Enterotoxigenic Escherichia coli virulence factors and vaccine ...

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Enterotoxigenic Escherichia coli virulence factors and vaccine approaches Donata R Sizemore†, Kenneth L Roland and Una S Ryan

CONTENTS Pathogenesis: toxins & colonization factors Vaccines Expert opinion & concluding comments Five-year view Key issues

Enterotoxigenic Escherichia coli (ETEC) is recognized as one of the major causes of infectious diarrhea in developing countries. Worldwide, the incidence of ETEC infections is estimated to result in 650 million cases of diarrhea and 380,000 deaths in children under 5 years of age. ETEC is also an important cause of travelers’ diarrhea in people traveling to endemic regions of the world. Although ETEC is an uncommon cause of infections in the USA, there have been 14 reported outbreaks of ETEC in the USA and seven on cruise ships over the 20-year period between 1975 and 1995. ETEC strains are comprised of a large number of serotypes that produce a variety of colonization factors and enterotoxins. On infection, ETEC first establishes itself by adhering to the epithelium of the small intestine via one or more colonization factor antigens or coli surface proteins. Once established, ETEC expresses one or more enterotoxin(s), which results in the production of secretory diarrhea. While the need for an efficacious, easily administered vaccine is great, there are currently no licensed ETEC vaccines available for use in endemic countries or for US travelers. Expert Rev. Vaccines 3(5), 585–595 (2004)

References Affiliations

†

Author for correspondence AVANT Immunotherapeutics, Inc., St Louis, MO, USA Tel.: +1 314 983 9050 Ext. 109 Fax: +1 314 983 9077 KEYWORDS: colonization factor antigens, enterotoxigenic E. coli, enterotoxins, heat-labile toxin, heat-stable toxin

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Escherichia coli is a facultative anaerobe that colonizes the human colon within hours of birth and comprises 0.1% of the total gut flora. E. coli maintains a harmless and beneficial relationship with the human host. However, there are seven E. coli pathotypes highly suited to cause infections of the gut. These pathogenic E. coli include: enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroaggregative (EAEC), enteroinvasive (EIEC), diarrhea-associated hemolytic (DHEC), cytolethal distending toxin (CDT)-producing, and enterotoxigenic (ETEC). This article will concentrate on the latter organism, which was first recognized in piglets and was described by DuPont and colleagues as the causative agent of diarrhea in human volunteers in 1971 [1]. ETEC is found in the stool of infected animals and humans, and can be spread if the contaminated stool comes into contact with food and water sources. Symptoms are a watery diarrhea that begins 15–50 h after consumption of the bacteria. The infectious dose is believed to be approximately 108 colony forming units (CFUs). At present, the best

treatment begins with prevention, by using proper hygiene procedures and avoiding potentially contaminated food and water sources. If infection does occur, it is important that electrolyte fluid replacement is started in order to prevent dehydration; antibiotics are only given in severe cases. Although ETEC is most often recognized as a cause of traveler’s diarrhea and an important pathogen affecting children and elderly individuals of developing countries, there were 14 ETEC outbreaks in the USA and seven on cruise ships between 1975 and 1995. These outbreaks were caused by 17 different serotypes and affected 5683 people. A more recent study found that there were 12 US outbreaks, defined as three or more ill people and no other isolated viral or bacterial pathogens, from 1996 through to 2003. It is believed that the epidemiology of ETEC outbreaks in the USA is decreasing. ETEC causes disease by colonizing the small intestine, utilizing antigenically distinct fimbriae (CFA/I, CFA/II and CFA/IV) and fibrillar or afimbrial colonization factors. Once

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Sizemore, Roland & Ryan

attached, production of either heat-labile (LT) and/or heat-stable (ST) enterotoxins leads to diarrhea. ETEC strains are antigenically diverse, exhibiting many different O:H serotypes, multiple fimbrial colonization factors and three different toxin phenotypes (LT-only, ST-only and LT plus ST). An ideal vaccine would stimulate protective immune responses directed at cell-surface antigens and the enterotoxins. The identification of 78 different O serotypes and 34 different H serotypes have precluded their use as effective antigens for eliciting broad-based protection, due to the large number of antigenically distinct possibilities. Therefore, the primary antigen targets for vaccine development have been identified as the colonization factors and enterotoxins, described in detail below. Steinsland and colleagues have reported that ETEC infections induced considerable protection against new infections when the ETEC strain was of the same toxin-colonization factor (CF) profile [2]. However, immunity to the CFs played a lesser role than that of toxin immunity. These findings, and other natural infection data, indicate that immunity can be achieved, since it is known that repeated exposure of infants, children and adults to ETEC can result in immunity that provides protection against future ETEC contacts. Steinsland’s data emphasizes the importance of the induction of strong and sustainable immunity to the toxins, which will be described in the proceding section. Pathogenesis: toxins & colonization factors Heat stable toxin

ETEC strains can produce one of two types of low-molecularweight heat stable toxins, STa or STb. STb is found primarily in a variety of animal isolates, although human isolates expressing STb have been reported. STa and STb differ in size, amino acid sequence, antigenic cross-reactivity, mode of action, methanol solubility and activity in an infant murine model. STa is produced exclusively by human and porcine isolates. For the purpose of this discussion, the authors will focus on STa. Two distinct forms of STa have been reported. StaH, isolated from human strains, and StaP, isolated from porcine and human strains. The mature forms differ slightly in size and amino acid sequence. They share identical, antigenically cross-reactive active sites and the capacity to retain full activity after incubation at 100oC for 30 min. The estA gene encodes the STa protein. It is initially translated as a 73 amino acid preproprotein, consisting of the preleader region responsible for SecA-dependent translocation to the periplasm, the proleader region, possibly required for transport across the outer membrane by an undefined mechanism, and the mature protein sequence [3]. After cleavage of the pre- and proregions, the mature form of the toxin consists of 19 amino acids (StaH) or 18 amino acids (StaP) having a molecular mass of approximately 2000 Da. The active site consists of a 13-amino acid subsequence, which is the minimum requirement for toxin activity. The mature form of the toxin forms a compact structure held together by three sets of disulfide bonds, which are required for full activity. ST exerts its toxic activity by elevating cGMP levels in villus epithelial cells. When ST is secreted,

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it binds to the intestinal epithelial-cell receptor, guanylate cyclase (GC)-C. As a result of this interaction, protein kinase C is activated via Ca2+ influx, leading to phosphorylation and activation of the catalytic domain of GC-C. The activated GC-C begins catalyzing the formation of cGMP from GTP. The subsequent elevation of cGMP levels in the intestinal epithelial cells leads to inhibition of sodium and chloride ion uptake, concomitant with increased secretion of chloride ions through activation of the cystic fibrosis transregulator chloride channel. The result of these complex interactions is net fluid accumulation in the intestinal lumen and secretory diarrhea. ST is structurally homologous to the endogenous host peptides guanylin and uroguanylin, which control intestinal fluid and electrolyte homeostasis. Approximately 75% of ETEC strains express ST, either alone or in conjunction with LT. While wild-type ST is not inherently immunogenic; a protective host antibody response can be induced in animals against engineered ST when it is linked to another protein [4–6]. Therefore, as an ETEC vaccine antigen, ST is likely to provide broad protection if immunity could be stimulated through vaccination with an antigenic form of the toxin. Heat labile toxin

The LT produced by ETEC is an ADP-ribosylating toxin consisting of a multisubunit complex of two proteins, LT-A and LTB, with the arrangement AB5. It is encoded by plasmid-borne etxA and etxB genes, arranged as an operon. The A and B subunits have respective molecular masses of 27,200 and 11,800 Da. Each subunit is translated with a short signal sequence, which directs it to the periplasm, where holotoxin assembly occurs. The holotoxin is then secreted to the exterior of the cell [7]. Activation of the toxin is accomplished when LT-A is nicked between amino acids 192 and 193 by a trypsin-like enzyme, possibly encoded by host enterocytes [8], resulting in A1 and A2 peptides. While the two peptides are not covalently linked, they are held together by a disulfide linkage between C187 and C199 of the mature protein. The A1 subunit carries the ADPribosylating activity. Mutations that eliminate the A1 portion are nontoxic. The A2 peptide serves to anchor A1 to the B subunit. The pentameric B subunit directs the toxin to the surface of epithelial cells by binding to the GM1 gangliosides present on most cells. A cluster of the toxin–GM1 complexes forms on the cell surface leading to the formation of smooth, apical endocytic vesicles. These vesicles are trafficked into the endoplasmic reticulum, possibly facilitated by the carboxy-terminal RDEL sequence of the A2 peptide, that mimics a known eukaryotic endoplasmic retention signal. Upon reaching the basolateral membrane of the cell, A1 catalyzes the ADP ribosylation of protein GSα, a component of the trimeric GTPbinding protein that activates adenyl cyclase. This leads to the activation of adenyl cyclase, increased levels of cAMP and an efflux of chloride ions resulting in a watery diarrhea [9]. Approximately 50% of ETEC strains produce LT and roughly half of these produce both LT and ST. Unlike ST, LT is highly immunogenic. Immune responses generated to LT-B appear to

Expert Rev. Vaccines 3(5), (2004)

ETEC vaccines

Table 1. Colonization factors found in human ETEC strains. Group

Group members

Characteristics

CFA/I

§

Highly genetically related. Supplementary proteins of one operon can substitute for another.

CS5

CS5, CS7, PCFO9 (CS13), PCFO20 (CS18), CS20

Sequence similarities to fimbriae of animal ETEC strains.

Bundle-forming

CFA/III (CS8), Longus (CS21)

Most closely related to Type IV pili.

CS15

8786 (CS15), CS22

Related to SEF14 fimbriae of Salmonella enterica serovar Enteritidis.

Distinct

§

§

§

CFA/I, CS1, CS2, CS4, PCFO166 (CS14), CS17, CS19

CS3, §CS6, 2230 (CS10), PCFO148 (CS11), PCFO159 (CS12) No known homology with any known fimbriae.

§

Colonization factors that have been expressed in attenuated strains of bacteria.

be protective, although the data are not conclusive. Certainly, short-term immunity can be generated through vaccination and anti-LT titers rise as natural immunity increases for individuals in developing countries that are repeatedly exposed to the organism. However, the acquisition of natural immunity most likely involves immune responses to many antigens in addition to LT. Cholera toxin (CT), elicited by Vibrio cholerae, is an AB5 toxin that is nearly identical to LT, sharing greater than 80% amino acid sequence identity for both the A and B subunits. It binds GM1 ganglioside, catalyzes ADP-ribosylation of the GSα protein, and is antigenically cross-reactive. Although antibodies elicited to CT can provide protection against LT-producing ETEC strains, this protection is temporary and not long-lived. The use of CT-B to protect against LT-producing ETEC strains will be discussed later when Dukoral™ (Chiron), a commercial vaccine against cholerae that provides some protection against ETEC is discussed. Colonization factors

Although many ETEC strains found in field studies express unidentified colonization factor antigens (CFAs), 21 different ETEC CFAs have been identified from human-associated strains [10,11]. Most of these are encoded on high-molecularweight plasmids and their expression is thermoregulated. They are classified either as coli surface antigens (CS) or as CFAs. The three most prevalent are CFA/I, CFA/II and CFA/IV, accounting for nearly 90% of the typeable strains. All three appear to be expressed as fimbriae, although the structure of CFA/IV has not been elucidated. The CFA/I fimbria is composed of a single structural subunit, encoded by the cfaB gene and forms a well-defined, rigid structure. Nomenclature for the CFA/II and CFA/IV groups is complex and represents multiple fimbrial types. Cells expressing these antigens can express several different CS subunits, with the exact composition varying among strains. Strains designated as CFA/II express CS3 alone or in combination with either CS1 or CS2. CFA/IV strains express CS6 either alone or in combination with CS4 or CS5, but never both. In general, strains expressing any one of these CFAs do not express the other two, although CS6 may be expressed with CFAs other than CS4 and CS5 [11]. www.future-drugs.com

To clarify the confusion in the nomenclature, Gaastra and Svennerholm reorganized the different ETEC CS factors into five groups based on their genetic and structural homologies (TABLE 1) [12]. CFA/I fimbriae are composed of a single structural subunit. The subunit at the tip contains the adhesive properties, while the remaining subunits are nonadhesive, probably due to conformational differences. Members of this group may share a limited number of common epitopes but are not antigenically cross reactive [11]. Members of the CS5 group are closely related to fimbriae commonly found in porcine isolates. CS18 (PFCO20) demonstrated 56% identity with 987P fimbriae from porcine ETEC strains, although they probably do not bind to the same receptor. CS5 exhibits approximately 25% identity with F41 and CS13 is closely related to K88. The bundle-forming group, also called type IV pili, is termed such because of their tendency to form large bundles of pili and their similarity to the bundle-forming pilus expressed by EPEC. The CFA/III fimbriae are 5–10 µ in length, while the longus pilus is over 20 µ long. The longus pilus has been associated with ETEC known to express other CS antigens. CS15 and CS22 are antigenically cross reactive and are closely related to the Sef14 fimbriae of Salmonella enteriditis. The remaining CS factors share no homology either with each other or with any known fimbriae. Vaccines

Although no licensed products are available in the USA, Dukoral is licensed in 15 countries worldwide including Sweden, Norway and New Zealand. Dukoral is composed of CT-B and whole-cell killed V. cholerae. Dukoral is a drinkable vaccine that protects against diarrhea caused by V. cholerae and subsequently evolved into an ETEC vaccine. This product has been tested in over 200,000 volunteers for safety, immunogenicity and/or efficacy to either V. cholerae or ETEC [101]. Early studies by Clemens and Holmgren noted that short-term cross protection (67% fewer cases of LT–ETEC episodes) of up to 3 months could be achieved against LT-producing ETEC strains due to the antigenic similarities of LT-B and CT-B [13]. As mentioned previously, LT-B and CT-B share greater than 80% amino acid sequence identity. While short-term protection was achieved, 587


protection was not detected at 9 months. In addition to the lack of long-term protection, this vaccine fails to protect against STproducing ETEC strains [14]. Recently, Chiron’s Dukoral vaccine for cholera has been licensed for use in the UK, thus making available an oral cholera vaccine that provides protection against ETEC as well [15]. To broaden and increase the duration of protection, AnnMari Svennerholm’s group in Göteburg (Sweden) has developed and tested in several clinical and field trials, an orally inactivated ETEC vaccine consisting of a mixture of five different formalin-killed ETEC strains in combination with recombinant CT-B subunit (produced by SBL Vaccin, Stockholm). The combination of five ETEC strains gives coverage to the most prevalent colonization factors – CFA/I, CFA/II and CFA/IV. This vaccine has been tested in Swedish adult volunteers [16–18]; Bangladeshi adult volunteers [19] and children 3–9 years of age [20]; Egyptian infants, 6–18 months of age [21], children, 2–12 years of age [22] and adults [16]; and young Israeli adults [23]. Lots 001, 003 and 005 have been found to be welltolerated in all age groups tested, with mild cramping, loose stool and abdominal distension most often reported. Immunoglobulin (Ig)A antibody-secreting cells (ASCs) have been demonstrated to all CFAs with a high percentage of responders while significantly increased serum levels of IgA and IgG have been demonstrated to the recombinant CT-B (rCT-B) component. Many of these studies were performed in collaboration with and funded by the US Department of Defense (DoD). A US Army Medical Materiel Development Activity (USAMMDA) information paper, dated 31 March 2004 [102], states that an orally administered vaccine consisting of killed, whole cells of five of the most common ETEC strains combined with CT-B failed to elicit significant protection against nonsevere ETEC diarrhea when given to over 300 Egyptian infants and young children in a Phase II clinical trial. Data for the trial have been published, and the conclusions drawn were that vaccinepreventable ETEC fecal excretion was highly associated with diarrhea, however, excretion of LT-ETEC was not related to diarrhea. Furthermore, excretion of antigenic types of vaccinepreventable ETEC other than LT-ETEC was highly associated with nonbloody diarrhea. Since vaccines are designed to prevent symptomatic infections, fecal excretion of a pathogen in a patient does not truly establish that the isolated organism played a role in the individual’s symptoms. The recommendation by the DoD was to terminate the vaccine program because of the low efficacy, which was estimated at 20% [24,25]. AVANT Immunotherapeutics, Inc. (MA, USA) [103], Microscience (Wokingham, UK) [104], and the Center for Vaccine Development (CVD) at the University of Maryland’s School of Medicine (MD, USA) have reported in the literature, at meetings, or on their respective web pages that they are involved in the development of ETEC vaccines utilizing live, attenuated bacteria as carriers to express ETEC antigens. Previously, several Salmonella typhi strains expressing foreign antigens have been tested in human trials with little evidence of induction of immune responses to the heterologous protein and no candidates have

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advanced to licensed products. While S. typhi is a systemic pathogen in humans, new efforts are concentrated on induction of gastrointestinal immune responses by utilizing carriers that limit themselves to the gut (e.g., Shigella, Vibrio and S. typhimurium). These new vectors may advance the bacterialvectored antigen technology for use against intestinal pathogens by improving antigen delivery to important immune cells located in the intestinal tract. AVANT’s approach has been to utilize a variety of bacterial vectors based on human-tested V. cholerae, S. typhi and S. typhimurium vectors. The clinical profile of their most advanced candidate, Peru-15, which produces CT-B, has been assessed in several clinical trials involving more than 250 adult volunteers and it was demonstrated to be a well-tolerated and immunogenic oral vaccine that affords protective immunity in a human cholera challenge model [26]. Their S. typhi vector, the phoPdeleted Ty800, has been tested in a Phase I study and was demonstrated to be safe [27]. A similar phoP-deleted S. typhimurium strain has been used to deliver a Helicobacter pylori antigen to humans [28]. In the study, five of six volunteers seroconverted to S. typhimurium antigens. Three of the five Salmonella-positive volunteers had detectable immune responses to the H. pylori antigen. Although the immune responses to the vectored antigen were not robust, these results demonstrate the feasibility of this approach. In addition, AVANT scientists have successfully used an attenuated S. typhimurium strain to deliver E. coli antigens to chickens for the prevention of the extraintestinal poultry disease, airsacculitis [29,30]. Microscience’s ETEC vaccine is based on their spi-VEC system. This technology is an extension of their typhoid vaccine in which Salmonella strains mutated in Salmonella Pathogenicity Island (SPI)-2 are defective in the ability to survive systemically. This group has reported human safety data evaluating live, attenuated S. typhi and S. typhimurium strains that carry combined mutations in aroC and SPI-2 [31]. Both strains were welltolerated. Attenuated S. typhi strain ZH9 was able to induce LPS-specific IgA ASCs in six of nine volunteers, with those receiving the higher doses eliciting higher serum antibody responses. Attenuated S. typhimurium strain WT05 demonstrated no signs of inducing diarrhea, but was shed in the stool for up to 23 days. Interestingly, only volunteers given the highest dose of 109 CFUs demonstrated detectable LPS-specific ASCs with variable serum responses seen through all three dosing groups. Both of these strains could be used as vectors of ETEC antigens. The latest trial results, described in a press release (8th March 2004) [104] were undertaken at the St George Institute at St George’s Hospital Medical School (London, UK) and demonstrated the safety and immunogenicity of an oral ETEC vaccine based on their spi-VEC technology. The trial was a dose escalation study involving 36 volunteers with the aim to assess safety, and serum and mucosal antibody responses. The vaccine was found to be both safe and highly immunogenic. A single dose gave rise to 50% seroconversion against LT, while two doses increased the seroconversion to 70%. The vaccine is now moving into a Phase II ETEC challenge study that is to


ETEC vaccines

take place in the USA in late 2004. In the study, 100 volunteers will be vaccinated and then challenged with ETEC. The results are expected to be available in 2005 and, if they are positive, the company plans to proceed to a Phase III program in advance of receiving the full 2004 data package. The group at the CVD is also involved in utilizing live, attenuated bacterial carriers. The CVD has developed attenuated Salmonella and Shigella strains expressing CFA/I [32], CFA/I and mutant LT [33], and CS2 and CS3 colonization factors [34]. Testing in mice and guinea-pigs demonstrated that these constructs elicit high serum and mucosal antibody titers to the expressed colonization factors. Additionally, Shigella-vectored constructs elicited elevated mucosal IgA and serum IgG directed against Shigella lipopolysaccharide (LPS) and animals were protected against Shigella flexneri 2a challenge, indicating the potential for a multivalent vaccine. The CVD is now utilizing a two-component plasmid maintenance system comprised of the hok-sok postsegregational killing system plus the parA plasmid partitioning system to minimize plasmid loss from the population of actively growing bacteria [35]. Cell lysis occurs when plasmids are lost from the population by segregation. In addition to the plasmid maintenance system, the investigators have added a detoxified variant of LT. Results following intranasal immunization of guinea-pigs with a mixed inoculum containing five Shigella strains each expressing a different ETEC colonization antigen, showed serum IgG and mucosal IgA to the S. flexneri vector LPS. These animals were also protected against Shigella challenge as measured by the Sereny test. Specific serum and mucosal IgA antibody was detected to CFA/I, CS2, CS3, CS4 and LT. Most notable was the fact that antibody responses against CFA/I and CS4 were able to inhibit hemagglutination by wild-type ETEC. Furthermore, the antibody titers generated when given as a multivalent vaccine were equivalent to those induced when the strains were given individually. In addition to the above-mentioned facilities, a group at the Beijing Institute of Biotechnology (China) has constructed attenuated S. typhimurium strains expressing an LT-B/ST fusion protein and CS3 [4]. Mice inoculated with the vaccine strain produced antibodies against all three antigens. Serum from vaccinated animals was able to neutralize ST in a suckling mouse assay. These results demonstrate that S. typhimurium can be used as a vector for ETEC antigens. When these data are considered together, the use of various attenuated bacteria as vaccines to deliver ETEC antigens shows great promise, allowing for easy oral delivery and perhaps even cross protection against the bacterial carrier, making this technology intriguing and the pursuit of an efficacious vaccine worthwhile. Attenuated bacteria have been studied for many years and have been through countless human trials evaluating the safety of the vector strain. Those expressing foreign antigens have been cited above. Vaccine development is also progressing in other technology areas, including the utilization of bovine hyperimmune milk products as a passive prophylactic treatment [36]. However, bovine hyperimmune milk, TravelGAM, was unable to provide


adequate passive protection against ETEC challenge when given with a standard meal, unlike in early studies when subjects were challenged after fasting [37]. ImmunCell (ME, USA) continues to perform research in this area [105]. A European Patent Application and a European Patent Specification describes the use and production of egg-yolk antibody specific for ETEC fimbrial antigens that have been used to protect piglets [201,202]. Egg-yolk antibody has also been used to protect neonatal and early-weaned piglets [38], and rabbits [39] against ETEC diarrhea. If passive protection can be achieved in such a simplified manner as drinking a solution, it could help eliminate or reduce the symptoms of disease during the time between vaccination and induction of protective mucosal immunity. Most cases of diarrhea occur in travelers and deployed troops within the first few days to weeks after entry or deployment to a developing country, at a time when immune response levels may not be adequate if vaccination took place just prior to departure. Drinks would need to be manufactured and stored in a convenient form and with a long shelf-life to be suitable for traveling and military field conditions. An initial attempt to create an ETEC DNA vaccine that fused cfaB, the structural gene for CFA/I, with glycoprotein D from herpes simplex virus Type I in pRE4, a eukaryotic expression vector, failed to induce serum antibody capable of agglutinating bacterial cells or inhibiting hemagglutination [40]. The use of a different vector, pBLCFA, resulted in the induction of antibodies that were able to block the adhesive properties of CFA/I [41]. Although the authors attribute this finding to the secretion of the CFA/I product when expressed by the CMV promoter of pBLCFA, it likely has more to do with the conformation of the protein when it is expressed alone and not fused with the herpes simplex virus glycoprotein. The pBLCFA construct preserved the important conformational epitopes required to induce antibodies able to inhibit adhesion. Injectable DNA vaccines may not be well-suited for protection of gut pathogens and further data are needed to demonstrate protective mucosal immunity. One technology that may be better suited for protection of intestinal pathogens such as ETEC, is the use of plants or food crops to express protective antigens (termed edible vaccines). Engineered food crops can then be fed to large numbers of people. Potatoes expressing ETEC LT-B antigen have been tested in a Phase I trial at the CVD. Volunteers received three doses of peeled raw potatoes over a 3-week period with the dose of LT-B ranging from 0.4 to 1.1 mg. Ten of the 11 volunteers demonstrated four-fold increases in serum antibody specific for LT-B, with six individuals having four-fold increases in intestinal antibody [42]. Prodigene (USA), a privately held biotechnology company that holds at least nine plant vaccine technology patents, lists an LT-B vaccine as part of its human oral vaccine products [106]. Their main corporate focus is on developing edible vaccines that will be practical to deliver (e.g., approximately 2 g of corn material containing the antigen of interest). The material delivered must be palatable and of a realistic amount, which was not the case in earlier systems, such as, the raw potato (STEPHEN STREATFIELD, PERS. COMM.).

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Yu and Langridge took the use of edible vaccines a step further by testing other ETEC targets to strengthen this approach [43]. Tests in mice demonstrated that potato tuber tissue expressing CFA/I fused with CT-B produced modest levels of serum IgG antibody to CFA/I following five oral inoculations of 3 g of tubers or 10 mg of recombinant protein. This was the first demonstration of a multicomponent edible vaccine. FaeG, the major subunit and adhesin of K88 fimbriae, was expressed in transgenic tobacco plants [44]. Subsequently, 0.5 mL of leaf extract emulsified in an equal volume of complete or incomplete Freund’s adjuvant was used to intraperitoneally vaccinate female KM mice on days 0, 14, 28, and 42. The transgenic FaeG induced antibodies to FaeG, as shown by immunoblot. In addition, sera from mice injected with the transgenic FaeG were able to significantly reduce fluid accumulation in the rabbit ileal loop ligation model far better than mice vaccinated with the nontransgenic extracts [44]. Corn has also been used to produce human and animal vaccines [45,46]. The application of transgenic corn or potatoes requires that the antigen level be high enough to allow for adequate antigen dosing in practical amounts of plant material. LT-B expression could be increased by four orders of magnitude by adjusting the subcellular location where it was produced. This group has previously shown that LT-B produced in corn could bind GM1 receptors and form a pentamer, making it more heat-resistant. Edible antigens have come a long way and continue to advance. One difficulty with this approach is the quantity of plant material that needs to be consumed in order to induce significant amounts of antibody that are protective. This may be overcome with improvements in plant expression technology. If so, oral feeding of transgenic plants may be an inexpensive way to boost established systemic and mucosal immune responses. This prime–boost strategy has been shown to enhance immune responses to measles vaccination by combining a DNA vaccine with multiple boosts of an edible vaccine [47]. Two companies, Antex (CA, USA) and Acambis (MA, USA), that had been involved in the production of vaccines for various travelers’ diseases including ETEC, are no longer working in this area. Effective of 31st May 2003, the assets of Antex Biologics, Inc. and Antex Pharma, Inc. were acquired by BioPort Corporation. Antex Biologics was developing a multicomponent travelers’ vaccine consisting of Campylobacter, Shigella and ETEC components. The stage of testing for the ETEC component of these vaccines is unclear, but press releases in 2003 indicate that BioPort was enhancing its long-term strategic plan by providing an exciting new product pipeline through the acquisition of the bankrupted Antex Biologics, Inc. and Antex Pharma [107]. Antex developed a technology known as nutriment signal transduction. Use of this technology enhances expression of antigenic proteins and/or virulence factors during in vitro growth. Acambis, once a DoD corporate collaborator, was developing live attenuated ETEC strains. They have since re-evaluated their product line and are no longer actively working in the area [108].

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Although the decision has been made to put this area of work on hold, their data are worth a brief discussion. E1392/75–2A, a spontaneously arising toxin-negative ETEC variant, was demonstrated to confer 75% protection against challenge in human volunteers [48]. Unfortunately, mild diarrhea was reported. If live, attenuated ETEC strains are to be used there must be a balance between immunogenicity and reactogenicity. To eliminate this unacceptable reactogenicity, different combinations of defined deletion mutations were introduced into the chromosome. Of these mutants, two strains, PTL002 (∆ompR and ∆aroC) and PTL003 (∆aroC, ∆ompC and ∆ompF), were selected for Phase I dose escalation studies in volunteers. The two strains were well-tolerated, with mild-to-moderate cramping and two cases of vomiting reported. Recipients receiving the highest dose (109 CFUs) demonstrated a significant increase in IgA specific for CFA/II, as measured in antibody lymphocyte supernatants. Further clinical evaluation of the strains was reportedly underway to determine if they could provide protection against challenge by wild-type ETEC. Certainly, it remains to be seen if this approach will be reinitiated by Acambis or another group of investors. At this stage, further data needs to be provided on protection without reactogenicity. The Iomai Corporation (MD, USA), has taken the unique approach of using the Langerhan’s cells of the skin to deliver antigen via transcutaneous immunization. A Phase I study to measure the safety and immunogenicity of CF CS6 with or without LT demonstrated that 68% of volunteers receiving patches containing CS6 and LT mounted IgG serum responses to CS6 with 53% having IgA responses [49,50]. Additionally, 90% were found to have serum responses to LT. IOMAI has announced an agreement with Berna Biotech Ltd (Bern) to pool their efforts and expertise to develop a number of therapeutic and prophylactic bacterial and viral vaccines [109]. Target organisms were not disclosed in the press release. Microencapsulation of CFA/II was and continues to be evaluated by the DoD. In early studies, volunteers each swallowed intestinal tubes on days 0, 7, 14 and 28 followed by challenge with 109 CFUs of ETEC strain E24377A [51]. Unfortunately, seven of the ten volunteers developed diarrhea. A study in pigs has demonstrated that peroral administration, which is the only practical method of delivery, of microencapsulated enterotoxigenic E. coli and detached fimbriae resulted in no significant serum antibody or reduction in colonization by ETEC [52]. This method of vaccination seems unable to stimulate protective gut mucosal immune responses in the form tested. However, improvements in the technology have led to the encapsulation of CS6 in a biodegradable polymer poly(D,L)-lactide-co-glycolide (PLG). This formula, administered orally in a solution of either normal saline or rice-based buffer, was demonstrated to be welltolerated by volunteers after three doses given at 2-week intervals. Four of five volunteers who received 1 mg of CS6 in PLG or in buffer showed significant IgA ASC development. Immune responses could also be detected when 5 mg of nonencapsulated CS6 in buffer was used to vaccinate. Further work is required to determine if the responses are sufficient for protection [53].


ETEC vaccines

A barrier to assessing the efficiency of these candidate vaccines is the lack of a good animal model that reflects the disease state observed in humans. This would be invaluable in screening potential candidates in order to eliminate those that may be too reactogenic early in the R&D process, or those that fail to raise serum and mucosal antibodies that provide protective immunity. The model must also be amenable to the vaccine technology being evaluated. Among the models most widely used for evaluation of STbased vaccines, is the suckling mouse model [54] and the ileal loop model [39,55]. In the suckling mouse model, sera from vaccinated animals (e.g., mice) are combined with ST and injected into suckling mice. After a few hours, intestines are harvested and fluid accumulation is evaluated. While this model detects the presence of ST-neutralizing antibodies in biological fluids, typically serum, it does not provide any evidence as to the effectiveness of the immune responses in the vaccinated animal to protect against ETEC-mediated disease. The ileal loop model utilizes live ETEC challenge, but requires surgical procedures to be performed on the test animal. The removable intestinal tieadult rabbit diarrhea (RITARD) model was developed over 20 years ago to study ETEC and V. cholerae and results in the development of severe and watery diarrhea in challenged animals [55]. This model, which has been used to study the action of many bacterial enterotoxins, allows for vaccination and blood sampling to study the kinetics of the immune responses of the animals prior to challenge. In 2002, Mond, Kokai-Kun and Cassels filed a patent describing a rat model in which the normal gut flora is cleared prior to challenge [203]. This model has the potential for evaluating protection directly in vaccinated animals, although any potential influences from the normal flora would be obscured. More recently, an intranasal murine model was described, in which BALB/c mice were inoculated with ETEC strains that

had been previously used in human challenge studies [56]. Although not the normal route of infection, it may be useful as a relatively inexpensive alternative for screening live vaccine candidates. The best models also allow for a challenge that results in symptoms that mimic human disease. Although no perfect model is available at present, many investigators continue to work to develop improved models that could lead us to a better understanding of the factors that provide protection. The next question is what surrogate markers are to be used to decide if a vaccine candidate has the potential to provide protection against wild-type infection in humans. At this time, one would expect a vaccine candidate to induce antibodies capable of neutralizing toxin activity. Although the development of immune responses to the CFs have recently been demonstrated to play a lesser role than those induced against toxins, one would expect mucosal immune responses to stop or reduce colonization of the intestinal tract by the organism and have an additive affect. The most valuable models for vaccine assessment would use animals that are immunologically mature to allow for the development of serum and mucosal antibodies (IgA ASCs in the blood or intestinal scrapings). Expert opinion & concluding comments

In this review we have provided a brief overview of ETEC pathogenesis and described the various vaccine technologies being investigated. Clearly, the development of an ETEC vaccine has been the goal of many researchers over several years (TABLE 2). Given the diversity of the organism and the lack of understanding of the virulence mechanisms, one needs to start asking difficult questions regarding the ability of the scientific community to develop a safe, efficacious, and inexpensive ETEC vaccine within the next 5–10 years. Furthermore, if a vaccine is found to be safe and efficacious in one part of

Table 2. ETEC vaccines undergoing development. Company/University

Technology

Phase of development

BioPort (Antex Biologics)

Nutriment signal transduction

Preclinical

Acambis

Live, attenuated ETEC

Phase I

IOMAI

Transdermal patch

Phase I

Chiron (Powderject)

Dukoral Inactivated ETEC

Phase III

AVANT Immunotherapeutics, Inc.

Live, attenuated bacterial carriers

Preclinical

Center for Vaccine Development at the University of Maryland School of Medicine


Preclinical

Prodigene/Boyce

Edible plants

Phase I

Microscience


Phase I

ImmunCell

Bovine hyperimmune milk

Phase I

DoD PLG microspheres

Encapsulated

Phase I

DoD: Department of Defense; ETEC: Enterotoxigenic Escherichia coli; PLG: poly(D,L)-lactide-co-glycolide.


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the world will it be in other regions? Molecular biological techniques first applied to this problem nearly 20 years ago have advanced quite rapidly and a wide variety of vaccine approaches have resulted, however, a reliable, commercial vaccine continues to elude us. Several approaches reported in the literature as successfully inducing immune responses to ETEC antigens, including ST, have not led to a vaccine [57,58]. Did these approaches fail and if so, at what point did this occur? Today’s journal and corporate philosophies do not support the reporting of negative results. However, public scrutiny of failed vaccines could provide valuable insight into why some approaches fail and perhaps provide clues that would point the way toward other technological approaches or experimental designs. Clearly, there is much that we do not understand regarding the infectious process of ETEC. Do we understand enough about the triggering of intestinal immunity to be able to use that information to develop a good ETEC vaccine? Do we fully understand the mechanisms that are protective? Can a single vaccine protect against ETEC strains seen in the various regions of the world? Are there alternative approaches, such as probiotics [59,60] that could be investigated further? In light of the fact that a number of companies have dropped their ETEC programs, what can be done to encourage companies to stay involved in this research? With a calculated market value of US$350 million [104], a few small companies continue to work in the area with the hope of producing a vaccine that can be used to protect travelers, military personnel and individuals in endemic areas. However, small companies can only focus on applying their proprietary technology to a problem. More collaborative work between academic and government laboratories must be encouraged and supported. These laboratories in many cases have the largest databases on pathogenesis and epidemiology; information that small companies do not have resources to generate themselves. In addition, small companies cannot afford to spend extensive amounts of time and resources developing disease models or to identifying target antigens without financial support.

Many large companies do not seem to have an interest in researching and developing travelers’ vaccines. Certainly, more funds being made available through both the private and public sectors would encourage small companies with the knowledge base and new innovative technologies to stay in the game for the long run. The DoD has supported much of the work on ETEC vaccine development, field surveillance and isolate typing. This stems from the need for deployed troops to be combat ready at all times in areas of the world where ETEC is prevalent and under conditions in which precautions normally given to travelers, such as drinking only bottled water and eating at reputable establishments, cannot be followed. Support from public and private agencies along with collaborative efforts between small companies and academic and government resources would undoubtedly increase the probability of producing an efficacious ETEC vaccine in the near future. Five-year view

The next 5 years will abound with testing of the vaccine candidates described above. Some will not progress beyond the preclinical stage, while others, some of which have already been tested in initial Phase I trials, will be tested further in Phase I and II clinical trials. Efficacy following challenge in small controlled clinical trials is possible within the timeframe, but it is extremely unlikely, given the processes required that few, if any, vaccine candidates will complete field-trial testing. These trials, which are performed as large studies in areas of the world where ETEC is endemic, are extremely expensive and require a lot of planning and manpower to coordinate the efforts of recruiting volunteers, patient follow-up, sample collections, transport and processing. The next 5 years should bring important insights into the new technologies described above (e.g., hyperimmune serum, eggyolk antibody, transdermal and edible vaccines), alone or in combination with other approaches (e.g., attenuated ETEC strains or live, attenuated bacterial carriers expressing ETEC proteins). During this time, we should be able to determine if a safe and efficacious vaccine against an organism with many survival tools such as; colonization factors, toxins and other yet to be identified virulence factors can be identified that are broadly protective.

Key issues • Achievement of a broadly protective enterotoxigenic Escherichia coli (ETEC) vaccine will require a better understanding of the infectious process in the intestinal tract, with emphasis on protective immune responses and a broader knowledge of the roles played by each virulence factor. • The predictive correlates for human immunity must be established, for example, antitoxin antibodies. The importance of the immune responses to toxins should be evaluated further to determine whether protection can be achieved in the absence of other immune responses, or whether immunity to other virulence components, such as colonization factors, add to long-term protection. • Strategies for developing new ETEC vaccines, including live, attenuated ETEC, bacterially vectored antigens, plant-based antigen delivery, transdermal delivery, microencapsulation and passive immunity hold promise for this area. • Development of relevant animal models is the key to facilitating a more rapid pace in evaluating new vaccine strategies. This will allow us to effectively narrow the number of candidates tested in clinical trials.

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ETEC vaccines

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29

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ETEC vaccines

Websites 101

102

103

104

105

Chiron Corporation www.chiron.com Accessed September, 2004 The US Army Medical Materiel Development Activity www.usammda.army.mil Accessed September, 2004 AVANT Immunotherapeutics, Inc. www.avantimmune.com Accessed September, 2004 Microscience www.microscience.com Accessed September, 2004 ImmunCell Corporation www.immucell.com Accessed September, 2004


106

Prodigene www.prodigene.com Accessed September, 2004

107

Bioport www.bioport.com Accessed September, 2004

108

Acambis www.acambis.com Accessed September, 2004

109

IOMAI Corporation www.iomai.com Accessed September, 2004

203

Affiliations •

•

Patents 201

Ghen Corporation, JP. EP-0 225 254 B1 (1986).

202

Medipharm, CX, s.r.o. EP-0 955 061 A1 (1999).

Biosynexus, Inc. and Walter Reed Army Institute of Research. WO 02/081653 A2 (2002).

•

Donata R Sizemore, PhD, PMP Senior Scientist III, AVANT Immunotherapeutics, Inc., St. Louis, MO, USA Tel.: +1 314 983 9050 Ext. 109 Fax: +1 314 983 9077 Kenneth L Roland, PhD Senior Scientist III, AVANT Immunotherapeutics, Inc., St. Louis, MO, USA Tel.: +1 314 983 9050 Ext. 108 Fax: +1 314 983 9077 Una S Ryan, PhD, OBE President and CEO, AVANT Immunotherapeutics, Inc., Needham, MA, USA Tel.: +1 781 433 0771 Fax: +1 781 433 0262

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