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Recent studies have confirmed older observations that the enterotoxins enhance enteric bacterial colonization and pathogenicity. How and why this happens remains unknown at this time. It appears that toxins such as the heat-labile enterotoxin (LT) from Escherichia coli can help overcome the innate mucosal barrier as a key step in enteric pathogen survival. We review key observations relevant to the roles of LT and cholera toxin in protective immunity and the effects of these toxins on innate mucosal defenses. We suggest either that toxin-mediated fluid secretion mechanically disrupts the mucus layer or that toxins interfere with innate mucosal defenses by other means. Such a breach gives pathogens access to the enterocyte, leading to binding and pathogenicity by enterotoxigenic E. coli (ETEC) and other organisms. Given the common exposure to LT+ ETEC by humans visiting or residing in regions of endemicity, barrier disruption should frequently render the gut vulnerable to ETEC and other enteric infections. Conversely, toxin immunity would be expected to block this process by protecting the innate mucosal barrier. Years ago, Peltola et al. (Lancet 338:1285-1289, 1991) observed unexpectedly broad protective effects against LT+ ETEC and mixed infections when using a toxin-based enteric vaccine. If toxins truly exert barrier-disruptive effects as a key step in pathogenesis, then a return to classic toxin-based vaccine strategies for enteric disease is warranted and can be expected to have unexpectedly broad protective effects.
Enterotoxigenic Escherichia coli (ETEC) infection and cholera are toxin-mediated enteric diseases that are leading causes of morbidity and mortality worldwide (25, 32, 36). Although cholera is generally less frequent than ETEC infection, both cholera and ETEC infection can result in severely dehydrating diarrheal disease (73). While there is a cholera vaccine that confers protection against cholera for up to 3 years (11), there is, by contrast, currently no licensed vaccine to protect humans against ETEC disease. The establishment of a vaccine against ETEC is a major unmet need for both travelers and children in the developing world, as ETEC disease creates an extensive disease burden in both children (94) and travelers (85) and ranks as a top global health priority.
The pathogenesis of cholera and that of ETEC disease are quite similar. Each begins with the ingestion of inocula, followed by elaboration of toxin, bacterial colonization, induction of profuse watery diarrhea, and dissemination of organisms back into the environment. The many similarities between cholera and ETEC disease are striking (13, 84), especially in regard to the centrality of the toxins to their pathogenicity. Vibrio cholerae secretes cholera toxin (CT), whereas ETEC elaborates both the heat-labile enterotoxin LT and the heat stable toxin ST. Both LT and CT are 86 kDa A:B5 ADP-ribosylating exotoxins that are functionally and structurally homologous (ca. 80%), highly immunogenic, and characterized by high levels of cross-neutralizing immunity. From a vaccine development perspective, this high level of homology may allow cholera and LT-containing ETEC disease to be addressed simultaneously in a single vaccine (10). However, ETEC disease differs from cholera in that many strains also produce the diarrheagenic heat-stable toxin (ST), a small, poorly immunogenic peptide. The question that remains is whether toxin-based vaccine protection against ST+ ETEC strains can be achieved (12, 69). The presence of ST toxin-containing strains complicates ETEC vaccine development, especially given that in some geographic regions ST-containing strains appear to be predominant (36, 72). However, efficacy data from vaccine trials have suggested that LT toxin-neutralizing immunity protects against LT+ ETEC and to some degree against both ST+ ETEC and non-ETEC organisms (9, 10, 68), indicating that LT toxin immunity may have unexpectedly broad effects. The goals of this review are to consider the role of LT toxin in ETEC pathogenicity in the context of the innate mucosal defenses and to consider how LT immunity may be protective in this context, leading to vaccine protection against non-LT-containing organisms.
The intact, innate gut defenses, a combination of mechanical, chemical, environmental, and innate and adaptive immune effectors, are generally sufficient to ward off microbial infections. Consequently, organisms causing enteric diseases have developed a series of strategies to penetrate, disrupt, or modify the innate mucosal barrier, rendering the gut wall susceptible to subsequent pathogen colonization or entry (65). CT and the heat-labile (LT) enterotoxins are exotoxins that have long been known to cause the secretory diarrhea characteristic of these diseases, but the survival advantage the microbe gains by producing these toxins is debated. It has long been suggested that CT aids efficient pathogen colonization in the intestine, and more recent studies have confirmed that LT toxins enhance enteric bacterial colonization and pathogenicity (1, 2, 15, 44). As enteric pathogens must overcome the innate mucosal barrier to cause disease, we suggest here that toxins such as LT and CT are specifically produced to overcome the innate mucosal barrier as a key step in enteric pathogen survival. Given the common exposure of humans visiting or residing in regions with CT-containing vibrios and LT-containing ETEC (see below), one would predict that toxin exposure and subsequent barrier disruption would frequently render the gut generally vulnerable to enteric infection as the toxin-mediated breach in the mucosa is exploited. The corollary is that elimination of localized toxin effects through antitoxin neutralizing immunity could be expected to interrupt a strategy central to the survival of ETEC and possibly other coinfecting enteric pathogens.
The surface of the gut, like the skin, is organized with functional layers that serve to effectively defend the host against the microbial world. The external surface of the gut cavity is coated with mucus (derived from goblet cells and submucosal glands), whose thickness is specific to the gut segment and to which a large, highly individual but relatively stable and complex permanent flora is attached (31). The mammalian gut becomes colonized early in life by these commensal bacteria that thrive in the mucosal microenvironment with progressively denser populations as they move from the small bowel to the colon. Their presence and complex interaction with the gut-associated lymphoid tissue contribute to the normal healthy function of the digestive system and the gut immune system. Destruction of normal flora, such as by the use of antibiotics, is known to enhance susceptibility to organisms of normally low pathogenicity, such as Clostridium difficile. The normal presence of this enormous and diverse collection of bacteria (31) represents a key barrier for pathogens through competition for nutrients and other factors.
The goblet cell-derived, gel form mucus (containing mucins) coating the intestine holds the normal flora and serves as a size-selective sieve excluding bacteria and viruses from the enterocyte surface (24). The goblet cell-derived mucus also plays a role in host defense through specific binding to organisms via mucins (53), for example, Salmonella enterica serovar Typhimurium (93), followed by mechanical clearance. Secretory immunoglobulin A (sIgA) and secreted enteric antimicrobial peptides (AMPs), either constitutively secreted or produced in response to bacterial signals (64), are concentrated in the upper mucus surface layer to provide an added layer of active defense (61). Cathelcidin (LL-37) and B-defensin 1 (HBD-1) are specific and relevant AMPs that have antimicrobial activity against a broad range of enteric pathogens (80). Membrane-anchored enterocyte surface mucin glycoproteins (glycocalyx) extend 100 to 500 nm beyond the cell surface and also bind to the surface of pathogens (53) (for example, Campylobacter jejuni), blocking their pathogenic effects by acting as a releasable decoy ligand for bacterial surface adhesion (57). Together, these layered host defenses appear to cope well with exposure to modest numbers of most pathogens by excluding, clearing, or killing them. Conversely, the relatively small toxins LT and CT readily move through the innate mucosal barrier to induce fluid secretion, as made clear by studies using oral toxin challenge (48, 50, 62, 79, 97).
The small intestinal enterocyte is lined with a brush border consisting of a dense wall of microvilli whose high surface area is designed for nutrient digestion and absorption. The brush border has multiple focal collections of membrane glycolipids, so-called lipid rafts, which stabilize digestive enzymes and thus are the normal route for efficient uptake of nutrients. In these membrane microdomains, glycosphingolipids account for >30% of the total lipids (8). These glycosphingolipids are additionally cross-linked by lectins to make the microvillar rafts stable (17). Although microvillar lipid rafts normally function as a special portal for nutrient absorption, this same portal is the key target for the entry and binding of a wide variety of pathogens, including fungi, parasites, bacteria, and viruses, and their toxins (19, 55, 76, 81, 87). For example, by targeting the lipid rafts, organisms such as S. enterica serovar Typhimurium and Shigella flexneri avoid degradative pathways (lysosomes) and simultaneously hijack intracellular signaling, resulting in membrane ruffling and increased macropinocytosis of organisms. Common enteric pathogens that exploit the lipid raft for survival include C. jejuni (96) and S. enterica serovar Typhi (5, 27). Significantly, the lipid rafts are also the molecular target for toxin binding by LT and CT (33). The antigen-sampling, follicle-associated epithelium (M cells) is also an important portal for enteric pathogen entry (43). Although the relative vulnerability of the gut mucosa to pathogen entry via the M cell versus the enterocyte lipid raft remains to be defined, the lipid rafts represent a key point of vulnerability exploited by enteric pathogens and are special targets for the enterotoxins.
While the microenvironment of the enterocyte brush border is coated with a wide variety of sIgA, IgG, and IgM antibodies, a large number of intestinal antibodies are self-antiglycosyl antibodies, primarily IgG, that bind to the glycosphingolipid component of the lipid rafts (35). The observations that the antiglycosyl antibodies make up 1% of the total circulating IgG and IgM antibodies in humans (26) and that plasma cells synthesize and actively transport IgG to the apical surface of the enterocyte via the FcRn transporter (18, 42) at a rate four times that of IgA (34) together suggest that the presence of lipid raft IgG antibodies is critical for keeping microbes from using the lipid raft as an easily accessed portal of entry. Similarly, this biological commitment to the synthesis of antiglycosyl antibodies reinforces the notion of gut vulnerability localized at the lipid raft. Hansen et al. (34) have proposed that these antibodies act essentially as “guardians of the lipid rafts,” blocking the binding of pathogens and toxins through steric hindrance or competition for binding sites (Fig. (Fig.11).
The microbial production of both CT and LT is most likely triggered by elements of the microenvironment of the gut, leading to a well-orchestrated production of the toxins and the factors required for binding and subsequent colonization. These events are well characterized for V. cholerae, which has an orchestrated expression of several genes as it transitions from its environmental reservoirs to the human host (56). Gut exposure triggers downregulation of genes encoding a type IV pilus, the mannose-sensitive hemagglutinin pilus that aids V. cholerae's persistence in aquatic environments but causes clearance of bacteria by host immune defenses. Conversely, gut exposure triggers upregulation of the toxin and genes encoding the toxin-coregulated pilus (TCP) that is required for intestinal colonization. The antagonistic effects, clearance versus colonization, are resolved transcriptionally by the regulator ToxT, which represses msh genes (clearance) while activating TCP genes (colonization) during infection (39). The coordinated proteolysis of the mannose-sensitive hemagglutinin pilus by the ToxT-regulated prepilin peptidase TcpJ is a regulatory mechanism in V. cholerae that coordinates this organism's transition from the environment to the host (39, 40, 59). The expression of TCP and CT in a spatiotemporal manner within the small intestine (78) suggests that the enterocyte environment is actively modified by the toxin, setting the stage for vibrio binding and colonization on the enterocyte surface via TCP. ETEC organisms likely follow a similar pattern of gene expression in response to environmental stimuli to render the organism suitable for colonization, although little is known regarding in vivo triggers for LT toxin expression at this time (20, 83, 92).
CT and LT are known to selectively bind to the GM1 ganglioside (37, 38, 45). GM1 is a ubiquitous cell membrane component preferentially located in the exoplasmic leaflet on the apical membrane and concentrated together with sphingomyelin and cholesterol in ordered microdomains (i.e., lipid rafts) (33). LT/CT binding to GM1 occurs with almost unprecedented affinity for biological molecules (7, 60), and thus it must be presumed that toxin-GM1 targeting is tied to microbial survival.
At the molecular level, CT and LT toxins interact with the GM1 in lipid rafts via the B subunit of their A:B5 homopentameric structure (16, 33). The B subunit's portion of the holotoxin avidly binds to the GM1 ganglioside, primarily or exclusively in the lipid rafts, and the toxin is endocytosed into the Golgi apparatus and moves into the endoplasmic reticulum, where the A-1 subunit is actively extruded into the cytosol (49). Via G proteins, ADP ribosylation leads to profoundly increased levels of cyclic (cAMP) and many subsequent downstream activation events, the most clinically apparent of which is translocation of the cystic fibrosis transmembrane conductance regulator to the enterocyte surface (29, 78), resulting in increased Cl− ion flux and brisk fluid secretion. Although enterocyte gross histological structure is unaltered by cholera and ETEC disease, other effects are apparent. In intestinal biopsy samples from cholera patients and rabbit models using LT (63), goblet cells are empty and markedly increased mucus is seen in the lumen (67). Some of the diarrheagenic effects of CT also appear to be due to stimulation of the enteric nervous system by CT (21). With respect to diarrhea, exposure to LT or CT in the immunonaïve may manifest itself in many liters of diarrheal stools, result in mild or subclinical infections, or be completely asymptomatic (46, 73, 94). In fact, passive and active surveillance studies indicate that the majority of infections are entirely asymptomatic (46, 72, 94). Given the high rate of symptomatic ETEC diarrhea in travelers to these regions, LT exposure must be common indeed. Animal models indicate that significant fluid secretion can occur in the gut after LT exposure without diarrhea (97), reinforcing the notion that field exposure to LT may result in fluid flux from the enterocyte that does not manifest itself as frank diarrhea.
At the mechanistic level, we suggest that toxin-mediated fluid secretion itself either mechanically washes away the mucus layer or hydrates and therefore weakens the barrier effects of the mucus layer and its constituents described above. By such means, local toxin effects on the enterocyte disrupt the innate mucosal barrier, leaving the gut surface vulnerable to subsequent colonization (Fig. (Fig.2).2). We suggest that the loss of the normal flora, mucus, defensins, and antibodies exposes the enterocyte for further colonization, production, and toxin binding, leading to cascading pathogen effects and frank diarrhea. Alternatively or in parallel, the CT and LT toxins may depress more specific barrier defenses, such as the AMP-driven killing of pathogens, through the downregulation of LL-37 and HBD-1 (6), thus weakening the innate mucosal defenses. Both CT and LT transcriptionally downregulate both LL-37 and HBD-1 (which are normally involved in broad-spectrum pathogen killing) via intracellular signaling pathways (6). Of important note, while LT demonstrated clear downregulation of AMP expression in the form of either a toxin or a clinical isolate, neither the ST toxin nor ST ETEC clinical isolates had the downregulating effects seen with LT (6). Suppression of LL-37 and/or HBD-1 has been observed in Shigella dysenteriae (41), Neisseria gonorrhoeae (3), and Cryptosporidium parvum (98), supporting the notion that AMP suppression is a general mechanism of pathogenicity used by mucosal pathogens to favorably alter the host environment for colonization. The theme of toxin impairment of innate immunity has also been described in the context of the mucosal pathogen Bacillus anthracis, whose lethal toxin downregulates interleukin-8 in the lungs, where the production of this proinflammatory cytokine involved in polymorphonuclear neutrophil recruitment would normally be a key step in pathogen killing (75). Finally, other barrier-disruptive mechanisms may come into play that extend below the innate intestinal mucosal barrier, as suggested by data obtained with mice, where it was shown that CT increases intestinal permeability to antigens (54). However, the intense, cAMP-dependent secretion of fluid induced by the toxin warrants consideration as the most important innate mucosal barrier-disruptive event.
Although most ETEC and cholera infections are asymptomatic, we propose that the common event of subclinical toxin exposure (72, 94) results in focal areas of innate intestinal barrier disruption (Fig. (Fig.2),2), exposing the microvillar surface for organism colonization and/or with focal suppression of AMP pathogen killing. On subsequent pathogen ingestion, the initial disruption allows colonization by ETEC, leading to cascading barrier-disruptive effects and progression from focal collections of organisms to a carpet of pathogens bound to the intestinal wall (66). From the organisms' standpoint, survival depends on the highly infrequent event of ingestion, as the pathogens' life cycle relies on successful attachment to and colonization of the small intestine, where they proliferate and set the stage for further distribution in the environment. Toxin-mediated innate mucosal barrier disruption, therefore, seems to be a key initial event for survival. Other factors may contribute to pathogen survival, such as the motility of ETEC and V. cholerae (95), or in the latter, the organism's production of the mucinase HapA (82), but the preclinical data suggest that the initial stages of infection are highly augmented by the presence of toxin. The steps involved in subsequent ETEC enterocyte binding and colonization are complex, not fully elucidated, and reviewed elsewhere (77, 91).
The observation that toxins condition the enterocyte for enhanced colonization was put forward more than 20 years ago by Pierce et al. (71). In the search for a live, attenuated cholera vaccine, V. cholerae strains lacking either the A or both the A and B genes (A− B+ or A− B−) were compared in rabbit models with fully virulent V. cholerae expressing CT. In these studies, it became clear that organisms expressing the intact CT had superior efficacy in the rabbit challenge model due to more effective colonization of the gut, creating more effective immunity (70). More importantly, vibrios containing the full toxin genes were significantly more efficient at colonizing the gut, and toxin-negative strains could be made to colonize the gut similarly by preconditioning the gut through preexposure to CT (71). Similarly, in studies using a respiratory pathogen with toxin-mediated pathogenicity, Bordetella pertussis, the absence of the adenlyate cyclase toxin was shown to impair colonization and infection (30).
Preclinical challenge studies have shown that exposure of enterocytes to LT, similar to the effects of CT, conditions the gut for initial colonization by ETEC and is a key step in the pathogenesis of ETEC disease (1, 2, 99). Studies with a pig enterocyte cell line with glycocalyx-bound mucin have shown that LT holotoxin, LT containing outer membrane vesicles, and LT-containing ETEC organisms condition the enterocyte surface for enhanced adherence by LT+, LT+ ST+, and LT− ST+ ETEC field isolates, as well as toxin-negative isogenic ETEC and non-ETEC E. coli (44). The enhanced binding was dependent on intact LT enzymatic activity and was mediated by cAMP, the primary intracellular mediator of LT toxin-mediated cellular activation, illustrating the centrality of LT to colonization and suggesting that the cAMP activation of the enterocyte confers an important survival advantage on ETEC organisms. LT gut preconditioning has also been shown to enhance the virulence of other non-ETEC pathogens (15), and together these data suggest that LT renders the gut generally susceptible to enteric pathogens.
These data are in line with the clinical finding that colonization by ETEC is associated with a significant risk of contracting diarrhea (94). In human travelers, high ETEC isolation rates are found in stools of asymptomatic individuals of all ages. In countries of endemicity, it is estimated that 50 million children aged 0 to 4 years are colonized with ETEC at any time (94). The high prevalence of asymptomatic cholera (46) or LT+ ETEC infections (94) might suggest that LT+ ETEC is not pathogenic. It is clear that LT+ ETEC (51) and the toxin itself are diarrheagenic (48, 62). We suggest here that asymptomatic exposure, a common event for travelers and children, is pathogenic in that it preconditions the gut for more efficient pathogen colonization, making the nonimmune host more prone to disease. However, the innate mucosal barrier disruption is likely a transient effect. Enterocytes are formed in the crypt, migrate up the villus, and are sloughed from the villus tip over several days (89); mucus, antibody, and defensin secretion is ongoing and can restore the mucosal barrier. Yet, as repeated ETEC exposure in regions of endemicity is very common, the transient nature of the toxin effects is counterbalanced by frequent exposure, creating a continuum of vulnerability.
In a recent field trial, Frech et al. (23) found that immunization with LT toxin in a transcutaneous patch led to protective efficacy against clinically significant diarrhea. The protective effects appeared to extend to ST+ ETEC and other pathogens, as well as LT+ ETEC. This finding is in concert with data obtained by Peltola et al. (68) by using the homologous cholera toxin B subunit (CTB)-based oral cholera vaccine. In this study, the expected protective efficacy against LT+ ETEC was accompanied by high levels of protective efficacy against mixed infections and mixed ETEC-Salmonella infections and reduction in ST-containing ETEC disease. The data from a large field trial using the same vaccine had similar surprising results, where the expected protective efficacy against LT+ ETEC was surpassed by the protection against LT+/ST+ disease (10). More recently, Bourgeois et al. have shown that CTB-based oral vaccination had both the expected effects against LT+ ETEC and a significant effect against C. jejuni and S. enterica serovar Typhi in a subset of subjects with high levels of antitoxin immunity (4). It may be of significance that the latter vaccine, where only modest protective effects were seen, contained purified recombinant CTB whereas earlier studies with robust cross-protection contained the purified form of CTB containing a small percentage of holotoxin (88). The promising results seen in initial field studies need to be extended in a well-controlled and powered field trial to confirm the efficacy of LT toxin immunity.
Taken together, these data suggest that antibodies that neutralize LT toxin have effects that extend beyond blocking the pathogenicity of LT containing ETEC alone. We suggest that antitoxin antibodies against LT prevent the disruption of the host defense mechanisms, a breach in the normal host defense that can be used by both ETEC and other pathogens. The corollary is that anti-LT antibodies neutralize LT, preventing the binding of LT to its target ligand, GM1, in the lipid rafts, thus preventing the secretory effects of LT and subsequent washing away or disruption of the mucosal barrier (Fig. (Fig.1).1). Ironically, it is possible that anti-LT IgG may be the most critical effector molecule and IgG is the primary antibody “guarding” the lipid raft (35). The importance of IgG is bolstered by passive-transfer experiments using anti-LT serum, which protects against intestinal toxin challenge (97). Thus, protection can be effective either in the “preconditioning” (asymptomatic) phase of exposure or during active exposure to larger diarrheagenic inoculums of ETEC (58). We suggest that the anti-LT antibodies can have important effects on maintenance of the integrity of the gut wall barrier in exposed individuals and that anti-LT immunity might thus prevent disease caused by LT+ ETEC, ST+ ETEC, and non-ETEC pathogens that exploit innate mucosal barrier disruptions.
With regard to the question of how ETEC organisms may have a sustained toxin-based survival strategy, it appears that ETEC disease, like other toxin-mediated diseases (e.g., C. difficile infection, tetanus), can infect without conferring significant protective antitoxin immunity, allowing repeat infections. Experimental human homologous ETEC challenge-rechallenge experiments readily confer protection after a single infection, likely due to the strong strain-specific, antilipopolysaccharide immunity or anti-colonization factor immunity (51, 52). By contrast, single experimental and natural infections with LT+ ETEC do not appear to confer protection based on toxin immunity (51, 74), whereas multiple infections or toxin exposure clearly do induce protective toxin immunity (62, 86), suggesting that the toxin immunity reaches a protective level after repeated exposure. Incomplete protection against ETEC is also seen in the face of preexisting, low-level anti-LT immunity in travelers, who must also have naturally induced anti-LT antibodies (22). Toxin and other ETEC antigen antibody responses derived from natural protection and vaccines wane over time (10, 90), data reinforced by the cholera-like ETEC disease seen in Bangladeshi adults (73). Our observations in challenge trials (58) and U.S. travelers to Mexico indicate that while LT immunity is generated after single LT+ ETEC infections, it is not robust compared to LT antibodies generated by patch immunization (14, 28, 58; unpublished data). It seems that LT+ ETEC infections confer very little immunity to the key and highly conserved pathogenic factor LT, allowing repeated use of toxin-mediated innate mucosal barrier disruption by the organism to provide for efficient colonization, and ensuring the continued utility of this survival advantage for the organism.
We suggest that robust antitoxin immunity is needed to neutralize the toxin and block toxin-mediated effects that disrupt the innate mucosal barriers, similar to diphtheria, where a certain level of antitoxin immunity is needed for protection (47). With respect to ETEC disease, the guardian antiglycosyl antibodies found on the lipid rafts that normally protect this region of the enterocyte targeted by LT are low affinity and in disease, these antibodies are displaced by the high-affinity binding of GM1-binding toxins (16, 33, 38). Protective antitoxin immunity must neutralize LT prior to binding to the lipid raft GM1, and the antitoxin antibodies must be both high titer and ubiquitous to provide clinical effects. As lipid raft protection is primarily IgG mediated and indicates lumenal IgG transport, then anti-LT IgG serum levels may provide the most reliable measure of protective immunity at the site of greatest vulnerability.
In summary, we suggest that the toxins LT and CT are central to the life cycles of ETEC disease and cholera and that antitoxin antibody-mediated protection against the toxin-mediated disruptive preconditioning of the gut's innate mucosal barrier may explain serial observations in the field showing the protective effects of toxin immunity that extend beyond LT+ ETEC (4, 10, 23, 58, 68). Furthermore, we suggest that “preconditioning” of the mucosal barrier by toxin or similar factors that can alter the host innate mucosal barrier for efficient entry may be a general biological mechanism employed by pathogens. Further studies to explore these mechanisms are needed to yield a clear picture of the role of enteric toxins in pathogenesis, but the assumption that toxins act in this manner should direct mechanistic investigations and the search for suitable enteric vaccine antigens. The role of mucus barrier disruption and AMP suppression should be explored in the context of disease and colonization models and may provide insight both for ETEC and concepts of general pathogenicity. In the context of toxin-mediated enteric disease, we suggest that toxin-based antigens should be considered preeminent, as their safe and effective delivery may lead to major advances in protective efficacy for this important global health problem.
G.G. is an employee of and a stockholder in Intercell USA, which is commercializing the LT ETEC patch.
Thanks to Matt Chansky of Momentum 18 for help with the figures and Wanda Hardy for manuscript preparation.
Gregory M. Glenn was born in Spokane, WA, and grew up mostly overseas in the shadow of tracking stations used for U.S. space exploration. He obtained his Biology-Chemistry degree from Whitman College, Walla Walla, WA, and his M.D. degree from Oral Roberts University in Tulsa, OK, and completed his pediatric residency at Madigan Army Medical Center and a Medical Research Fellowship at the Walter Reed Army Institute of Research, where he followed his interests in vaccine adjuvants, delivery systems, and enteric diseases. He founded IOMAI, a biotech company focused on vaccine delivery through the skin, and is currently the Senior Medical Officer at Intercell and an Associate at the Johns Hopkins School of Public Health. Pondering his father's mission at JPL to search for interplanetary life, and the spectacular success of the exploration program that failed to reach that goal, has inspired him to aim high and enjoy the view.
David H. Francis obtained his B.S. (1971) and M.S. (1974) degrees from Brigham Young University and his Ph.D. in microbiology from the University of Missouri in 1978. Currently, he is the director of the Center for Infectious Disease Research and Vaccinology at the South Dakota State University in Brookings. He has a long-standing interest in host-pathogen interactions, disease mechanisms of E. coli enteric diseases, and the role of toxins and E. coli intestinal receptors in pathogenesis. He is also an expert in the prevention of enteric disease and dissemination of food-borne pathogens.
E. Michael Danielsen was born in 1953 in Copenhagen, Denmark, and obtained his M.Sc. in biochemistry from the University of Copenhagen in 1978. He spent a sabbatical in 1979 and 1980 at the Department of Biochemistry, University of Leeds, England, in John Kenny's laboratory, which at the time was an international hot spot for research on membrane peptidases. Returning to Copenhagen, he resumed his work on gut mucosal membrane biology at the Panum Institute in Hans Sjöström's and Ove Norén's laboratory. He obtained his doctorate in medicine in 1986 and became a faculty associate professor of biochemistry in 1991. Throughout his career, the gut brush border has been his main research interest. Off duty, he enjoys running with the local Danish Heart Society exercise club and plays decent left-handed tennis. With wife Gillian, he frequently retreats from the dark Scandinavian winters to enjoy life in their casa on the Spanish south coast.
Editor: J. B. Kaper
Published ahead of print on 8 September 2009.