 7A), heterophiles (FIG. 7B), vascular abnormalities (FIG. 7C), and thrombus formation (FIG. 7D). Bars represent standard errors.
FIG. 8 is a graph showing rabbit serum IgG titers specific to MBP-Int280 following immunization with RDEC-l Δeae. Bars designate standard errors.
FIG. 9A shows the nucleotide alignments of ler and upstream regions of hEPEC (0127:H6, strain E2348/69, GenBank accession no. AF022236; SEQ ID NO: 14), EHEC (O157:H7, strain Eα933, GenBank accession no. AF071034; SEQ ID NO:16), C. rodentium (strain DBSlOO, GenBank accession no. AF311901; SEQ ID NO:18), rEPEC RDEC-I (GenBank accession no. AF200363; SEQ ID NO:20), and rEPEC O103:H2, strain E22; SEQ ID NO:22) showing the ler coding sequence (bold). The 300-bp deletion fragment generated in the ler of strain E22 is underlined. FIG. 9B shows the alignment of the predicted amino acid sequences of Ler proteins of EPEC (SEQ ID NO:15), EHEC (SEQ ID NO:17), C. rodentium (SEQ ID NO:19), RDEC-I (SEQ ID NO:21), E22 (SEQ ID NO: 23) and E22Δler (SEQ ID NO: 24). Identical sequences are indicated by dots. Gaps introduced for alignment are represented by dashes. Numbers on the right side of the sequence indicate the position in the relevant LEE PAI (A) or deduced Ler (B).
FIG. 10 is a gel showing secreted protein profiles of WT rEPEC (E22) and its derivative mutant E22Δler. Proteins were separated by 12% SDS-PAGE and visualized by silver staining. The positions of protein standards (in kDa) are indicated on the left.
FIGS. 11A-11D are graphs of a pathogenicity study comparisons of fecal bacterial shedding (Fig. HA), cumulative weight change (Fig. HB), percentage survival (Fig. HC), and percentage of mucosal surface (cecum) with adherent bacteria (Fig. HD) between groups of rabbits inoculated with WT rEPEC strain E22 (6×105 CFU) or its derivative ler mutant (1×108 CFU). Averages were derived from six rabbits in each group at the start of experiment. By the end of the experiment, averages in the WT E22 group were derived from the number of survivors (survivor numbers shown next to average data points in Fig. H A and HB). Bars designate standard errors.
FIGS. 12A and 12B are micrographs (Giemsa stain, magnification 400×) of cecal tissues from rabbits infected for five days with WT rEPEC (FIG. 12A) or E22. Δler (FIG. 12B). Intimate mucosal attachment of bacteria with effacement of the apical brush border (arrows) representing typical A/E lesions in shown in FIG. 12A. This process involves 40% of the mucosal surface in this micrograph. Intact intestinal mucosa and normal brush borders (arrow heads) are shown in FIG. 12B. Note the normal appearance of the brush border. Although scattered adherence of organisms was seen (white arrow head), bacterial attachment was non-intimate and brush borders appeared normal.
FIG. 13A-13D are graphs of a protection study showing cumulative weight gain (FIG. 13A), fecal bacterial shedding (FIG. 13B), percent survival (FIG. 13C), and fold increase of serum IgG against whole bacterial cells (FIG. 13D) in rabbits previously-immunized with a single orogastric dose of E22Δ2er and challenged with the WT parent E22 (day of challenge shown as day 0). Bars represent the standard errors.
FIG. 14 is a schematic illustration of single-overlap extension PCR (SOE PCR).
FIG. 15 is a schematic representation of native intimin and truncated intimin.
FIG. 16 is a timeline of the vaccination protocol.
FIGS. 17A and 17B are graphs of adherence to HeLa cells showing recovered percentage of CFU (FIG. 17A) and adherence pattern (FIG. 17B) following 6 h incubation. Bars represent standard error.
FIGS. 18A and 18B are micrographs showing microcolonies by the WT EHEC 0157:H7 strain 86-24 (FIG. 18A) and its derivative mutant (FIG. 18B). Microcolonies were seen for the WT EHEC (arrow heads) but rarely seen for the isogenic mutant.
FIG. 19 is a graph showing fecal bacterial shedding following primary and booster vaccination with 86-24 ΔeaeΔstx2AB by oral or rectal route.
FIG. 20 is a graph showing a comparison of clearance of 0157:H7 in the intestinal tract of mice following oral and rectal immunization with 86-24 ΔeaeΔstx2AB with those receiving PBS. Challenge with 86-24 Δstx2AB was performed at 2 weeks (A) or 4 weeks (B) post booster immunization.
FIGS. 21A and 21B are graphs showing comparison of sera IgG titers specific to 0157:H7 LPS at day 0, 14, and 28, among groups receiving oral (FIG. 21A) or rectal (FIG. 21B) vaccination of 86-24 ΔeaeΔstx2AB. Dashed line indicates the cutoff value.
FIG. 22 is a graph showing a comparison of sera IgG titers specific to 0157:H7 LPS at day 28 following immunization among mouse groups receiving oral and rectal immunization of 86-24 ΔeaeΔstx2AB with naïve mice. Dashed line indicates the cutoff value.
FIG. 23 is a graph showing a comparison of IgA titers specific to 0157 LPS from intestinal lavage. Bars represent the standard errors.



DETAILED DESCRIPTION OF THE INVENTION
STEC/EHEC strains cause bloody diarrhea and potentially fatal systemic sequelae in humans. A successful human vaccine would need to elicit anti-bacterial immunity to prevent bacterial adhesion or anti-toxin immunity to neutralize the Shiga toxin. While there is indirect evidence that human vaccination against STEC may be effective in preventing illness, at present, there are no human vaccines for STEC or effective intervention strategies. The difficulty in identifying such vaccines and interventions is hampered by the fact that human volunteers cannot be challenged with either virulent STEC or Shiga toxin preparations and that there are not enough incidences for field trials. Moreover, because of the sporadic nature of EHEC outbreaks, it is unlikely that universal vaccination of susceptible human populations will be adopted as a public health measure.
The present invention overcomes these above limitations and develops a novel vaccination approach, which will eliminate STEC strains from the food chain, where the major reservoirs of EHEC are cattle. Asymptomatic cattle are colonized by and shed these organisms leading to contamination of livestock-derived food products and water, and subsequent human STEC infections. Vaccination of cattle, which eliminate, inhibit or reduce the level of STEC shedding, can greatly impact the number of foodborne outbreaks. Immunization of cattle with live attenuated EHEC vaccine is expected to induce effective mucosal immunity, thus preventing or inhibiting EHEC colonization in the gut. Such immunization would provide safer meat and a cleaner environment.
The laboratory of the present inventors have developed a rabbit model for acute symptomatic STEC infection by introducing the Stx1-producing bacteriophage from a human 026 isolate into the rabbit enteropathogenic E. coli strain (REPEC) RDEC-I (Cantey et al., 1977; and Hicks et al., 1998). The resulting bacterium, RDEC-H19A, lysogenic for phage H19A, produces an illness in rabbits which closely resembles the hemorrhagic colitis induced by STEC in humans in both clinical course and histopathological findings (Hicks et al., 1998; and Sjogren et al., 1994).
A number of isogeneic mutants in REPEC defective in selective virulence genes, e.g., the regulatory genes ler and luxS and the genes coding for proteins involved in bacterial adherence (eae, tir and lifA), were constructed in the laboratory of the present inventors. LuxS is involved in quorum sensing in EPEC and EHEC bacterial cells (Sperandio et al., 1999 and 2001). The HfA is a lymphotoxin in human EPEC which down-regulates host immune responses (Klapproth et al., 2000). The HfA homologue in STEC/EHEC 0111:H- is designated as efal (EHEC factor for adhesion) which has been ascribed an adherence function independent of the LEE (Nicholls et al., 2000). The efal/lifA is present in all A/E organisms of human and animal origin (Badea et al., 2003). However, in human EHEC 0157:H7, the efal/lifA homologue is smaller (9507 nt) and located on the pO157 plasmid and named toxB (Badea et al., 2003 and Tasuno et al., 2001). The phenotype alterations of these mutants have been examined in vivo and in vitro. The potential of these mutants as vaccine candidates has been tested by experimental challenge with the WT REPEC strain RDEC-I (O15:H-) or E22 (O103:H2). Three classes of mutants have been observed: 1) fully attenuated, including the ler, eae, tir mutants; 2) partially attenuated, the HfA mutant; and 3) no in vivo attenuation, the luxS mutant. Only the data for the eae and ler mutant in the first class are presented in Examples 1 and 2 hereinbelow.
The laboratory of the present inventors has generated a substantial amount of information on the level of attenuation in virulence and mechanisms of homologous protection by using the rabbit STEC model and has demonstrated full attenuation of bacterial virulence by inactivation of the ler gene encoding a global regulator, the eae gene encoding intimin, or the tir gene encoding translocated intimin receptor Tir. The results presented in Example 1 hereinbelow demonstrate that intimin plays a critical role in A/E, and the intimin truncation mutation induced significant amounts of sera IgG against intimin and also induced effective protection. Likewise, the ler mutants in Example 2 hereinbelow are attenuated, and immunization with ler mutants provided protection against STEC of the same serotype. Thus, the studies in Examples 1 and 2 show that mutation in the eae and ler genes attenuated bacterial virulence while retaining their immunogenicity. The laboratory of the present inventors have also created tir mutants of REPEC and demonstrated attenuation in the virulence of the tir mutants. The immunogenicity of the tir mutant is expected to be similar to the eae and ler mutants.
The eae mutant in which the C-terminal Tir binding domain (TBD) is truncated or deleted would retain the immunodominant region of intimin and be able to induce antibody production that has been shown to be protective. Moreover, the eae truncation will not affect the expression of proteins encoded on the LEE and outside the LEE, which may enhance immune responses against EHEC because a full array of protein encoded by wild-type EHEC would be expressed with the exception of the trancation at the C-terminal portion of intimin. Because Ler is a central regulator for the genes encoded on the LEE, it is expected that the attenuated mutant will not express ler-regulated protein expression, such as Tir and other secreted proteins. The same strategies used in Examples 1 and 2 for REPEC can be applied to construct STEC/EHEC vaccines for cattle.
The present invention is directed to a live, attenuated enterohemorrhagic Escherichia coli (EHEC) in which the Shiga toxin Stx1 or Stx2 coding sequence, stx1A/stxB or stx2A/stx2b, is mutated, preferably deleted, to abolish Shiga toxin production, and one or more of the nucleotide sequence coding for bacterial adhesion protein intimin (eae), the locus of enterocyte effacement (LEE)-encoded regulator (ler), and the translocated intimin receipt (tir) are mutated, preferably by creating a deletion mutant, to inactivate the virulence-associated activity of the encoded protein(s).
The deletion of stxAB genes (stx1AB and/or stx2AB) will make EHEC non-toxic, and inactivation of either eae, ler, or tir will abolish A/E capacity of EHEC strains. In addition, modifying genes in the LEE such as eae, ler, or tir may have little impact on the function of accessory molecules involved in STEC colonization and persistence in the gut. Thus, a double mutation will fully attenuate bacterial virulence and make the live, attenuated EHEC strains safe for use as a vaccine in cattle. It will be appreciated by those of skill in the art that, while a double mutant is preferred, triple or quadruple mutants in which two or more of eae, ler, or tir are mutated are also encompassed in the present invention.
The present invention is also directed to a vaccine for reducing or inhibiting carriage and shedding of enterohemorrhagic E. coli in cattle, which vaccine contains a pharmaceutically acceptable carrier and an immunogenically effective amount of the live, attenuated enterohemorrhagic E. coli. The term “immunogenically effective amount” is meant to be the amount of live attenuated EHEC sufficient to induce in the host an effective immune response to the virulent EHEC strains of the same serogroup. This immunogenically effective amount is in a range of IxIO3 to IxIO10 CFU of the live attenuated bacteria, more preferably in a range of IxIO5 to IxIO10 CFU, and most preferably in a range of IxIO8 to IxIO9 CFU. The live attenuated EHEC belongs to serogroup 0157, 026 or 0111, preferably of serotype 0157:H7 and 011:H-. In the vaccine of the present invention, live attenuated EHEC of serogroups 0157, 026, and 0111 can be used alone or in combination to induce immunity against the most prevalent STEC serogroups.
The particular pharmaceutically acceptable carrier or diluent employed is not critical to the present invention. Examples of diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) (Levine et al., 1987; and Black et al., 1987), or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame (Levine et al., 1988).
Optionally, one or more compounds having adjuvant activity may be added to the vaccine. Adjuvants are non-specific stimulators of the immune system. They enhance the immune response of the host to the vaccine. Examples of adjuvants known in the art are Freund's Complete and Incomplete adjuvant, vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulating complexes, see for instance European Patent EP 109942), Saponins, mineral oil, vegetable oil, and CarbopolTM. Other suitable adjuvants are for example aluminium hydroxide, aluminium phosphate or aluminium oxide, oil-emulsions (e.g. of BAYOLFTM or MARCOL 52TM, saponins or vitamin-E solubilizate.
Other examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk and buffers (e.g. phosphate buffer). Especially when such stabilizers are added to the vaccine, the vaccine is very suitable for freeze-drying.
The present invention is further directed to a method for reducing or inhibiting carriage and shedding of enterohemorrhagic Escherichia coli in cattle, which involves immunizing cattle with the vaccine of thβ ‘present invention to reduce or inhibit carriage and shedding of enterohemorrhagic E. coli in the immunized cattle.
The mucosal and systemic immune systems are compartmentalized (Mesteky, 1987; Newby, 1984; and Pascual et al., 1994). Thus, antigens delivered to mucosal surfaces elicit mucosal and systemic responses, whereas parentally delivered antigens elicit mainly systemic responses but only stimulate poor mucosal responses (Mesteky, 1987). Moreover, mucosal stimulation at one mucosal site (for example the intestine) can result in development of immunity at other mucosal surfaces (for example genital/urinary tract) (Mesteky, 1987). This phenomenon is referred to as the common mucosal system and is well documented (Mesteky, 1987; and Pascual et al., 1994).
Mucosal surfaces comprise the largest surface area of the human and animal body and are the first line of defense against many pathogens. The oral route of vaccination has been widely used in the vaccination practice, both in humans and animals. The disadvantage of oral administration of vaccines for cattle is that, in these animals gastric acid provides a formidable barrier against microorganisms, and is highly-effective in killing orally administered, attenuated live bacterial vaccines. In addition, the complex rumen, with its own flora, represents an additional barrier to establishment of live vaccine strains given orgogastrically. Another well-established avenue for effective induction of the gut-associated immunity is intrarectal immunization. Studies conducted in mice demonstrated that rectal immunization elicits high levels of specific immune responses (Hopkins et al., 1995; Kawahara et al., 2002; Mitchel et al., 2003a; Zhou et al., 1995). The sub-epithelium at the bovine terminal rectum contains a high concentration of lymphoid follicles (Naylor et al., 2003) and has all the characterisitics of a classical inductive site for mucosal immunization. It is noteworthy that this organized lymphoid tissue has been proposed as the predominant site of carriage of STEC strains in cattle. In one study, the majority of tissue-associated EHEC 0157:H7 were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction (RAJ) having a high density of lymphoid follicles. For this reason, rectal immunization in cattle may be uniquely able to directly induce local immunity in the sites of colonization (Low et al., 2003).
Because the mucosal surface is the primary site for STEC attachment, the vaccine of the present invention is preferably administered to induce mucosal immunity. The vaccine is more preferably administered orally (with prior administration of sodium bicarbonate, for example, to neutralize stomach acidity) or intrarectally.
A still further aspect of the present invention is directed to a method for producing the isolated live, attenuated enterohemorrhagic Escherichia coli of the present invention, which involves deleting any Shiga toxin sequences coding for active Shiga toxin in an enterohemorrhagic E. coli of serogroup 0157, 026 or 0111 to abolish Shiga toxin production and mutating, preferably by introducing a deletion in, one or more of the nucleotide sequences coding for the bacterial adhesin protein intimin (eae), the loss of enterocyte effacement-encoded regulator (ler), and the translocated intimin receptor (tir) to inactivate the virulence-associated activity of the encoded protein (s).
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting of the present invention.
Example 1
Protection Against Hemorrhagic Colitis in an Animal Model by Oral Immunization with an Isogeneic Rabbit Enteropathogenic Escherichia coli Expressing Truncated Intimin
A rabbit model for EHEC infection by introducing Stx1-converting phage into a rabbit A/E pathogen RDEC-I to obtain strain RDEC-Hl 9A was previously developed in the laboratory of the present inventors (Sjogren et al., 1994). The rabbit EHEC model reproduces both the A/E pathology and the vascular damage and inflammation attributable to Stx and is a suitable model for evaluating various EHEC interventions (Sjogren et al., 1994). In this example, an eae mutant of RDEC-I was constructed and examined for both the level of attenuation in virulence and the effectiveness of this eae mutant as a vaccine candidate. RDEC-lΔeae is shown to be fully attenuated and well tolerated in rabbits. It is further shown that immunization of rabbits with RDEC-lΔeae protected animals from occurrence of diarrhea, weight loss, and tissue damage induced by virulent RDEC-H19A. Thus, the attenuation of an AEEC strain by the truncation of intimin is expected to provide an effective vaccine for the prevention of EHEC infection.
Materials and Methods
Bacterial Strains and Culture Conditions.
Strains used in this study are listed in Table 1. The prototype RDEC-I (015:H-) was originally isolated from rabbits with diarrhea (Cantey et al., 1977). Strain RDEC-H19A is a RDEC-I derivative containing the Stx-I-producing phage H19A from human 026 EHEC (Sjogren et al., 1994). The laboratory E. coli strain DH5α was used for plasmid transformation except in the case of suicide plasmids (pCVD442 and its derivatives)—which were maintained in E. coli SY327 or SMlO (Donnenberg et al., 1991). Bacterial strains were stored at −70° C. in Luria-Bertani (LB) containing 20% glycerol and grown on LB agar or broth or otherwise described. Appropriate antibiotics were supplemented to the media when needed at the following concentrations: ampicillin (Amp), 50 μg/ml; nalidixic acid (NaI), 20 μg/ml; tetracycline (Tet), 25 μg/ml.







TABLE 1







Bacterial strains and plasmids used in this study (Example 1)









Strains & plasmids
Relevant features
Source or reference










Strains:









DH5α
Laboratory E. coli strain



RDEC-I
O15:H-, NalR
Cantey et al., 1977


RDEC-Hl 9A
RDEC-I transduced with phage H19A, NalR TetR
Sjogren et al., 1994


SMlO
SMlO λpir, recipient for suicide vector pCVD442
Donneπberg et al., 1991


TSAOl
SMlO containing pM3 8 1
This study


RDEC-\Aeae
RDEC-IΔeae, NalR
This study







Plasmids:









pALT417-3
PALTER-I ::RDEC-le αe, AmpR



p368
Derived from pALT417-3 with one bp deletion in the
This study



eae, AmρR


pCVD442
Suicide vector, AmpR
Donnenberg et al., 1991


pM381
pCVD442:: Δeae, KanR, AmpR
This study









Construction of an Eae Truncation Mutation in RDEC-I.
A 4.36 kb HindIII DNA fragment containing RDEC-I eae gene (excluding the first 100 bp) and downstream sequences was cloned into pALTER-1 and site-directed mutagenesis of the eae gene was achieved using the single stranded phagmid protocol and mutant oligo 224832C (5′-GATGCCGAAAACAACTGTAAGACAAATAGCGCAA-S′; SEQ ID NO:1) following manufacturer's guidance (Promega Inc., Madison, Wis.) to obtain plasmid p368. This oligo contains a single base pair deletion at the position of 2565 nt in the eae coding sequence (FIG. 3A) thus resulting in a frame shift which generates a stop codon twenty-three base pairs downstream of the deletion (FIG. 3A). A DNA fragment containing the mutated eae gene was excised from pALTER plasmid by Xbal digestion and cloned into Xba I-digested suicide plasmid pCVD442, resulting in plasmid pM381. Plasmid pM381 was transformed into E. coli strain SY327 and the resultant plasmid transformed subsequently into strain SMlO to obtain strain TSAOl (Table 1). A chromosomal eae mutation in RDEC-I was generated by allelic exchange as described by Donnerberg and Kaper (Donnenberg et al., 1991). The Amps, NalR, sucrose-resistant bacteria that underwent allelic exchange were screened for FAS (fluorescence actin staining) activity on HEp-2 cells as previously described (Karaolis et al., 1997). Strains that failed to induce actin aggregation on HEp-2 cells were further examined by sequencing the intimin C-terminal portion using PCR-amplified DNA with a pairs of primers (Agin1, 5′-CCAGTATTACTGAGATTAAG (SEQ ID NO: 2), 27351-2737lnt; Agin2, 5′-TCCGGGATTTGAGATGTAAT (SEQ ID NO:3), 28223-28204 nt) derived from RDEC-I LEE (GenBank Accession #AF200363) (Zhu et al., 1995). Expression of the truncated intimin by RDEC-lΔeae was examined by separation of bacterial outer membrane preparations by SDS-PAGE as described (Zhu et al., 1995).
Examination of In Vivo Virulence of RDEC-lΔeae and Protection Following Immunization.
The in vivo virulence of RDEC-l Δeae was determined in 2 month-old New-Zealand White rabbits. Bacteria strains were streaked onto MacConkey agar supplemented with NaI and an individual colony was then inoculated into 100 ml Penassy broth (PAB; antibiotic medium 3 Difco) and cultured at 37° C. overnight without shaking. Bacteria were washed once and suspended in sterile PBS and adjusted to the concentration of OD6oo 0.10 (approximately 1×108 cfu/ml). Bacterial viable counts were determined by standard plating methods. Rabbits were fasted overnight and unsedated animals were inoculated intragastrically via pediatric feeding tube with 10 ml 10% bicarbonate solution to neutralize gastric contents followed by the inoculum. Inocula consisted of a total volume of 3 ml of PBS containing RDEC-I or its derivative RDEC-l Δeae. The tube was then flushed with 5 ml sterile PBS to ensure that the total inocula were delivered into the stomach. Rabbits were sacrificed fourteen days post inoculation.
To determine if RDEC-l Δeae immunization would protect animals from experimental challenge, rabbits were orogastrically inoculated with RDEC-l Δeae as described above and boosted with the same dose 14 days later. Rabbits orogastrically inoculated with PBS served as controls. Fourteen days post boost, rabbits were challenged with 5×lO7 CFU of RDEC-H19A, a Stx1-producing RDEC-I derivative, which is highly virulent to its natural rabbit hosts (Sjogren et al., 1994). Rabbits were sacrificed seven days post challenge.
Animals were observed daily for clinical signs of disease. Rabbit weights were recorded and stool consistency scored daily. Fecal shedding of inoculated bacteria was determined for each rabbit by semi-quantitative cultures of rectal swabs as previously described (Sjogren et al., 1994). In brief, rectal swabs are rolled onto MacConkey plates (supplemented with Tet and NaI or NaI alone for RDEC-Hl 9A or RDEC-l Δeae, respectively) and grown overnight at 37° C. and graded as 0 (no CFU), 1+ (1-50 CFU), 2+ (50-200 CFU), 3+ (>200 CFU), or 4+ (confluent growth).
Rabbits were euthanized according to standard protocols. At necropsy, the abdominal organs were inspected for serosal hemorrhage and bowel edema, and the degree of liquidity of cecal contents was observed and recorded. Transmural sections from the distal ileum, cecum, proximal colon, and distal colon were excised and fixed in 10% buffered formalin for sectioning. Tissues were stained with hematoxylin and eosin (H&E) or with Giemsa. Microscopic examination of histopathology was based on ten sequential, well-oriented, 400× fields from each sample. Bacterial enteroadherence was graded as the percentage of surface area in the field which is covered by closely adherent bacteria (Sjogren et al., 1994). Edema depth was quantitated with an ocular micrometer by measuring the distance from the muscularis mucosa to the muscularis propria. Heterophils are counted in the mucosa, in fields immediately adjacent to the muscularis mucosa extending luminally to the limits of the 250 μm diameter field (4O× objective). Counts from ten fields are tabulated, averaged and expressed as heterophils per high power field. Vascular changes, including endothelial swelling, adherent heterophiles, endothelial denudation and thrombus formation were measured separately in sequential vessels in the mucosa, submucosa, and serosa and graded from 0 to 4 scales (Sjogren et al., 1994). A composite score for vascular changes based on summation of the scores for the four individual parameters was calculated for each of the three tissue compartments.
Detection of Antibodies Specific to RDEC-I Intimin.
For serum and mucosal antibody detection, sera were prepared and bile was aspirated from the gallbladders. A maltose-binding protein (MBP)-intimin fusion protein was constructed by cloning PCR-amplified 843-bp fragment containing the C-terminal 280aa portion of RDEC-I intimin into the pMAL-p2 (New England Biolabs, Beverly, Mass.) according to manufacturer's instructions. The MBP-intimin fusion protein was isolated from B. coli periplasm by affinity chromatography (Again et al., 1997). Sera IgG or bile IgA specific to RDEC-I intimin was determined by ELISA as previously described (McKee et al., 1996). Briefly, microtiter wells (LabTeck) were coated with MBP-intimin fusion protein at the concentration of 4 μg/ml in bicarbonate buffer at pH 9.6. Serial dilutions of rabbit sera or bile were added, and bound antibodies were detected with HRP-conjugated sheep anti-rabbit IgG or goat anti-rabbit IgA and developed with ABT peroxidase substrate (KPL, Gaithersburg, Md.).
Statistical Analysis.
Values for differences in rabbit weight gain, bacterial adherence, and histological findings between experimental groups were compared by the Student T-Test
Results
Frame Shift Mutation Resulted in Intimin Truncation.
A single nucleotide deletion was made at 2565 nt in the eae coding sequence of RDEC-I (SEQ ID NO: 4). As a result of the frame shift, a stop codon is introduced 24 bp immediately after deletion (FIG. 3B). Thus, the intimin C-terminal 81 residues (860-939aa) containing a disulfide loop was truncated and replaced by a series of new eight residues (NVRQIAQI, residues 18-25 of SEQ ID NO:6; FIG. 3A). Therefore, the predicted truncated intimin is composed of 866 residues, which is 73 aa shorter than the native intimin molecule.
Conjugation of RDEC-I with conjugative strain TSAOl (SMlO containing suicide plasmid pCVD442; Donnenberg et al., 1991) yielded numerous colonies, one of which was cultured overnight in LB broth without antibiotics and subsequently plated on LB containing 5% sucrose. Individual colonies grown on LB supplemented with sucrose were examined for antibiotic resistance profile. The Amp-resistant bacteria were selected to further characterized by the fluorescent actin staining (FAS) test on HEp-2 cells (Karaolis et al., 1997). Four isolates that were negative by FAS assay (data not shown) were recovered. Nucleotide sequencing of PCR amplicons using primers Agin1/Agin2 for the FAS-negative isolates showed a nucleotide deletion at the position of 2565 nt in the eae gene. One of these isolates was designated as RDEC-lΔeae and used in subsequent study.
Because the truncated intimin has 73 fewer residues than the native intimin, a molecular weight shift is expected for the truncated intimin. The predicted sizes for the native and truncated intimins are 101.7-kDa and 97.0-kDa, respectively. Since post-translational modification of intimin eliminates the signal peptide of 39 aa (Zhu et al., 1995), the mature intimins of RDEC-I or RDEC-l Δeae are 97.1-kDa or 89.2-kDa, respectively. Consistent with previous observations (Zhu et al., 1995), Coomassie brilliant blue staining of SDS-PAGE-separated OMP extracts from the WT strain RDEC-I revealed an intimin protein band with estimated molecular mass of 97-kDa (FIG. 3C). However, this 97-kDa-protein band was missing in strain RDEC-l Δeae. Rather, a protein band with estimated molecular mass of 90-kDa was present for RDEC-l Δeae (FIG. 3C), confirming a molecular weight shift from native intimin expressed by the WT RDEC-I to the truncated intimin expressed by RDEC-l Δeae.
Effect of the Intimin Mutation on In Vitro Adherence to HEp-2 Cells and In Vivo Pathogenicity
The intimin C-terminal 76-aa disulfide loop has been shown to be essential for Tir-bing (Frankel et al., 1995; Hicks et al., 1998; and Luo et al., 2000). Thus, elimination of this disulfide loop is expected to abolish Tir-binding capacity of RDEC-l Δeae. When examined by HEp-2 cell adherence assay, the mutant strain RDEC-l Δeae is unable to induce actin accumulation on HEp-2 cells as had been observed for the parent strain RDEC-I (data not shown).
The level of in vivo virulence of the WT RDEC-I and its isogeneic ler mutant, RDEC-l Δeae was compared. Rabbits inoculated with RDEC-l Δeae remained clinically normal and they gained an average weight of 488 g by day 14 post inoculation (FIG. 4A). Fecal shedding of RDEC-l Δeae occurred within 24 hrs and persisted for nearly ten days (FIG. 4B). Light microscopic examination of tissues from ileum, cecum, and colon revealed no mucosal Iy adherent bacteria and normal morphology (data not shown).
All rabbits receiving WT RDEC-I developed mild diarrhea ranging from soft stools to watery discharge. These rabbits initially gained as much body weight as those inoculated with RDEC-lΔeae until day three post inoculation but gained less weight than RDEC-lΔeae-inoculated rabbits thereafter. They gained an average weight of 22O g by day 14 post inoculation which is significantly (p<0.006) less than RDEC-l Δeae group (FIG. 4A). They shed high numbers of inoculated organisms during the whole observation period (FIG. 4B). Consistent with previous studies, typical A/E lesions with cupping of adherent bacteria and effacement microvilli were observed among rabbits inoculated with the WT RDEC-I (data not shown). This initial comparison clearly indicates the attenuated virulence of RDEC-l Δeae.
Protection of Rabbits Following RDEC-lΔeae Immunization.
Further experiments were carried on to determine the level of protection provided by RDEC-l Δeae. All RDEC-lΔeae-immunized rabbits discharged normal stool (FIG. 5A) and had an average cumulative weight gain of 17O g by day seven post RDEC-H19A challenge (FIG. 5B). In contrast, all non-immunized rabbits manifested abnormal stools ranging from soft stools to frank watery diarrhea (FIG. 5A). They lost an average of 35O g body weight (50 g/day) during the same period (FIG. 5B). Fecal shedding of inoculated organisms was observed in rabbits of both RDEC-l Δeae-immunized and PBS control groups. However, the level of fecal bacterial shedding was greatly reduced in RDEC-lΔeae-immunized rabbits compared to PBS control rabbits (p<0.01) (FIG. 5C). While confluent growth of Nal/Tet-resistant RDEC-H19A was observed in fecal pellets of un-immunized rabbits, only approximately 50 CFU per swab inoculation were seen for the RDEC-lΔeae-immunized rabbits.
Major differences in gross appearance of the intestinal tissues were observed between immunized and non-immunized rabbits challenged with virulent RDEC-H19A. All immunized and challenged rabbits exhibited normal appearance of intestinal mucosa (ileum, cecum, and colon). However, non-immunized rabbits showed varying degrees of gross pathological changes, including pale cecum and proximal colon, watery intestinal contents, and edematous and thickened cecal walls.
Microscopically, mucosally closely adherent RDEC-H19A were seen covering on 10% surface area of the cecal mucosa (FIG. 5D). The severity of A/E lesions induced by RDEC-Hl 9A varied from extensive attachment/effacement to small-scattered focal lesions with a small cluster of adherent bacteria and irregularity of intestinal mucosa. In areas of adherence, the epithelial cells became irregular with reduced cytoplasm. Frequent clusters of cells were observed to be desquamated from the mucosal surface (FIG. 6). The A/E lesions, when observed, were most severe in the cecum and in the proximal colon (data not shown). However, less than 1% of the cecal surface had observable adherent bacteria among the RDEC-lΔeae-immunized rabbits (FIG. 5D). Where bacteria attached, the integrity of epithelial cells remains unchanged.
The WT RDEC-Hl 9A induced marked submucosal edema in rabbits of all groups (FIG. 7A). This pathology correlates with the thickened cecal wall seen on gross examination (Sjogren et al., 1994). RDEC-H19A induced inflammatory infiltrates of polymorphonuclear of heterophiles among unimmunized rabbits but not immunized ones (FIG. 7B). Vascular changes in the mucosa, submucosa, or serosa were seen among unimmunized rabbits (FIG. 7C). However, decreased vascular changes were observed in the mucosa of immunized rabbits as compared to those observed in the un-immunized/challenged rabbits. The vessels in the submucosa and serosa appeared as normal as in the immunized-non-challenged rabbits. Thrombus formation (rare) was only observed in the submucosa of immunized rabbits (FIG. 7D).
Immune Responses.
Serum and bilary antibodies specific to RDEC-I intimin were measured by ELISA using RDEC-I intimin-MBP fusion protein. Among PBS control rabbits, the intimin-specific IgG titer maintained the same low level during sampling period. However, intimin-specific IgG sharply increased following immunization with RDEC-lΔeae but did not show further increase following boost (FIG. 8). Bilary IgA specific for intimin was not detected in any group (data not shown).
Discussion
Interaction of intimin to its receptor, Tir, plays a key role in intimate adherence of AEEC to the host intestinal mucosa (Jerse et al., 1990; Kaper et al., 2004; and Kenny et al., 1997). Early studies demonstrated that the isogeneic intimin mutants are deficient in virulence as examined in human volunteers (Donnenberg et al., 1993a), in piglets (Donnenberg et al., 1993b) and in rabbits (Marches et al., 2000). Further supporting this is the fact that intimin immune responses can modulate the outcome of A/E organism infection as antibodies specific to intimin blocked intimin-mediated attachment (Dean-Nystrom et al., 2002 and Ghaem-Maghami et al., 2001). These results establish that intimin may serve as a putative candidate protein for attenuation as well as a crucial vaccine component. Functional and structural studies at molecular level on intimin further suggest that it is feasible to attenuate bacterial virulence while retaining its immunogenicity by one single step to eliminate the Tir-binding domain located in the D3 region of native intimin.
To test this hypothesis, the laboratory of the present inventors have constructed in the current study an eae mutant by a single nucleotide deletion to truncate the C-terminal 81 residues containing a disulfide loop essential for intimin function. It was demonstrated that RDEC-lΔeae is unable to induce A/E lesions in vitro and in vivo indicating successful attenuation of virulence of the WT RDEC-I. The results in this Example are in full agreement with previous reports that defined mutation in the eae attenuated virulence of human EPEC, EHEC, and rEPEC strains (Sonnenberg et al., 1991; Donnenberg et al., 1993a and 1993b and Marches et al., 2000). Donnenberg and Kaper created an internal 1848-bp deletion in EPEC eae gene (Donnenberg et al., 1991) and demonstrated that while all of the human volunteers received WT EPEC developed diarrhea, the isogeneic eae mutant caused diarrhea among only 4 of 11 individuals (Donnenberg et al., 1993a). In a separate study, the isogeneic eae mutant of EHEC strain 86-24 (0157:H7) generated by replacing the internal 1.1-kb eae DNA with a 2.9-kb NDA fragment containing a Tet marker was unable to colonize in the experimentally inoculated piglets (Donnenberg et al., 1993). In another rEPEC strain belonging to the serogroup O103:H2, an insertion of aphT encoding Kan resistance in the eae gene (between 993 nt and 994 nt) disrupted the expression of intimin and abolished bacterial virulence when tested by experimental inoculation of it natural rabbit host (Marches et al., 2000).
One of the unique features of the eae mutant constructed in the current study is the preservation of the remaining intimin molecule unaltered while the intimin functional D3 domain is truncated. This novel strategy retains immunogenicity of intimin which constitutes an important component of the attenuated live bacterial vaccines. The elevated serum IgG levels specific to intimin only seen among rabbits immunized with the isogeneic RDEC-lΔeae is an indication that the truncated intimin retains sufficient immunogenicity to boost immune responses to intimin. This specific immunity, combined with immunity to serotype-specific antigens and other as-yet unidentified factors, may contribute to the protection of rabbits from A/E pathological effects caused by virulent RDEC-H19A. Although a significant increase of specific IgA responses in biles of immunized rabbits was not observed it may be necessary to recover antibodies from the biles of immunized animals prior to challenge to determine the level of secretory IgA immunity.
Although New Zealand White Rabbits lack Stx receptors in the kidney glomeruli, Stx receptors are present in the microvascular endothelium of the intestine, in particular in the cecum, and Stx-induced vascular lesion are evident in the intestinal mucosa following RDEC-H19A challenge (Sjogren et al., 1994) Thus, this RDEC-H19A infection model is appropriate to evaluate the effectiveness of RDEC-lΔeae as vaccine candidate to prevent toxin-induced vascular lesions. The presence of HC and Stx-related histopathologuical alterations in non-immunized rabbits but the absence of clinical illness and lesions in RDEC-lΔeae-immunized rabbits indicates protection of rabbits from Stx-induced pathological effects by RDEC-H19A. This protection likely results from the prevention of bacterial intimate adherence to the intestinal mucosa.
Furthermore, although intimin mutation abolishes the capacity of RDEC-I to attach closely to the intestinal epithelial cells, RDEC-lΔeae are still able to persist in the rabbit intestinal tract for over ten days. This persistence suggests that multiple adhesive factors, in addition to the intimin, are involved in RDEC-I colonization. RDEC-I express plasmid encoded AF/Rl fimbriae which mediates a species-specific mucosal attachment by interacting with a sialo-glycoprotein complex on the microvillus (Berendson et al., 1983; Cantey et al., 1999; and Rafiee et al., 1991). Studies also implicate the filamentous EspA-containing surface appendages in attachment of A/E organisms (Ebel et al., 1998). In addition, in nearly all AEEC, a large molecular weight protein Efal (EHEC factor for adherence)/LifA (lymphocyte inhibitory factor) has been shown to be an adhesive factor (Badea et al., 2003; Klapproth et al., 2000; and Nicholls et al., 2000), RDEC-I express EspA and LifA/Efal homologue (Zhu et al., 2001). In a separate study, the laboratory of the present inventors have demonstrated that the LifA/Efal plays a crucial role in in vivo colonization of RDEC-I because deletion mutation in the lifA/efal gene resulted in significantly reduced bacterial colonization by 100-fold (Mao et al., 2003). Thus, these adhesive factors may contribute collectively to the prolonged presence in the intestinal tract by the RDEC-I intimin mutant, independent of intimin-Tir binding. This persistence of the RDEC-lΔeae in the intestinal tract is favorable to promote the development of local immunity against subsequent infection.
The hallmark of pathogenicity of EHEC is the production of Stx implicated in the development of HUS (Griffin et al., 1991; Kaper et al., 2004; Noel et al., 1997; and O'Brien et al., 1992). Eliciting active immunity against Stx represents another an attractive option for the development of an EHEC vaccine. Challenge studies showed effective protection induced by immunization with Stx-toxoid (Ludwig et al., 2002), the A or B subunit (Bielaszewska et al., 1997), STX-liposome conjugates (Uchida et al., 2003), DNA vaccines (Capozzo et al., 2003), or purified mutant form of the toxin (Ishikawa et al., 2003). Another approach to prevent the toxin-induced consequences of EHEC infection is the use of toxin binding agents. Several such agents, with Stx neutralizing capacity, have been developed utilizing the trisaccharide moiety of globotriaosyl ceramide which can bind and inhibit Stx cytotoxic activity. One such neutralizer, called Synsorb PK, has been the subject of clinical trials (Armstrong et al., 1995). The Stx binding agents, based on carbosilane dendrimer, a series of carbosilane dentrimers having silicon core and branch points and, trisaccharides of GB3 at their terminals, referred to as SUPER TWIG, has been shown to markedly inhibit Stx-binding and stx-cytotoxicity in vivo (Nishikawa et al., 2002). In another report, a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), has been shown specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins (Watanabe et al., 2004). However, these binding agents have limitations, since they need to be continually given to patients to provide toxin-neutralizing activity. Although approaches in eliciting immunity specific to or in neutralizing the toxin activity would protect against death caused by Stx(s) produced during EHEC infection, they could not prevent the spread of infection. However, an attenuated live EHEC vaccine, such as the eae mutant constructed in this Example is expected to provide more effective protection against EHEC infection by preventing bacterial colonization by a majority of clinically relevant EHEC strains. A truncated attenuated eae mutant has more attractive features that make it an effective attenuated mucosal vaccine candidate. They are noninvasive enteric pathogens in nature and are well studied at the molecular level. They are able to colonize in the intestinal tract and induce potent mucosal and systemic humoral responses; and they are safe and immunogenic. Thus, this attenuated EHEC strain shows great promise as an effective vaccine candidate for the prevention of EHEC infection.
Example 2
A LEE Encoded Regulator (ler) Mutant of Rabbit Enteropathogenic Escherichia coli is Attenuated, Immunogenic, and Protects Rabbits from Lethal Challenge with the Wild-type Virulent Stain
The nucleotide sequence of ler and the upstream regions of rEPEC strain E22(O103:H2) was determined, a defined deletion mutation in the ler gene in the wild-type strain was constructed and the role of ler on virulence and on immunogenicity was examined by in vitro and in vivo assays. The protective efficacy of the rEPEC ler mutant strain was further determined by challenging rabbits with the WT virulent strain following immunization with the isogeneic ler mutant. The results demonstrate that the Ler of EPEC O103:H2 is critical for both in vitro pathogenic effects and in vivo virulence and that immunization with this isogeneic ler mutant protected rabbits from fatal challenge with the virulent WT parent strain.
Materials and Methods
Bacterial Strains and Culture Conditions.
Bacterial strains and plasmids used in this study are listed in Table 2. The rEPEC strain E22 (O103:H2) was originally isolated from a rabbit with diarrhea (Marches et al., 2001). A nalidixic acid (NaI) resistant derivative of E22 was selected as described by Hane (Hane et al., 1969) and designated E22N. The laboratory E. coli strain DH5α was used for plasmid transformation except for suicide plasmids (pCVD442 and derivatives), which were maintained in E. coli SY327 or SMlO (Donnenberg et al., 1991). Bacterial strains were stored at −80° C. in Luria-Bertani (LB) broth containing 20% glycerol and grown on LB agar, LB broth, or MacConkey agar supplemented with appropriate antibiotic (s) at the following concentrations: Ampicillin (Amp), 50 μg/ml; kanamycin (Kan), 50 μg/ml, NaI, 50 μg/ml.







TABLE 2







Strains and plasmids used in this study (Example 2)









Strains or plasmids
Relevant characteristics
Source or reference










E. coli:









E22
rEPEC O103:H2
Marches et al., 2001


E22N
E22 derivative, NalR
This study


SY327
SY327 λpir, intermediate recipient for suicide
Donnenberg et al., 1991



vector pCVD442


SMlO
SMlO λpir, recipient for suicide vector
Donnenberg et al., 1991



pCVD442 to serve as donor strain, KanR


ECB132
SMlO (pECBl 19), AmpR, KanR
This study


E22Δ/er
An isogeneic ler mutant of E22N, NalR
This study







Plasmids:









pCR2.1 Topo
PCR cloning vector
Invitrogen


pECB049
pCR2.1:: Δ/er
This study


pCVD442
Suicide vector, AmpR
Donnenberg et al., 1991


pECB119
pCVD442:: Δ/er, Amp′′
This study









DNA Sequence Determination of rEPEC ler Region.
The ler gene and upstream −600 bp region were cloned using primers B750f and B751r (Table 3). For PCR amplification, PCR SUPERMIX high fidelity mixture (Gibco BRL, Rockville, Md.) was mixed with template DNA (E22N bacterial suspension in distilled water, 94° C. for 10 min) and the primers, while amplification was performed on the PTC-200 DNA Engine (MJ Research Inc., Waltham, Mass.) using the following protocol: 33 amplification cycles of denaturation at 94° C. for 60 s, primer annealing at 55° C. for 60 s, and elongation at 72° C. for 90 s, followed by a final extension step at 72° C. for 10 min. The entire reaction mixture was then analyzed by agarose gel electrophoresis and the DNA bands excised and purified with QIAquick gel extraction kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. The purified DNA was then sent for automated sequencing and analysis of nucleotide and amino acid sequences were performed with BLAST programs offered by the National Center for Biotechnology Information (NCBI, NIH, Bethesda, Md.)
Generation of a Defined rEPEC ler Mutant.
Recombinant DNA techniques were performed according to standard procedures (Sambrook et al., 2001). Oligonucleotides (Table 3) were designed to generate a 300 bp internal deletion in the ler gene by single-overlap extension PCR (SOE PCR) using denatured bacteria as DNA template (Senanayake et al., 1995). Two pairs of primers (B650f/B648r and B647f/B649r) were used to independently amplify the 5′- or 3′-region of ler including flanking sequences, respectively. Primers B648r and B647f contain 18 bp stretches complementary to each other. PCR amplicons from the above two separate PCR reactions were gel-purified as described above, mixed, and used as DNA templates for the second PCR amplification to achieve assembly using primers B650f and B649r. The resultant 1,350-bp PCR product, containing an internal deletion of 300 bp (from 44 to 343 nt) in the ler gene, was purified and subsequently cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) to generate plasmid pECB049. The nucleotide sequence of the insert was determined by DNA sequencing as described above. Plasmid pECB04 9 was then digested with Sac I and Xba I endonucleases and the DNA fragment containing the mutated ler was then ligated with the suicide plasmid pCVD442 (Donnenberg et al., 1991) digested with Sac I and Xba I to yield plasmid pECB119 which was transformed into E. coli SY327 (λpir) containing the pir gene necessary for replication of pCVD442 Plasmid pECB119 was then prepared from SY327 and subsequently transformed into strain SMlO (λpir) (which contains the conjugal functions of plasmid pCVD442 so that the plasmid can be transferred to the recipient strain for mutagenesis) and plated onto LB agar supplemented with Amp and Kan to yield strain ECB132.
Conjugation between the recipient WT E22N and the donor ECB132 was performed to achieve the desired defined mutation of Ler (Donnenberg et al., 1991). Replacement of the chromosomal ler gene with this in-frame, non-polar mutated ler was confirmed by PCR using two sets of primers (B650f/B649r, or B650f/B810r). Primers B650f and B649r yield fragments of 1650 bp or 1350 bp for the intact or mutated ler, respectively, whereas primers B650f and B810r yield a 778 bp fragment only in the presence of the intact ler but yield no product for the mutated ler.








TABLE 3







Oligonucleotides used in this study (Example 2)













Position in the



Oligo
Sequence
RDEC-I LEE





B750f
5′-ccggaattc/
EcoRI/3979-3996




cgaatggtacggttatgc



(SEQ ID NO: 7)


 


B751r
5′-cgcggatcc/
BamHI/4931-491 1



agttcagttatcgttatcatt



(SEQ ID NO: 8)


 


B650f
5′-gggatagatatgggaata
3951-3968



(SEQ ID NO: 9)


 


B648r
5′-cttcggtgtccttcacaa/
4868-4851/4550-4533



tgtgcgaattagtttcca



(SEQ ID NO. 10)


 


B647f
5′-ttgtgaaggacaccgaag
4851-4868



(SEQ BD NO: 11)


 


B649r
5′-attacgagtagaactact
5600-5583



(SEQ ID NO: 12)


 


B810r
5′-cgagcaaggccatcatcagg



(SEQ ID NO: 13)
4728-4709









Examination of Secreted Protein Profiles.
Bacterial secreted proteins were prepared as described (Sperandio et al., 1999). Briefly, overnight culture of bacteria in LB were adjusted to an optical density of 1.0 at 600 nm and 100 μl of such bacterial suspensions were added into 100 ml DMEM containing 5% fetal bovine serum. After overnight growth at 37° C., the bacterial cells were removed by centrifugation at 4° C. and phenylmethylsulfonyl fluoride was added to the supernatants to a final concentration of 1 mM (Zhu et al., 1995) Supernatants were then concentrated using a Filtron Stirred Cell with MW cutoff of 3-kDa (Filtron Technology Corporation, MA) and resuspended in PBS to a final volume of 1 ml. A total of 20 μl of such a preparation was separated by SDS-PAGE on 12% gel system and protein bands were visualized by silver staining as described (Zhu et al., 1995).
Initial Examination of In Vivo Virulence.
The in vivo virulence of the rEPEC isogeneic ler mutant was examined by experimental inoculation of two-month old New Zealand White rabbits. Rabbits were fasted overnight and un-sedated animals (six per group) were inoculated intragastrically via a pediatric feeding tube with 10 ml 10% bicarbonate solution to neutralize gastric contents followed by the inoculum. Inocula consisted of a total volume of 3 ml of PBS containing WT parent rEPEC E22N or E22Δ2er or PBS (control group) (Table 4). Finally, the tube was flushed with 5 ml sterile PBS to ensure complete delivery of inocula into the stomach.
Rabbits were examined daily for clinical signs of diarrhea and their weights were determined daily. Fecal samples were graded as normal pellets, soft stools, and watery or bloody diarrhea. Rabbits that lost greater than 20% body weight or demonstrated severe watery or mucoid diarrhea or bloody diarrhea were euthanized and recorded as non-survival s. Fecal bacterial shedding was determined semi-quantitatively on MacConkey agar supplemented with appropriate antibiotics. In brief, rectal swabs were rolled onto agar plates and grown overnight at 37° C. and scored as 0 (no CFU), 1+ (1-50 CFU), 2+ (50-200 CFU), 3+ (>200 CFU), or 4+ (confluent growth).
At sacrifice, rabbits were euthanized according to standard protocols to obtain histological sections of the intestine and quantitative bacterial counts of the inoculated strains. For comparison of virulence of WT E22 and its isogeneic ler mutant, animals were sacrificed at five days, since extreme weight loss in the group receiving strain E22N demanded sacrifice at this time. At necropsy, the degree of gross cecal and colonic edema, and degree of liquidity of cecal contents were observed and recorded. Counts of E. coli in cecal contents were determined by plating serial 10-fold dilutions of weighed cecal contents on MacConkey agar supplemented with appropriate antibiotics and expressed as CFU/g. For histological examination, a fragment of cecum was fixed in 10% neutral buffered formalin, processed, cut into 5-micron sections, and stained with hematoxylin-erosin (H&E) or Giemsa (Zhu et al., 1994). Ten sequential, well-oriented, 400× fields were examined from at least two sections from each slide. Enteroadherence was graded as percent surface area covered by closely adherent bacteria (Sjogren et al., 1994 and Agin et al., 1999).
Immunization and Challenge Studies:
To determine if vaccination with the isogeneic ler mutant would provide protection, two groups of rabbits (8 in each group) were immunized with a single orogastric dose of the isogeneic ler mutant, or received PBS. Rabbits were monitored for clinical signs of disease and bacterial shedding as described above. Two weeks following immunization, serum was drawn to determine antibody titers and two rabbits in each group (preselected at random) were sacrificed for histopathological evaluation. The remaining rabbits (6/group) were challenged with the parent WT E22N (FIGS. 13A-13D). Rabbits were euthanized if demanded by severe weight loss or observed for an additional eleven days prior to sacrifice.
Examination of Immune Responses.
Sera were collected from rabbits one day prior to, and two weeks post immunization. Specific serum IgG immune responses against the C-terminal 280aa portion of jS-intimin or whole bacterial cells were tested by ELISA. The maltose-binding-protein (MBP)-intimin fusion was purified as described (Agin et al., 1999). For ELISA, microtiter plates (Immulon, Dynatech) were coated with 100 μl of purified MBP-intimin fusion protein (4 μg/ml) in 50 mM bicarbonate coating buffer (pH 9.6) and dried overnight at 37° C. For the whole bacterium ELISA, overnight-grown bacteria were suspended in the coating buffer to an optical density of 1.0 at 660 nm and diluted four times (Zhu et al., 1994). A volume of 100 μl of the diluted bacterial suspension was added to the wells of microtiter plates and dried overnight at 37° C. The ELISA procedure was performed as described (Zhu et al., 1994).
Statistical Analysis.
Values for differences in rabbit weight gain, enteroadherence (% surface area) and antibody titers between experimental groups were compared by the Student T-Test.
Nucleotide Sequence Accession Number.
The nucleotide sequence for rEPEC E22 ler (FIG. 9) and upstream region determined in this Example has been assigned GenBank accession number AF328682.
Results
Nucleotide Sequence Analysis of Ler Region of rEPEC O103.H2.
The laboratory of the present inventors demonstrated previously that proteins (Tir, intimin and Esps) encoded in the LEE of rEPEC strains RDEC-I (015:H-) and O103-.H2 share high homology (Zhu et al., 2001). However, the nucleotide sequence of the ler region of rEPEC strain E22 (O103:H2) was unknown. A DNA fragment containing the ler and its upstream region of strain E22 was obtained by PCR using primers derived from RDEC-I LEE. Direct sequencing of the 938-bp DNA PCR product showed high homology (99%) to the corresponding region of the RDEC-I LEE (4003-4933 nt, FIG. 9A). In contrast, this 938-bp DNA fragment from rEPEC O103:H2 shares 86% and 85% to the corresponding LEE regions of hEPEC (E2348/69, GenBank accession no. AF022236), and EHEC (EDL933, GenBank accession no. AF071034), respectively (FIG. 9A).
The structural ler gene of strain E22 demonstrated over 95% identity at the nucleotide level to the ler of hEPEC, EHEC, or rEPEC strain RDEC-I. However, E22 ler shares only 87% identity at the nucleotide level with the ler of C. rodentium (Deng et al., 2001). The deduced peptide sequence of Ler of rEPEC O103:H2 contains 129 residues and shares 98% identity with the RDEC-I Ler, 95% with the Lers of EPEC and EHEC (FIG. 9B).
Construction and Characterization of Ler Mutation in rEPEC O103.
A two-step SOE-PCR was used to generate a 300-bp in-frame deletion internal to the ler gene (Senanayake et al., 1995). A 1350 bp DNA fragment containing the ler deletion was cloned into pCR2.1-Topo vector to generate pECB04 9, which was used for confirmatory sequencing and subsequent cloning. Nucleotide sequencing of pECB049 indicated a 300-bp deletion in ler (from 44 to 343 nt of ler coding sequence) corresponding to 4551-4850 nt of the RDEC-I LEE (Zhu et al., 2001) without alteration of the remaining sequences (FIG. 9A). Thus, a deletion of 100 aa (from aa 15 to 114) was generated in the Ler central region (FIG. 9B).
The ler deletion mutation was subsequently introduced into the strain E22 chromosome by site-directed mutagenesis (Donnenberg et al., 1991). Colonies obtained following selection on LB plates supplemented with 5% sucrose were verified for ler mutation by PCR using two pairs of primers, B650f/B649r or B650f/B810r (Table 3). PCR amplification using primers B650f and B649r yielded a 1650-bp fragment for the intact ler from the WT parent strain E22 or a 1350-bp fragment for the mutated ler. Whereas PCR using primers B650f and B810r yielded a fragment of 778 bp for the intact ler, no amplification product for the ler mutant was seen because primer B810r is derived from the sequences internal to the deleted ler fragment. One such ler mutant strain designated as E22ΔJer, was selected for further characterization.
Effect of Ler Mutation on the Production of Bacterial Secreted Proteins.
As previously described for EPEC, disruption of Ler markedly abrogated the secretion of LEE-encoded proteins (Mellies et al., 1999). To investigate the role of Ler of rEPEC, secreted protein profiles of WT E22N and its derivative E22Δler were compared using standardized culture supernatants. Silver staining (FIG. 10) of SDS-PAGE-separated proteins from strain E22N revealed at least twenty-four protein bands including EspB/D and EspA. The secreted protein profile for E22Δ2er demonstrated markedly decreased expression of the majority of these bands including the LEE-encoded EspB/D and EspA, as identified by their relative molecular mass. These results for the LEE-encoded secreted proteins are in accordance with the findings of Elliott et al. and Mellies et al. (Elliott et al., 2000 and Mellies et al., 1999).
Effect of ler Mutation on In Vivo Virulence.
The level of in vivo virulence of the WT E22N and its isogeneic ler mutant E22Δler was compared. Following inoculation with 6×105 E22N large numbers of NaI-resistant E. coli were shed in stools (Fig. HA) such that confluent growth was observed within three days post inoculation. Loss of body weight began within 48 h and continued until sacrifice (Fig. HB). The average body weight loss was over 180 g (Fig. HB) such that all the animals required euthanasia by day five post inoculation (Fig. HC). One rabbit required euthanasia on day 3, three on day 4, and the remaining two on day 5. Although rabbits inoculated with E22N remained clinically normal for 24 h post inoculation, by day 2, two thirds of the rabbits had soft stools or watery diarrhea. Subsequently, all rabbits developed severe clinical illness characterized by severe diarrhea with weight loss requiring euthanasia (Table 4). At necropsy, severe colonic and cecal edema and liquid cecal contents were seen. Bacterial counts of WT rEPEC in cecal contents averaged 4×109 CFU per gram (range from 3×108 to 1×1010). Microscopic examination confirmed cecal submucosal edema and demonstrated extensive focal lesions with mucosally adherent bacteria covering approximately 50% of the mucosal surface (FIG. 12A). Extensive bacterial attachment to the intestinal mucosa was associated with severe effacement of the apical surface and distortion of the architecture of the epithelial cells (FIG. 12A). This light microscopic appearance is characteristic of A/E lesions (Marches et al., 2001). These lesions were seen in all animals receiving the WT strain (Table 4). These results are consistent with previous reports that the WT parent rEPEC strain cause high morbidity and mortality in weaned rabbits (Marches et al., 2001).







TABLE 4







Summary of clinical presentations and pathognomonic A/E


lesions observed among experimentally inoculated rabbits













Cumulative
No. of rabbits
No. of rabbits showing


Inoculation Strain
Challenge strain
weight gain
with diarrhea ′′/
A/E lesions c/total no.


(dose, CFU)
(dose, CFU)
or loss (g) ′′
total rabbits (%)
examined (%)










Pathogenicity study:











E22N (6 × 105)
N/A
−217 ± 57 d
6/6 (100%)
6/6 (100%)


E22Δ/er(1 × 108)
N/A
 168 ± 16 ′′
0/6 (0%)
0/6 (0%)







Protection study:











E22Δ/er(1 × 108)
E22N (2 × 105)
 382 ± 14 e
0/6 (0%)
0/6 (0%)


PBS
E22N (2 × 105)
−213 ± 54 f
6/6 (100%)
6/6 (100%)





′′ Watery or bloody diarrhea


b Cumulative weight gain (+) or loss (−) post inoculation. Mean ± standard errors


c As determined by light microscopy


d Rabbits sacrificed 3-5 days post challenge


e Rabbits sacrificed 11 days post challenge


N/A, not applicable






Following inoculation with 1×108 E22Δ2er rabbits shed the inoculated bacteria from the second day post inoculation to the end of the observation period (Fig. HA). All six rabbits remained clinically normal showing normal rates (35 g/day) of weight gain (Fig. HB) and normal stool consistency. Rabbits inoculated with E22Aler were then sacrificed five days post inoculation in order to permit comparison with rabbits inoculated with WT strain. Intestinal tissues, from ileum to distal colon, of all rabbits inoculated with E22Δ2er remained grossly normal. Bacterial counts of E22Δler in cecal contents averaged 3×10s CFU per gram (range from 1×104 to 1×106). Microscopically, the brush border appeared intact and deformation of intestinal mucosal architecture was not observed in the cecum. Small, scattered bacterial clusters were occasionally observed associated with normal brush borders resulting in an estimate of only 0.18% (p<0.0027 vs. WT) of the surface of the cecum covered by non-intimately adhering bacteria (FIG. 12B). This association was clearly distinct from the intimate adherence pattern exhibited by the WT parent E22N (FIG. 12A). This initial comparison clearly shows the diminished virulence of E22Δler.
Single Dose Vaccination with E22ΔLer Protects Rabbits from Challenge with Virulent Parent Strain.
It was next determined if vaccination with the attenuated E22Δ2er would induce immune responses and protect rabbits from lethal challenge with the virulent WT. All rabbits (8 per group) orogastrically inoculated with either a single dose of 1×108 E22Δ2er or PBS appeared normal following immunization. They consistently gained body weight and discharged normal stools. Fecal shedding of NaI-resistant bacteria was observed for all rabbits receiving E22Δ2er but not for rabbits receiving PBS (data not shown). Examination of tissues from two E22Δ2er or PBS-inoculated rabbits sacrificed two weeks post immunization revealed no abnormalities. These data confirmed with the initial study in the laboratory of the present inventors that E22Δ2er is well tolerated in rabbits.
Following challenge with 2×105 WT E22 two weeks post immunization, E22Δle:r-vaccinated rabbits continued to gain weight with an average of 32 g/day (FIG. 13A, Table 4). Only low level fecal bacterial shedding of the challenge strain was observed in these rabbits (FIG. 13B). All immunized animals remained clinically normal without evidence of diarrhea, and all survived until being sacrificed at day eleven post challenge (FIG. 13C).
All rabbits in the non-immunized PBS group began to lose weight on the second day post challenge and lost an average of 43 g/day until sacrifice (FIG. 13A). These animals all shed high levels of E22N by day 2 following challenge (FIG. 13B). Two days post-challenge, all rabbits in the PBS control group shed soft stools and subsequently all developed watery or bloody diarrhea. Severe diarrhea and loss of body weight necessitated sacrifice of these rabbits at day 5 as in our initial study.
The bacterial challenge strain was recovered from the cecal contents of all PBS control rabbits (ranging from 1×107 to 1.6×lO8), but from only one rabbit receiving E22Δ2er (data not shown). Microscopically, cecal tissues from E22Δler-immunized or PBS group rabbits collected before challenge, and from all E22Δ2er-immunized rabbits following challenge, revealed no adherent bacteria (Table 4). The brush borders appeared intact and deformation of intestinal mucosal architecture was not seen. In contrast, PBS control rabbits challenged with WT E22 developed cecal submucosal edema and demonstrated extensive focal lesions with bacteria intimately adherent to the mucosa following challenge with the WT parent strain (Table 4). The severe diarrhea and extensive A/E lesions observed in PBS control rabbits are consistent with the pathogenicity study and with previous reports from the laboratory of the present inventors that the WT E22 is highly virulent (Marches et al., 2001).
Detection of Immunoglobulin Specific to Bacterial Surface Antigens and to Intimin.
Antibodies present in sera were measured by ELISA using the whole bacterial and purified MBP-intimin (FIG. 13D). Rabbits immunized with E22Δler exhibited three to twenty-seven fold increases of serum IgG specific to bacterial surface antigens expressed on whole cells of E22. In contrast to the positive results for antibodies to whole bacteria, rises in serum antibody titer specific to the C-terminal domain of β-intimin was not observed among rabbits immunized with E22Δ2er or receiving PBS (data not shown).
Discussion
In the current study in this Example, the characterization of a defined ler mutant of rEPEC and evaluation of such isogeneic ler mutant as vaccine candidate are reported. Inactivation of Ler decreases secretion of LEE-encoded proteins. More importantly, it was demonstrated here that the in frame deletion mutation in the ler gene abolishes the in vivo capacity of rEPEC to cause disease in rabbits. This is the first in vivo demonstration of the effect of a Ler mutation of an A/E E. coli strain.
Ler belongs to the H—NS family of DNA-binding proteins that play a crucial role in the global gene regulation of enteric bacteria (Mellies et al., 1999). Although H—NS does not exhibit a high DNA sequence specificity, a number of H—NS-responsive promoters have been shown to contain regions of intrinsic DNA curvature located either upstream or downstream of the transcription start point (Rimsky et al., 2001). Bustamante et al showed that Ler acts as an antirepressor protein that overcomes the H—NS-mediated repression of LEE-encoded genes (Bustamante et al., 2001). To accomplish this, Ler displaces H—NS bound to a DNA fragment upstream of LEE operons thereby-increasing transcriptional activity (Haack et al., 2003 and Sanchez-Sanmartin et al., 2001). Thus, inactivation of Ler promotes down-regulation of LEE-encoded virulence factors by H—NS, resulting in diminished TTSS-secreted effector proteins as observed in the current study. Similarly, an in-frame non-polar deletion mutation in the ler decreased protein secretion, altered the profile of secreted proteins, and strongly diminished adherence to cultured HEp-2 cells (Mellies et al., 1999).
In the current study, the reduction in the density of secreted LEE-encoded proteins together with the disappearance of several other secreted protein bands in the ler mutant was observed. This suggests that Ler has profound regulatory effects which may alter the expression of the genes encoded on the LEE and outside the LEE. In hEPEC, Ler activates the expression of espC (encoding an autotransportor/enterotoxin) contained within a second PAI (Elliott et al., 2000). In human EHEC inactivation of ler upregulated some fimbrial gene expression (Elliott et al., 2000 and Ogierman et al., 2000).
The in vivo study here demonstrated that inactivation of Ler prevents rEPEC from adhering intimately to the rabbit intestinal mucosa in vivo. Animals receiving the ler mutant demonstrated no clinical evidence of disease even though the experimental inoculation of the mutant dose was over two logs greater than the WT. These results indicate a profound effect of the Ler in up-regulating in vivo virulence. These in vitro results are in full agreement with previous studies that Ler is essential for in vivo intimate A/E adherence. In a naturally occurring ler mutant of an 0157:H-EHEC with a single base substitution resulting in a change of H e57 to Thr, the adherence of bacteria to HEp-2 cells was significantly reduced (Ogierman et al., 2000). Furthermore, a ler mutant of murine A/E pathogen C. rodentium failed to induce A/E lesions in mice (Deng et al., 2004). Taken together, these results suggest a common mechanism of LEE-encoded virulence gene regulation by Ler among hEPEC, EHEC, C. rodentium and rEPEC (Elliott et al., 1998; Perna et al., 1998; and Deng et al., 2001).
It is well known that the specific binding of intimin and Tir play a key role in the formation of A/E lesions by A/E organisms. However, additional chromosomal and plasmid factors may serve as accessory factors in the mechanism of infection. rEPEC O103:H2 expresses the chromosomally encoded adhesive factor/rabbit 2 (AF/R2), which is a member of K88 adhesin family (Fiederling et al., 1997), and distinct from the adhesin AF/R1 found in the rEPEC 015:H-strain RDEC-I (Cantey et al., 1999 and Wolf et al., 1988 and 1990). Moreover, another plasmid-encoded rEPEC adherence locus (ral) has been identified in rEPEC strain 83/39 (O15:H-) (Adams et al., 1997). Although the isogeneic ler mutant of rEPEC has lost the ability, present in the WT, to adhere intimately to rabbit intestinal mucosa and induce effacement of microvilli, it is nevertheless able to colonize the rabbit intestine and shed at a measurable level for 10 days indicating that there may have been some in vivo replication resulting in their prolonged persistence. Since inactivation of Ler has markedly decreased the expression of virulence factors involved in intimate bacterial attachment, the observed prolonged bacterial persistence is likely independent of Tir interaction with intimin. This suggests that in the ler mutant there may be an upregulation of expression of additional accessory molecules which may facilitate colonization by bacterial attachment to the intestinal mucosa or other mechanisms (Nataro et al., 1998 and Elliott et al., 1997)
The immunization studies in this Example indicate that the defined isogeneic ler mutant of rEPEC is immunogenic after a single dose immunization. The significant increase in serum IgG titers directed to the whole bacterial cells in E22Δler vaccinated rabbits, but not in rabbits receiving PBS, suggests that the mucosal delivery of the attenuated rEPEC ler mutant induces host immune responses which protected rabbits from the lethal effects of the WT virulent strain. Because inactivation of Ler resulted in decreased production of proteins encoded on the LEE, including intimin, it is not unexpected that no antibody titers specific to intimin was detected. It is likely that the target of the immunization strategy in this study was bacterial-associated antigens independent of the LEE such as serogroup-specific lipopolysaccharide.
Previous studies on vaccination against AEEC organisms involved the inactivation of the eae or tir genes of hEPEC (Donnenberg et al., 1993) EHEC (Donnenberg et al., 1993), rEPEC O103:H2 (Marches et al., 2001), or C. rodentium (Ghaem-Maghami et al., 2001). As specific binding between intimin and Tir plays a key role in the intimate attachment of A/E organisms to host cells, inactivation of either intimin, or Tir, or both, attenuated in vivo virulence of these vaccine candidates. However, since multifactorial determinants are involved in the mechanisms of A/E infection, full attenuation may not be achieved by inactivation of intimin and/or Tir. For example, an eae mutant of hEPEC still caused diarrhea in 4 of 11 volunteer individuals, compared to 11 of 11 who ingested the WT (Donnenberg et al., 1993). Many rEPEC strains, including E22 (O103:H2), also express the novel effector Cif (for cycle inhibiting factor), which blocks the cell cycle G2 to M transition, and induces the formation of stress fibers through the recruitment of focal adhesions (Bustamante et al. 2001). Although it is not encoded in the LEE, Cif is a type III effector (Marches et al., 2003). Thus, full attenuation of A/E organisms may require inactivation of additional virulence determinants. In this study, we targeted the global regulator ler in order to down-regulate not only intimin and tir, but also the TTSS and secreted proteins so that such mutants have diminished capacity to inject bacterial proteins into the host cells. Compared to the eae/tir mutants previously studied, a ler mutant may provide a safer vaccine candidate. On the other hand, inactivation of Ler might be expected to provide more limited protective immunity because of the diminished production of virulence-associated proteins encoded on the LEE or other TTSS-secreted proteins encoded out side the LEE. These virulence proteins may be required to elicit cross protective immunity to infections caused by A/E organisms of differing serotype.
Example 3
Live, Attenuated Bacterial Vaccine Against Shiga Toxin-Producing Enterohemorrhagic Escherichia coli (STEC/EHEC) for Cattle
The present inventors are developing live attenuated bacteria for use as vaccines against Shiga toxin-producing E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC), in cattle. The approach is to construct isogeneic double mutants targeting both the stx and the eae, ler or tir genes to fully attenuate STEC/EHEC. Along with the mutation to inactivate the Shiga toxin gene to abolish production of the Shiga toxin, inactivation of Ler (locus of enterocyte effacement-encoded regulator) and/or modification of the translocated intimin receptor would sufficiently attenuate bacterial virulence but retain good immunogenicity. Thus, defined isogeneic mutants will be constructed among STEC strains representing the most prevalent serogroups 0157, 026, and 0111.
General Materials and Methods and Experimental Systems
Bacterial Strains:
STEC strains (Table 5) are selected with maximum available phenotypic and genotypic information to ensure obtaining the desired results. The primary 0157:H7 strain to be used for construction of vaccine candidate will be strain 86-24 (0157:H7) which was isolated from a case of HUS in Walla Walla, Wash. EHEC 86-24 is a Streptomycin (Str)-resistant Stx2-producing strain, which has been well characterized for its virulence mechanisms and genetic background and used in a variety of animal models of pigs, cattle, and sheep (Donnenberg et al., 1993b; Sperandio et al., 2001; Torres et al., 2003; and Tzipori et al., 1995). Strain 86-24 carries the long polar (LP) fimbria homologue which has been shown to participate in the interaction of E. coli 0157:H7 with eukaryotic cells by assisting in microcolony formation (Stevens et al., 2002 and Torres et al., 2003). The eae mutant of 86-24 was deficient in inducing F-actin accumulation in HEp-2 cells and was incapable of attaching intimately to colonic epithelial cells in a newborn piglet model of infection (Donnenberg et al., 1993b). In one-week-old calves, 86-24 was shed in the feces for a mean of 30 days (Sanderson et al., 1999). STEC strains belonging to 026 and 0111 were human isolates. The prevalence of these strains in STEC outbreaks has been increasingly recognized (Bopp et al., 1987). STEC 026 strain was isolated from human HC. It carries Stx2-converting phage and the LEE pathogenicity island (Marques et al., 1986 and Zhu et al., 2001). The laboratory of the present inventors have previously demonstrated high homology of eae gene between STEC 026 strains and REPEC strain RDEC-I (Zhu et al., 2001). However, STEC 026 strain 08566 appears to carry γ-type intimin of STEC 0157:H7 (strain EDL933) and demonstrated adherence to HEp-2 cells in a localized manner and were positive by the fluorescence actin staining (FAS) test that is considered to correlate with the ability to cause attaching and effacing lesions in vivo (Scotland et al., 1990). STEC strain E45035, serotype 0111:H-, was isolated from the feces of a patient with HUS and it carries eae and stx1, but not stx2 (Willshaw et al., 1992). Strain E45035 is able to adhere to cultured cells and to colonize and persist in the intestinal tract of calves (Nicholls et al., 2000 and Stevens et al., 2002). The strains are obtained from the STEC Center at the National Food Safety and Toxicology Center at Michigan State University (shigatox.net/stec). The STEC Center is designed to facilitate research on STEC by providing a standard reference collection of well-characterized strains and a central on-line accessible database.







TABLE 5







List of strains











Strain
Serotype
Origin snd relevant features







86-24
O157:H7
Human, Stx2, γ-intimin



08566
O26
Human, Stx2



E45035
O111:H-
Stx1, γ-intimin










Genetic Methods:
It is desirable that the vaccines strains contain no antibiotic resistant marker. Therefore, deletion mutations will be constructed by single-overlap extension PCR(SOE PCR) as illustrated in FIG. 14. In this technique, primers derived from the targeted gene(s) are designed for desired deletion mutation. Primers B and C contain a 20-bp stretch complementary to each other. SOE-PCR is a simple two-step PCR technique for generating deletions of a wide range of sizes. This technique was used to generate deletion mutations of up to 8,000 base pairs in the HfA gene in RDEC-I. In the first step PCR amplification, PCR SUPERMIX high fidelity mixture (Invitrogen, Carlsbad, Calif.) will be mixed with template DNA (bacterial suspension in distilled water, 94° C. for 10 min) and the primers (A and B for 5′-fragment, or C and D for 3′-fragment), while amplification is performed on the PTC-200 DNA Engine (MJ Research Inc., Waltham, Mass.) using the following protocol: 33 amplification cycles of denaturation at 94° C. for 60 s, primer annealing at 55° C. for 60 s, and elongation at 72° C. for 90 s, followed by a final extension step at 72° C. for 10 min. The entire reaction mixture will then be analyzed by agarose gel electrophoresis and the DNA bands excised and purified. PCR amplicons from the above two separate PCR reactions will then be used as DNA templates for the second PCR amplification using primers A and D to achieve assembly. The resultant PCR product containing an internal deletion will be purified and subsequently cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.). The resultant plasmid will be prepared and sent for automated sequencing. The plasmid containing the desired deletion mutation will be digested with Sac I and Xba I endonucleases and the DNA fragment containing the mutated gene will then be cloned into suicide plasmid pCVD442 (Donnenberg et al., 1991) digested with Sac I and Xba I. The plasmid pCVD442 will be transformed into E. coli SY327 (λpir), which contains the pir gene necessary for replication of plasmid pCVD442. Plasmid from B. coli SY327 will be prepared and subsequently transformed into strain SMlO (λpir) which contains the conjugal functions of plasmid pCVD442 so that the plasmid can be transferred to the recipient strain for mutagenesis. Conjugation between the recipient WT STEC and the donor SMlO (pCVD442::Δxgene) will be performed to achieve the desired defined mutation (Donnenberg et al., 1991). To verify the desired mutation, primers (A and B) overlapping the entire deletion are used in PCR amplification and the amplicon size is compared between the WT and the mutant strains. Additional primer (E) derived from the deleted fragments will be used in PCR at the same time. Thus, PCR amplification using primers A and E will yield a product for the WT but no PCR product for the mutant. All these mutants are in-frame and non-polar.
Preparation and Examination of Outer Membrane Proteins.
Bacterial outer membrane proteins (OMP) will be examined to determine the molecular weight shift of intimin and compare between the WT and isogenic eae mutant. OMP will be prepared by the method as previously described (Zhu et al., 1995). Briefly, bacterial cells will be lysed by two passages through a French press cell. The cytoplasmic membrane proteins will be solubilized with 1.67% N-lauroylsarcosine, followed by centrifugation at 200,000×g for 90 min. The protein content of the resuspended pellet containing bacterial OMPs will be determined by the Micro BCA protein assay reagent. The OMP pellet will then be examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous buffer system by the method of Laemmli in a mini-gel apparatus. Separated protein bands will be viewed by Commassiae BlueTM staining (Zhu et al., 1995).
Preparation and Examination of Secreted Proteins.
The level of secreted protein production by the isogenic ler mutants will be determined by examination of secreted proteins. Bacterial secreted proteins will be prepared from overnight culture in DMEM containing 5% fetal bovine serum. After removal of bacteria by centrifugation at 4° C. phenylmethylsulfonyl fluoride will be added to the supernatants to a final concentration of 1 mM (Zhu et al., 1995). Supernatants will then be concentrated using a Filtron Stirred Cell with MW cutoff of 3-kDa (Filtron Technology Corporation, MA). The concentrated proteins will be suspended in PBS to a final volume of 1 ml. A total of 20 ul of such a preparation was separated by SDS-PAGE on 12% gel system and protein bands will be visualized by Commassiae BlueTM staining (Zhu et al., 1995).
In Vitro Bacterial Adhesion to Epithelial Cells.
For both qualitative and quantitative adhesion assays, STEC strains and their isogeneic mutants will be evaluated for their ability to adhere to HeLa or CHO (for STEC strain 0111) cell monolayers as described (Torres et al., 2002). Cells are seeded at 1×105 cells/ml to yield a semi-confluent cell monolayer. Bacterial strains are grown in LB broth overnight at 37° C. and added to tissue culture cells replenished with fresh DMEM at a concentration of 107 bacteria per well for 3 or 6 h at 37° C. Then the cell monolayers are washed, fixed and stained with Giemsa and viewed by light microscopy, or bacteria are recovered with 0.1% Triton X-100TM in PBS and plated on LB agar or MacConkey agar plates containing proper antibiotic for quantification. Data are expressed as the percentage of the original inoculums recovered from duplicate wells and are the mean of at least three separate experiments. A modified protocol that can be used for the HeLa cell adherence assay is as follows below:
HeLa Cell Adherence Assay
Day 1


    
    
        Pour 1 ml of a HeLa cell suspension (1×105 cell/ml) into 24-well plates containing cover slips)
        Culture bacteria (including positive and negative controls) in LB containing appropriate antibiotics at 37° C. O/N

Day 2

        Wash all wells 3× with antibiotic-free DMEM media
        Add 1 ml complete media (DMEM containing 10% FBS and appropriate antibiotics, 1% mannose)
        Add 20 ul of each O/N culture bacteria to respective well (duplicate or triplicate the sample)
        Swirl to mix
        Incubate for 3 h in 5% CO2
        Take off media, add fresh media containing appropriate antibiotics
        Incubate for additional 3 h
        Wash cells 4× with PBS to remove non-adherent bacteria (For bacteria counting, lyse the cell with 200 ul 0.1% Triton-100TM in PBS, count bacteria by limited dilution, data are expressed as the percentage of the original inocula recovered from triplicate wells)
        Fix cells with methanol for 5 min at RT/pour off
        Stain with 10% Giemsa stain for 15 min at RT/pour off
        Rinse wells with dH2O until clear (5-6 times)
        Leave the last dH2O in the wells
        Remove coverslips using tweezers (be careful to remove the coverslips)
        Place on copy of grid to dry (air dry or use hair drier)
        When dry label slides
        Fix coverslips to slides using clear nail polish
        Let dry/read slides and record assay.
    
    


Stx Assay.
Shiga toxin production by the WT STEC and their isogeneic mutants will be tested by cytotoxicity assay as described (Bitzan et al., 2003). Bacterial whole lysates will be prepared from overnight cultures of STEC strains and their isogenic mutants by two passages through a French Press. Protein content of extracts will be determined by using BCA Protein Assay in a 96 microwell plate (BioRad). Confluent cells (Vero, or HeLa) in 96-well plates will be grown in the presence of serial-diluted toxin preparations for 48 h. Quantitative cytotoxicity will be determined by crystal violet staining (Bitzan et al., 2003). Specifically, the protocol for the Stx assay is as follows below:
Stx Assay
Preparation of Bacterial Cell Lysates:
1. Bacteria for analysis will include EHEC wild type and their isogenic mutants.
2. Grow bacteria in 5O mIs of Penassay Broth overnight at 37° C.
3. Harvest bacteria by centrifugation at 5,000 RPM for 15 minutes. Remove supernatant and weigh wet pellets.
4. Spin supernatant at 5,000 RPM followed by passage through a 0.2 micron filter to sterilize. Keep samples refrigerated at 4 degrees.
5. Wash pellet with 5 mls of PBS twice. Resuspend in PBS at a final volume of 4 mls.
6. Disrupt bacteria by two passages through a French Press at 15,000 psi. Wash press between each sample with 70% ETOH and DI water.
7. Weigh out 3.9 grams of each lysate sample and store the remaining material (approximately 2 mls). Ultracentrifuge the weighed lysate at 100,000 g (70,000 RPM) for 60 minutes.
8. Determine protein content of extracts using BCA Protein Assay in 96 microwell plate.
Cytotoxicity Assay:
1. Preparation of cells (HeLa or HEp-2). Add 100 ul HeLa cells (at a concentration of 2×105 cells/ml) to 96-well microtiter plate.
2. Incubate at 37° C. in 5% CO2 for 24 h so that they become just confluent.
3. Make serial dilution of Stx preparation (use commercial Stx as control at the same time) in complete tissue culture medium.
4. Tissue culture medium alone (toxin control) in wells of a microtiter plate and positive Stx will be used as controls
5. Incubate at 37° C. in a 5% CO2 incubator.
6. Observe at 24 h and 48 h.
7. Rinse wells with PBS, aspirate all liquid. Fix cells with 70 ul of 70% methanol per well for 1 min.
8. Remove methanol. Add 70 ul of crystal violet stain for at least 20 min. Rinse microtiter plate generously with tap water until no more dye is flowing off and air-dry.
9. Elute bound stain from cells with 200 ul 50% (v/v) ethanol.
10. Read absorbance at 490 nm or 550 nm in microplate reader.
Oral and Rectal Inoculation of Mice.
Mice have been extensively used in the studies to evaluate the induction of systemic and local immunity and experimental results demonstrate that rectal immunization could be an effective new vaccination method (Kelanthous et al., 1998; Mitchell et al., 2003a; and Nakase et al., 2001). As mice model for rectal immunization provides the simplest and valuable means to assess the immunogenicity, mice will be used to examine the immunogenicity of the STEC vaccine constructs. BALB/c female mice, 4-6 wks, will be grouped as listed in Table 8. For each immunization, a single colony of STEC mutant strain will be grown in Luria-Bertani (LB) broth for 15 h at 37° C. The cultures will be diluted at 1:100 in the same medium and incubated for 4 h at 37° C. The bacteria in logarithmic growth will be harvested by centrifugation for 20 min at 2,000×g and resuspended into sterilized PBS. The bacterial concentration in the suspension will be estimated from the optical density at 600 nth and adjusted with PBS to yield approximately 5×1010 CFU which will be further confirmed by plating dilutions. A 20-μl bacterial suspension will be used for immunizations, with approximately 109 CFU per inoculum. For oral immunization, mice are deprived of food 2 h before and 1 h after immunization. They will be fed with 30 μl of 10% sodium bicarbonate to neutralize stomach acidity 5 min prior to immunization and then given 20 μl of inoculum. For rectal immunization, mice will be fasted for 12 h before immunization. Mice will be anesthetized by intraperitoneal injection with 100 μl (per 10 g of body weight) of both 0.2% RompunTM in PBS (Bayer) and 10 mg of KetavetTM (Parker Davis) per ml. Inoculum (20 μl) will be gently introduced with a yellow tip into the rectum. Mice will be positioned with the rectum facing upwards for about 45 min to reduce leakage of the inoculum.
Determination of Bacterial Numbers in Feces.
Feces from mice will be collected and suspended in PBS. The w/v ratio will be recorded as dilution factor. 10 ul of bacterial suspension will be plated on MacConkey agar containing appropriate antibiotics.
Measurement of Antigen-Specific IgG and IgA Antibodies:
Sera IgG and fecal IgA will be examined by ELISA for specific antibodies to intimin and to the whole bacteria. To prepare γ-intimin for ELISA, the coding sequence for the C-terminal portion, the last 280 amino acid, of STEC will be amplified by PCR using primers, 5′-ttctacacaaaccgcatag (SEQ ID NO: 27) and 3′-tcaaaccaaggccagcatta (SEQ ID NO:28), derived from O157:H7 LEE sequence and cloned into pBAD-topo vector. The intimin-tag fusion protein will be induced with arabinose and purified.
Blood samples will be taken from the tail veins or orbital sinus of live animals and from the portal veins of killed animals. Sera will be collected pre-inoculation and fourteen days after last inoculation. For fecal IgA, three to six pieces of freshly voided feces will be collected into 1.5-ml centrifuge tubes, frozen at −20° C., and subsequently vacuum dried in a Speed Vac Concentrator. After net dry weighs are recorded, PBS containing 5% nonfat dry milk and protease inhibitors is added to samples at a ratio of 20 ul per mg dry feces. Following extensive vortexing and centrifugation at 16,000×g for 10 min, the clear supernatants will be collected and stored for ELISA (Hopkins et al., 1995). IgA antibody in mouse intestinal secretions will be determined by the method described by Elson, et al (Edson et al., 1984). Each mouse will be placed on a 12 cm×12 cm square of galvanized wire mesh placed within a Petri dish which contains a solution of protease inhibitors (0.1 mg/ml soybean trypsin inhibitor). The lavage solution will be given intragastrically at 15 min intervals using a blunt tipped feeding needle. Thirty minutes after the last dose of lavage solution the mice are given 0.1 mg pilocarpine I/P. A discharge of intestinal contents will be collected and vortexed vigorously, then centrifuged. The supernatant will be collected with addition of PMSF (Edson et al., 2004).
ELISA will carried out as described by Zhu et al (104) with minor modifications. Briefly, microtiter plates (Immulon, Dynatech) will be coated with 100 μl of purified intimin antigens (4 μg/ml) in 50 mM bicarbonate coating buffer (pH 9.6) and dried overnight at 37° C. For the whole bacterium ELISA, overnight-grown bacteria (the eae mutant strain) will be suspended in the coating buffer to an optical density of 1.0 at 660 nm and diluted four times. A volume of 100 μl of the diluted bacterial suspension will be added to the wells of microtiter plates and dried overnight at 37° C. The plates will then be washed and blocked with 5% skimmed milk in TTSB. Sera and fecal IgA preparations at various dilutions will be added to the wells containing intimin or bacterial antigens followed by conjugated anti-mice IgG or IgA (KPL). A phosphatase substrate system (pNPP, KPL) will be added and the reaction will be stopped by addition of 0.5 M NaOH, and read at 405 nm using a microplare reader (Zhu et al., 1995).
Statistical Analysis.
Statistical significance will be determined by Student's t-test for bacterial counts and antibody titers.
Proposed Experiments
Deletion of Genes Encoding Shiga Toxin in EHEC
Stx(s) is a major virulence factor implicated in the pathogenesis of STEC. Both Stx1 and Stx2 are bacteriophage encoded. The live attenuated vaccine strains according to the present invention will be non-Stx-producers. Thus, any genes encoding Stx(s) (stx1AB and stx2AB) will be mutated in STEC 0157, 026, and 0111.
To abolish the production of Stx1 and Stx2, the Stx1 and Stx2-coding sequences, stx1A/stx1B and stx2A/stx2B will be deleted. Because immunity specific to Stx is not desired for STEC vaccine for cattle, the StxAB genes will be deleted in their entirety. A deletion mutation by single-overlap extension PCR (SOE PCR) as illustrated in FIG. 14. Primers are designed to delete the coding sequence for stx1A/Stx1B, stx2A/stx2B including the flanking sequence between A and B gene (Table 6). Thus, a 1228-bp or 1260-bp deletion will be made for stx1AB or stx2AB, respectively. Either the stx1AB or the stx2AB genes, whichever is present in a particular strain, will be deleted from all the strains. Genotypic characterization of the mutants will be performed by PCR using primers flanking the deletion region. Single copy of deleted gene will be verified further by PCR including a primer derived from the deletion fragment. To ensure the accuracy of PCR, the WT STEC will be used as a positive control and an E. coli K-12 strain will be used as a negative control.








TABLE 6







Primers used to create deletion mutations



Note: *, f-forward, r-reverse; #, primers derived sequence data













Strain
GenBank





Primer
name
accession #
Position
Primer Location
















Stx2AB








2AP
EDL933
Y 10775
671 1-6730
aggaaggtgcgaccgtaatt
(SEQ ID NO: 29)


2Br


 7383-7364
atacaggtgttccttttggc
(SEQ ID NO: 30)


2Cf


 7364-
tgccaaaaggaacacctgtat
(SEQ ID NO: 31)





 7383/
ggcataacctgattcgtgg





 8625-8644


2Dr


 9300-9281
gtgcctggctcctctggtgt
(SEQ ID NO: 32)


2Ef


 7861-7881
tcatatctggcgttaatgga
(SEQ ID NO: 33)


 


Stx1A


1Af
EDL933
AE005442
 5800-5819
catcaccttctgcgacaatc
(SEQ ID NO: 34)


1Br


 6475-6466
cttagaatagctcagtgaaa
(SEQ ID NO: 35)


1Cf


 6475-6466/
tttcactgagctattctaagattacac
(SEQ ID NO: 36)





 7703-
a atactccttg ag





 7722


IDr


 8377-8358
gtgctctgacacctgtatag
(SEQ ID NO: 37)


IEf


 6781-6800
tgtatattttaagtattgca
(SEQ ID NO: 38)


 








Intimin (RDEC-I)














eae1Af
RDEC-I
AF200363
27201-
tcccccgggggaggggcaaaagt
(SEQ ID NO: 39)






27220
gccagaac


eae1Br


27956-
tttgttttcggcatcaaaat
(SEQ ID NO: 40)





27937


eae1Cf


27956-
attttgatgcc gaaaacaaagtc ga
(SEQ ID NO: 41)





27937/
c taatttaattacatctcaaatc





28197-





28216


eae1Dr


28901-
tcccccgggggattcttcctgttatc
(SEQ ID NO: 42)





28920
gggata


eae1Ef


27651-
ggtgataaagtgaccgtaat
(SEQ ID NO: 43)





27671


 








Intimin (EHEC)














eaeAf
EDL933
AF071034
15781-
acgccaggagttgcaggatg
(SEQ ID NO: 44)






15800


eaeBr


16460-
cctattatgctgatgctatggtcg
(SEQ ID NO: 45)





16480/
actaattccataaccaccccggc





16721-





16740


eaeCf


16721-
catagcatcagcataatagg
(SEQ ID NO: 46)





16740


eaeDr


17321-
ggttatattttttgatcaaa
(SEQ ID NO: 47)





17340


eaeEf


16501-
tagacatttggagtattaac
(SEQ ID NO: 48)





16520


 


Ler


lerAf
EDL933
AF071034
39308-
ttgctggactcagtgtctct
(SEQ ID NO: 49)





39327


lerBr


39812-
tgaatatggaaaataattcataac
(SEQ ID NO: 50)





39793/
atgaaataattaaatg





40161-





40142


IerCf


40142-
tgaattattttccatattca
(SEQ ID NO: 51)





40161


IerDr


40718-
caggttagtgctggctgtag
(SEQ ID NO: 52)





40699


lerEr


39981-
tgcctgatgatggactcgct
(SEQ ID NO: 53)





39962


 


Tir


tirAf
EDL933
AF071034
19292-
caggcgcatcggatttaca
(SEQ ID NO: 54)





19930


tirBr


19953-
gtaaatccgatgcgcctggtcgac
(SEQ ID NO: 55)





19934/
atatatccataatcatttta





21402-





21420


tirCf


21402-
caggcgcatcggatttaca
(SEQ ID NO: 56)





21420


tirDr


22003-
ctggtgtatagcatggcctt
(SEQ ID NO: 57)





22022


tirEf


20741-
ccagataatcaaaaagttaa
(SEQ ID NO: 58)





20760





Fully attenuating EHEC virulence by modifying the bacterial adhesion protein intimin (encoded by the eae gene) or the translocated intimin receptor (tir), or by inactivating the regulator Ler (lex-), which up-regulates the expression of virulence genes.






The studies in Examples 1 and 2 show that mutation in the eae and ler genes attenuated bacterial virulence while retaining their immunogenicity. The eae mutant appears ideal since the truncation of the C-terminal Tir binding domain (TBD) will retain the immunodominant region of intimin and be able to induce antibody production which has been shown to be protective. Moreover, the eae truncation will not affect the expression of proteins encoded on the LEE and outside the LEE, which may enhance immune responses against EHEC. Because Ler is a central regulator for the genes encoded on the LEE, it is expected that the attenuated mutant strains will not express ler-regulated protein expression, such as Tir and other secreted proteins.
A second deletion mutation targeted on the LEE PAI (eae or ler) will be generated.
1) The eae mutation. The previously constructed eae-mutant as described in Example 1 contains one single nucleotide deletion and selection of such mutants by either PCR or by cell adherence assay is laborsome and not practical. Instead, a 228-bp deletion mutation will be generated which will eliminate the C-terminal binding domain for easier identification by PCR using primers derived from the LEE nucleotide sequence. As a result, the defined truncated intimin retains its immunogenicity while losing the functional domain (FIG. 15). The complete eae nucleotide sequence is known for EHEC strains. The primers for the generation of eae mutants are derived from STEC strain O157:H7 strain EDL933 (Table 6). For strain 08566 and E45035, the direct sequences of eae genes are not known. However, it has been shown that the intimins of STEC 026 are β-intimins. Thus, primers were designed based on RDEC-I LEE sequence and they will be used for strain 08566 (Agin et al., 1996 and Zhu et al., 2001). The β- and γ-intimins are predominant among serogroup 0111 (China et al., 1999; Tarr et al., 2002; and Zhu et al., 2001). It can only be assumed that the eae gene of strain E45035 is homologous to RDEC-I. Therefore, primers derived from RDEC-I LEE will be used to generate eae deletion mutation for 0111.
2) The ler mutation. The studies in Example 2 demonstrated that deletion mutation in ler attenuated virulence of REPEC. The same strategy will be used to generate 330-bp in the ler of STEC strains using primers derived from STEC 0157 (Table 6).
The deletion of stxAB genes (stx1AB and/or stx2AB) will make EHEC non-toxic, and inactivation of either eae or ler will abolish A/E capacity of EHEC strains. Thus, a double mutation will fully attenuate bacterial virulence and make them harmless to cattle and safe for the environment.
Although the defined deletion mutations contain no selective marker, PCR-based identification of mutants is simple and straightforward. According to the previous studies with the rabbit EPEC strain in the laboratory of the present inventors, the percentage of occurrence of desired mutants selected from LB containing sucrose can range from 2 to 8 percent. If the eae and ler genes of 026 and 0111 are divergent from EHEC 0157 or RDEC-I, the eae and ler genes and flanking sequences will be determined from strain 08566 and E45035 using PCR products and the primers for the construction of defined deletion will be designed accordingly.
The nucleotide sequences of the stx, eae, ler and tir genes and their GenBank accession numbers for various E. coli strains are listed in Table 7.







TABLE 7







Stx, eae, ler and tir sequences










SEO ID NO:
GenBank Accession no.













Stx1AB
62
AE005174


Stx2AB
63
Y10775


RDEC-I eae
4
AF200363


EHEC 0157 eae
59
AF071034


RDEC-I ler
20
AF200363


EHEC 0157 ler
16
AF071034


EPEC 0127 ler
14
AF022236


REPEC 0103 (E22) ler
22
AF328682


RDEC-I tir
60
AF200363


EHEC 0157 tir
61
AF071034





Characterization of the mutants by examination of protein profiles and in vitro assays for Stx production and adherence to cultured cell lines






Deletion of Stx coding sequence would make the EHEC non-Stx-productive. Truncation of the C-terminus of intimin will result in a molecular weight shift for the intimin molecule. The deduced size for the native and truncated STEC intimins are approximately 101.7-kDa and 97.0-kDa, respectively. Since post-translational modification of intimin eliminates the signal peptide of 39 aa (Zhu et al., 1995), the mature intimins of STEC or their isogeneic eae mutants are 97-kDa for WT STEC or 89-kDa for truncated intimin, respectively. Inactivation of Ler will have a profound negative effect on the LEE-encoded secreted protein. Reduced production of secreted proteins, such as EspA, espB, EspD, is expected. As intimin and other LEE-encoded proteins play a key role in bacterial adherence, inactivation of eae or ler would abolish adherence of the mutants to cultured cell surface.
1) Stx assay. Stx assay will be performed at the time the stx1AB and/or stx2b mutants are obtained.
2) Examination of intimin. OMP will be prepared and examined for STEC and their isogenic eae mutants. Intimin expressed by the WT and their isogeneic eae mutants will be compared to view the molecular weight shift of intimin.
3) Examination of secreted protein profile. Secreted protein will be prepared and examined for STEC and their isogenic ler mutants. Secreted protein profile will be compared between the WT and their isogeneic mutants.
4) Adherence assay. Bacterial adherence to cultured cells will be performed qualitatively and quantitatively for all the mutants. HeLa cells will be used for the adherence assay, except that CHO cell line will be used for STEC O111. Cells are seeded at  ̃1-2×105 cells/ml to yield a semi-confluent monolayer and further cultured for 3 to 6 h in the presence of bacteria and are subjected for Giemsa staining or recovered with 0.3% Triton X-100TM in PBS for numeration (Torres et al., 2003). The above in vitro characterizations of isogenic mutants are aimed to characterize the relevant phenotypes of the mutants. These experiments will provide relevant information to confirm their genotypic alternations.
Evaluation of Immunogenicity of Live, Attenuated Vaccine Strains by Experimental Oral and Intrarectal Immunization in Mice
In Examples 1 and 2, it was demonstrated that protection of rabbits following immunization with attenuated isogeneic mutants is correlated with immune responses specific to pathogenic A/E organism. To test all the mutants for their immunogenicity in cattle is not practical. Studies demonstrated that STEC may induce relatively high levels of antigen-specific fecal IgA antibody (Funatogawa et al., 2002; and Nagano et al., 2003a and 2003b) and that rectal immunization could be an effective new vaccination method (Kleanthous et al., 1998; Mitchell et al., 2003a; and Nakase et al., 2001). Therefore, a mouse model is chosen to assess the immunogenicity of the vaccine candidates. STEC strains of serogroups 0157, 026, or 0111, containing double [ΔeaeΔstx (Ior2) AB, or ΔlerΔstx (Ior2) AB] mutations will be given to mice to evaluate immune response to bacterial LPS and/or to intimin.
1) Immunization of Mice. Groups of mice will be used for each mutant strain or vaccination route (Table 8). Sera will be collected pre-vaccination and 2 weeks after final boost (FIG. 16).







TABLE 8







Treatment groups of mice
















No of



Group
Strain
Serotype
Genotype
mice.
Immunization















1
86-24
O157:H7
ΔStx2AB/Δeae
16
Oral


2


ΔStx2AB/Δeae
16
Rectal


3


ΔStx2AB/Δler
16
Oral


4


ΔStx2AB/Δler
16
Rectal


5
Control

PBS
16
PBS


6
08566
O26
ΔStx2AB/Δeae
16
Oral


7


ΔStx2AB/Δeae
16
Rectal


8


ΔStx2AB/Δler
8
Oral


9


ΔStx2AB/Δler
8
Rectal


10
Control

PBS
16
PBS


11
E45035
O111:H-
ΔStx1AB/Δeae
16
Oral


12


ΔStx1AB/Δeae
16
Rectal


13


ΔStx1AB/Δler
8
Oral


14


ΔStx1AB/Δler
8
Rectal


15
Control

PBS
16
PBS









2) Determination of fecal bacterial shedding following vaccination. Fecal bacterial shedding will be determined once a week to monitor bacterial colonization. Bacterial suspension will be plated on MacConkey agar with appropriate antibiotics.
3) ELISA for sera IgG and fecal IgA: ELISA will be used to determine antigen-specific levels of Igs in sera and fecal samples, focusing on the immunogenicity of STEC 1157:H7 isogenic mutants. Therefore, for 0157:H7 derivatives, the IgG and IgA antibodies titers specific to the whole bacteria and intimin will be determined in sera and intestinal secretion, respectively, at day 0, 14, and 28 following first immunization. Sera will be collected from the remaining mice at day 0 and day 28. IgA samples from the intestinal secretion and feces will be collected at the time of sacrifice for the remaining mice.
Challenge of Vaccinated Mice with Wild-Type EHEC to Determine the Level of Protection
Once immunogenicity is determined, the optimal immunization regimen will be used in the challenge studies. Reduced bacterial shedding in feces among vaccinated mice will indicate the level of protection as described for passive administration of hyperimmune colostrums (Dean-Nystrom et al., 2002).
Challenge with a StrR, non-Shiga toxin producing 0157 strain will be performed two weeks after boost immunization. Mice immunized with vaccine candidate orally or intrarectally will be challenged. To promote colonization, mice will be given drinking water that contains streptomycin for 3 days before orogastric administration of the challenge strain (as described for oral vaccination). Un-inoculated mice will be used as controls.
Feces will be collected from each mouse for ten days. Feces will be weighed and then suspended in PBS. The fecal wt/final volume ratio will represent the dilution factor. lO μl of fecal suspensions will be plated on MacConkey agar containing appropriate antibiotics. To determine bacterial numbers in cecal contents, excised cecum will be vigorously washed three times with 10-ml PBS serially diluted and plated on MacConkey (str). Mouse weight will be recorded daily.
Statistical significance will be determined by Student's t-test for bacterial counts, antibody titers and weight changes.
Example 4
In this example, an attenuated live STEC vaccine candidate deficient in cell adherence and Shiga-toxin production was constructed. Oral or rectal immunization of mice with this attenuated mutant strain induced measurable serum IgG and fecal IgA specific to 0157 lipopolysacharide antigen. Following experimental challenge with an intact intimin 0157:H7 strain, vaccinated mice demonstrated significant reduction of intestinal colonization when compared to the naive mice. Immunization of animals with this double mutant represents a novel and practical vaccination strategy to induce effective local mucosal immunity, thereby reducing or preventing STEC colonization in the gut.
Materials and Methods
Bacterial Strains, Plasmids, and Cultural Conditions.
Bacterial strains and plasmids are listed in Table 9. The prototype STEC 86-24 (0157:H7) was obtained from the STEC Center at the National Food Safety and Toxicology Center at Michigan State University. The laboratory E. coli strain DH5α was used for plasmid transformation except for suicide plasmids (pCVD442 and derivatives), which were maintained in E. coli SY327 or SMlO (Donnenberg, 1991). Bacterial strains were stored at −80° C. in Luria-Bertani (LB) containing 20% glycerol and grown on LB agar or LB broth or MacConkey agar. Appropriate antibiotics were added to the media when needed at the following concentrations: carbenicillin (Car), 50 μg/ml; Nalidixic acid (NaI), 50 μg/ml; Streptomycin (Str), 25 μg/ml.







TABLE 9







Strains and plasmids used in this study (Example 4)









Strains or plasmids
Relevant characteristics
Source or reference










E. coli:









86-24
EHEC O157:H7, Stx2, StrR
Zhu et al., 1995


SY327
SY327 λpir, intermediate recipient for
Donnenberg et al., 1991



suicide vector pCVD442


SMlO
SMlO λpir, recipient for suicide vector
Donnenberg et al., 1991



pCVD442 to serve as donor strain, KanR


O157Δeae
An isogeneic eae mutant of 86-24, StrR
This study


0157 AeaeAstx2′AB
An isogeneic eaelestx2Ab mutant of 86-
This study



24, StrR







Plasmids:









pCR2.1 Topo
PCR cloning vector
Invitrogen


pCVD442
Suicide vector, AmpR
Donnenberg et al., 1991


pE525
pCR2.1::Δeαei57
This study


pE538(SM10)
pCVD442: :Aeael57
This study


pE462
VCR2A::Astx2AB
This study


pE488(SM10)
vCVO442::Astx2AB
This study









Generation of a Defined Deletion Mutation.
Recombinant DNA techniques were performed according to standard procedures (Sambrook, 2001). The defined deletion mutations in the eae or stx2AB genes were generated by single-overlap extension PCR (SOE PCR) (Senanayake, 1995). For PCR amplification, PCR SUPERMIX high fidelity mixture (Gibco BRL, Rockville, Md.) was mixed with template DNA (86-24 bacterial suspension in distilled water, 94° C. for 10 min) and the primers, while amplification was performed on the PTC-200 DNA Engine (MJ Research Inc., Waltham, Mass.) using the following protocol: 33 amplification cycles of denaturation at 94° C. for 60 s, primer annealing at 55° C. for 60 s, and elongation at 72° C. for 90 s, followed by a final extension step at 72° C. for 10 min (Zhu, 2005a). To create the intimin truncation, a 240-bp fragment at the intimin C-terminus was deleted by PCR using primers (Table 10) derived from the eae gene of EHEC 0157:H7. The initial PCR used primers eaeAf/eaeBr or eaeCf/eaeDr, respectively. Primers eaeBr and eaeCf contain a 20 bp stretch overlapping each other. The subsequent PCR using initial PCR amplicon with the primers eaeAf/eaeDr generated a DNA fragment yielding a 240-bp deletion at the C-terminal domain of intimin. The entire coding sequence for stx2A and stx2B was deleted by SOE PCR using primers (Table 10) derived from known sequence of 0157:H7 (Perna, 2001). The initial PCR amplification used primers 2Af/2Br or 2Cf/2Dr, respectively. The subsequent PCR using 2Af/2Dr generated a DNA fragment yielding a 1241-bp deletion. The primers 2Br and 2Cf contain a 20 bp stretch overlapping each other for assembling DNA fragments obtained by initial PCR. The resultant DNA fragment harbors an internal deletion of 1,241 bp to eliminate the entire Stx2A and Stx2B coding sequences








TABLE 10







Primers used to create defined deletion



mutations












GenBank






acces-




Primer
sion #
Position
Primer Location





eae






eaeAf
AF071034
15781-45800
acgccaggagttgcaggatg





(SEQ ID NO: 44)


eaeBr

16460-
cctattatgctgatgctatggtcg




16480/
(SEQ ID NO: 45)




16721-16740
actaattccataaccaccccggc


eaeCf

16721-16740
catagcatcagcataatagg





(SEQ ID NO: 46)


eaeDr

17321-17340
ggttatattttttgatcaaa





(SEQ ID NO: 47)


eaeEf

16501-16520
tagacatttggagtattaac





(SEQ ID NO: 48)


 


STX2AB


2aF*
Y10775
 6711-6730
aggaaggtgcgaccgtaatt





(SEQ ID NO: 29)


2Br

 7383-7364
atacaggtgttccttttggc






(SEQ ID NO: 30)


2Cf

 7364-7383/
tgccaaaaggaacacctgtat





(SEQ ID NO: 31)




 8625-8644
ggcataacctgattcgtgg


2Dr

 9300-9281
gtgcctggctcctctggtgt





(SEQ ID NO: 32)


2Ef

 7861-7881
tcatatctggcgttaatgga





(SEQ ID NO: 33)


 


tir


tirAf
AF071034
19292-19930
caggcgcatcggatttaca





(SEQ ID NO: 54)


tirBr

19953-
gtaaatccgatgcgcctggtcgac




19934/
(SEQ ID NO: 55)




21402-21420
atatatccataatcatttta


tirCf

21402-21420
caggcgcatcggatttaca





(SEQ ID NO: 56)


tirDr

22003-22022
ctggtgtatagcatggcctt





(SEQ ID NO: 57)


tirEf

20741-20760
ccagataatcaaaaagttaa





(SEQ ID NO: 58)





Note:


*, f-forward, r-reverse; #, primers derived sequence data






The SOE PCR products for eae and stx2AB harboring defined deletion mutation were purified and subsequently cloned into cloning vector pCR2.1-TOPO (Invitrogen), which were then prepared and being sequenced. Subcloning of DNA fragments containing deleted eae or stx2AB to the suicide vector pCVD442 was performed and transformed into strain SY327 and then into SMlO as previously described (Zhu, 2005a). Conjugation between the recipient WT strain 86-24 and the donor SMlO (pCVD442:Δeae) or SMlO (pCVD442::Δstx2AB) was performed to achieve the desired defined mutation (Zhu, 2005a). To verify deletion mutations in the WT background, primers (A and D) overlapping the entire deletion and an additional primer (E) derived from within the deleted fragments were used for PCR amplification and the amplicon size was compared between the WT and the mutant strains. Thus, PCR amplification using primers A and E yielded a product for the intact genes but yielded no PCR product for the mutated genes.
In Vitro Adherence Assay.
The cell adherence assay for the WT O157:H7 and its isogeneic mutant was performed on HeLa monolayers as described (Tories, 2002; Zhu, 1994). Briefly, HeLa cells were seeded at 1×105 cells/ml and grown overnight to yield a semi-confluent cell monolayer. Bacterial strains were grown in LB broth overnight at 37° C. and added to tissue culture cells replenished with fresh DMEM at a concentration of 107 bacteria per well and grown at 37° C. for 3 or 6 h. The cell monolayers were then washed, fixed and either stained with Giemsa and viewed by light microscopy, or treated with 0.1% Triton X-100TM in PBS in order to lyse the cells and disperse the adherent bacteria. This cell lysate was plated on MacConkey agar plates supplemented with Str for viable bacteria counting.
Cytotoxicity Assays.
Bacterial whole lysates were prepared from overnight cultures by two passages through a French Press. The bacterial pellets were removed by centrifugation. Protein content of extracts was determined by using BCA Protein Assay (BioRad) according to manufacturer's instructions. Microcytotoxicity was analyzed on HeLa cells and compared to the parent WT strain as described (Gentry, 1980). The last dilution of the sample in which 50% of the cells detached from the plate surface was considered the 50% cytotoxic dose (CD50).
Oral and Rectal Immunization of Mice.
BALB/c female mice, 4-6 week-old, were used for vaccination studies. For each immunization, a single colony of STEC mutant strain was grown in LB broth for 15 h at 37° C. The cultures were diluted at 1:100 in the same medium and incubated at 37° C. for additional 4 h to obtain logarithmic growth. The bacterial cells were harvested by centrifugation at 2,000×g for 20 min and resuspended with sterilized PBS. The bacterial concentration in the suspension was determined by optical density at 600 nm and adjusted with PBS to yield approximately 5×1010 CFU/ml. The viable counts were determined by limited dilution method. A 20 μl bacterial suspension was used for vaccinations, with approximately 109 CFU per inoculum. For oral immunization, mice were deprived of food 2 h before and 1 h after vaccination. They were fed with 30 μl of 10% sodium bicarbonate to neutralize stomach acidity 5 min prior to immunization and then given 20 μl of inoculum. For rectal immunization, mice were fasted for 12 h before immunization. Mice were anesthetized by intraperitoneal injection with 100 μl (per 10 g of body weight) of both 0.2%. RompunTM in PBS (Bayer) and 10 mg of KetavetTM (Parker Davis) per ml. The inoculum (20 μl) was gently introduced with a yellow tip into the rectum. Mice were positioned with the rectum facing upwards for about 45 min to reduce leakage of the inoculum.
Determination of Bacterial Numbers in Feces.
Feces from mice were collected and suspended in PBS. The w/v ratio was recorded as dilution factor. 10 ul of bacterial suspension was plated on MacConkey agar supplemented with appropriate antibiotics.
Preparation of Serum and Intestinal Secretions by Wash Out.
Sera were collected pre-inoculation and fourteen or twenty-eight days after last inoculation. Blood samples were taken from portal veins of killed animals. IgA antibody in mouse intestinal secretions was prepared by the method described by Elson et al (Elson, 1984). Each mouse was placed on a 12 cm×12 cm square of galvanized wire mesh placed within a Petri dish which contained a solution of protease inhibitors (0.1 mg/ml soybean trypsin inhibitor). The lavage solution was given intragastrically at 15 min intervals using a blunt tipped feeding needle. Thirty minutes after the last dose of lavage solution the mice were given 0.1 mg pilocarpine I/P to promote peristalsis. Discharged intestinal contents were collected and vortexed vigorously, then centrifuged. The supernatant was collected with addition of PMSF.
Measurement of Antigen-Specific IgG and IgA Antibodies:
0157 LPS was prepared by the phenol extract method. Sera IgG and fecal IgA specific antibodies to 0157 LPS was examined by ELISA as described (Agin, 2005; Zhu, 1995) with minor modifications. Briefly, microtiter plates (Immulon, Dynatech) were coated with 100 μl of 0157 LPS (10 μg/ml) in 50 mM bicarbonate coating buffer (pH 9.6) and dried overnight at 37° C. The plates were blocked with 10% skimmed milk in PBS. Sera and IgA preparations at various dilutions were added to the wells followed by conjugated anti-mice IgG or IgA (KPL). A phosphatase substrate system (pNPP, KPL) was added and the reaction was stopped by addition of 0.5 M NaOH and read at 405 nm using a microplate reader (Zhu, 1995; 2005b).
Statistical Analysis.
Comparisons of the values for in vitro adherence, antibody titers, and neutralization titers between experimental groups were made with Student's t test. Differences were considered significant when the P value was ≦0.05.
Results
Generation of Defined Deletion Mutation in the eae and stx2AB Genes.
As previously reported, a truncated intimin mutant retains its immunogenicity and protectivity (Agin, 2005). The laboratory of the present inventors thus created an intimin mutant by deleting the C-terminal 80 aa, which is responsible for binding Tir. Initial PCR amplification using primers eaeAf/eaeBr or eaeCf/eaeD obtained amplicons of 720 or 620-bp, respectively (data not shown). Subsequent PCR using primers eaeAf/eaeDr obtained a PCR product of 1,320 bp, which was then cloned into pCR2.1-topo vector to obtain plasmid pE525. Nucleotide sequencing of the insert indicated a 240-bp deletion at the C-terminal domain of intimin without alternating the remaining flanking sequences.
By using the same strategy, a deletion of 1,241-bp was made in the stx2AB gene. The initial PCR using primers 2Af/2Br and 2Cf/2Dr obtained 673 and 696-bp, respectively, whereas subsequent PCR using primers 2Af/2Dr obtained a 1,349 bp fragment which was cloned into pCR2.1 vector to obtain plasmid pE462. Nucleotide sequencing indicated deletion of the entire coding sequence for Stx2A and Stx2B including the flanking sequence between stx2A and stx2B while the remaining sequences flanking the stx2AB remained unchanged.
The DNA fragments containing the defined deletion mutation (Δeae or Δstx2AB) were obtained from pE525 and pE462, respectively, by digesting with endonucleases Sad and Xbal and subcloned into delivery suicide plasmid pCVD442 digested with Sacl and Xbal, thus obtaining plasmids pE538 (Δeae) or pE488 (Δstx2AB). Both of these plasmids were maintained into strain SY327 and subsequently transformed into conjugative strain SMlO.
Construction of Isogenic eae and stx2AB Mutants of 0157:H7.
An isogenic eae mutant on the WT EHEC strain 86-24 background was first generated and subsequently an additional stx2AB mutation was generated. Conjugation between 86-24 and SMlO harboring suicide delivery plasmid pE53 8 containing defined eae deletion mutation yielded numerous colonies. PCR amplification using primer eaeAf/eaeDr indicated that these resultant conjugates contained both the intact and mutated eae. One conjugate was selected to grow in LB broth without selection and subsequently selected on LB containing 5% sucrose. Allelic exchange of the native eae with mutated eae was confirmed by PCR using two sets of primers (eaeAf/eaeDr, or eaeFf/eaeDr) as described (Zhu, 2005a). Primers eaeAf/eaeBr and eaeFf/eaeDr yielded respective 1,560 bp or 840 bp fragment for the intact eae, respectively, whereas yielded only a 1,320 bp fragment for the mutated eae (data not shown). One such mutant was designated as O157 Δeae which was used for further studies.
The additional stx2AB mutation was generated in a similar manner except that conjugation was made between O157_leae and SMlO harboring pE488 containing the mutated stx2AB fragment. Following selection on LB supplemented with sucrose, allelic exchange of native stx2AB by mutated stx2AB was confirmed by PCR using two sets of primers (2Af/2Dr, or 2Ff/2Dr). Primers 2Af/2Dr and 2Ff/2Dr yielded fragments of respective 2,590 bp or 1,440 bp for the intact stx2AB, whereas they yielded only 1,349 bp for the deletion-mutated stx2AB (data not shown). One such final double mutant was designated as O151ΔeaeΔstx.
Intimin Molecular Shift.
The truncation of 80 residues at the C-terminal portion of intimin was further verified. The predicted sizes for the native and truncated intimins are approximately 101.8 or 93.1-kDa, respectively. Because post-translational modification of intimin eliminates a signal peptide of 39 aa (Zhu, 1995), the mature intimins of EHEC 0157:H7 WT or its isogenic eae mutant are 97.3-kDa or 88.6-kDa, respectively. Coomassie brilliant blue staining of SDS-PAGE-separated OMP extracts from the WT 86-24 revealed an intimin protein band with estimated molecular mass of 97-kDa (Data not shown). However, this 97-kDa-protein band was absent in strain O157Z\eae and a protein band with an estimated molecular mass of 89-kDa appeared.
Adherence on HeLa Cells.
When examined on HeLa cells, O157 Δeae showed significant (p<0.013) reduction on bacterial adherence when compared to the WT parent strain (FIGS. 17A-18B). While the WT 86-24 form microcolonies on about 47% of cells, the isogenic eae mutant form microcolonies on only 5% of cells.
Cytotoxicity.
Cytotoxicity to HeLa cells was compared between the WT and its isogenic stx2AB mutant. While the WT strain 86-24 demonstrated a LD50 of 1.2×104/mg, the isogenic eae mutant showed nearly two log reduction of the toxicity to HeLa cells (data not shown).
Colonization of 0157ΔEaeΔStx2AB Following Vaccination.
All mice were negative for the Str-resistant E. coli before vaccination. Nearly all mice, vaccinated either via oral or rectal route, shed Str-resistant E. coli 24 h following initial inoculation of 0157 ΔeaeΔstx2AB (FIG. 19). The total viable counts in the fecal pallets reached 1×109 CFU/ml (oral vaccination) or 1×109 CFU/ml (rectal vaccination) at day six post vaccination. The pattern of fecal shedding of vaccinated bacteria was similar, since day 8 post vaccination between animals receiving the vaccine strain orally or rectally reaching 3×107 CFU/ml. PCR of random selected Str-resistant CFUs using primers for the eae (eaeAf/eaeDr) or stx2AB (2Ar/2Dr) revealed two DNA bands corresponding to the mutated eae or stx2AB genes indicating they were vaccinated 0157 ΔeaeΔstx2AB.
The pattern for fecal bacterial shedding following boost oral or rectal vaccination at day 14, either via oral or rectal route, appeared very similar too. At day 22 post vaccination, only one mouse in the oral vaccination group shed some Str-resistant bacteria. While other mice were negative for Str-resistant bacteria, three out of seven mice shed nearly 2×108 bacteria at day 29 post vaccination. Fecal shedding was observed among two rabbits at day 25, 28, or 30 days post vaccination.
Bacteria Recovery Following Challenge with O157Δstx2AB.
Fecal shedding of challenged O157Δstκ2AB occurred the following day of challenge (FIG. 20). The mice in the naive group shed the highest numbers reaching nearly 1010 CFU/g within the first 6 days following challenge. Mice vaccinated with O151 ΔeaeΔstx2AB either by the oral or rectal route shed significantly less (p<0.05) of the challenge bacteria than those in naïve group. Six days following challenge, similar numbers of bacteria was recovered from all groups.
Detection of 0157 LPS-Specific IgG in Serum and IgA in Intestinal Secretion.
Mice in the oral vaccination group showed two-fold increases of serum IgG at day 28 following vaccination (FIG. 21A). The serum IgG titers at day 0, 14, and 28, in rectal vaccination were similar (FIG. 21B). Comparison of serum IgG titers at day 28 showed two fold increases among oral vaccination group over the remaining two groups (FIG. 22). IgA specific to 0157 LPS showed slight increase among oral vaccination mice over the remaining two groups (FIG. 23).
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.
All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.
Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
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## Claims

1. An isolated, live attenuated, double mutant of enterohemorrhagic Escherichia coli (EHEC) O157:H7, E. coli O26, or E. coli O111 in which the Shiga toxin coding sequence is deleted to abolish its Shiga toxin production and the nucleotide sequence coding for its bacterial adhesion protein, intimin or Eae, is mutated by deletion of the C-terminal Tir-binding domain to eliminate intimin's Tir binding activity while retaining rest of the intimin that comprises the intimin's immunodominant regions.

2. The live attenuated double mutant of claim 1, which contains no antibiotic resistance marker.

3. The live attenuated double mutant of claim 1 comprising a pharmaceutically acceptable carrier.

4. The live attenuated double mutant of claim 1 comprising an adjuvant.

5. The live attenuated double mutant of claim 2 comprising a pharmaceutically acceptable carrier.

6. The live attenuated double mutant of claim 2 comprising an adjuvant.