1. Field of the Invention
The present invention relates generally to the field of microbiology, immunology and vaccine development. More specifically, the present invention discloses flagellin and secreted two-partner secretion proteins as target antigens in the development of vaccines for flagellated human pathogens such as enterotoxigenic escherichia coli (ETEC).
2. Description of the Related Art
Infectious diarrhea is a leading cause of mortality in developing countries accounting for more than one fifth of all deaths under the age of 5 years (Kosek et al., 2003). In this population, enterotoxigenic Escherichia coli (ETEC) commonly cause diarrhea with annual estimates of diarrheal illness approaching one billion episodes (WHO, 2006), resulting in hundreds of thousands of deaths. Enterotoxigenic Escherichia coli cause an estimated 500,000 deaths per year in young children and are perenially the most common causes of diarrheal illness in travellers (Sack, 1990; Jiang et al., 2002) and soldiers deployed to developing countries (Hyams et al., 1991; Bourgeois et al., 1993). Enterotoxigenic Escherichia coli have also emerged in several recent large-scale outbreaks in the United States (Beatty et al., 2006; Daniels, 2006).
Illness caused by ETEC may range from mildly symptomatic to severe, life-threatening cholera-like diarrhea (Sack et al., 1971; Vicente et al., 2005). In the classic paradigm for enterotoxigenic Escherichia coli pathogenesis, these organisms utilize fimbrial colonization factors (CFs) to colonize the intestine where they elaborate heat labile (LT) and/or heat stable (ST) enterotoxins that elicit watery diarrhera.
Painstaking ETEC vaccine development efforts, which have largely targeted colonization factors (CFs) (Boedeker, 2005), have been hindered by the fact that these antigens are neither cross-protective nor well-conserved, and in some cases identified only in a minority of strains even after diligent searches for more than 20 colonization factors that have been discovered to date (Peruski et al., 1999). Similarly, the toxins produced by enterotoxigenic Escherichia coli are either poorly immunogenic in the case of the heat stable toxin (ST) or appear to offer limited protection despite robust antigenicity in the case of the cholera toxin-like heat labile toxin (LT) (Clemens et al., 1988). This later phenomenon may be explained by the ability of enterotoxigenic Escherichia coli to deliver its toxin payload in a fashion that circumvents immune neutralization (Dorsey et al., 2006).
Recently EtpA, identified in a search for novel ETEC antigens (Fleckenstein et al., 2006) is a large glycoprotein secreted by the prototypical enterotoxigenic Escherichia coli H10407 strain that was originally isolated from a patient in Bangladesh with severe cholera-like diarrheal illness (Evans et al., 1975). EtpA is a member of a large family of potential virulence proteins that are secreted by two-partner secretion (TPS) systems (Jacob-Dubuisson et al., 2001). In two-partner secretion systems, one partner (generically referred to as TpsB proteins) transports a second secreted (TpsA) protein through the bacterial outer membrane. Although EtpA, like other TPS exoproteins, promotes adhesion to epithelial cells, its precise mechanism is not clear.
Furthermore, many other elements of the pathogenesis of enterotoxigenic Escherichia coli remain poorly understood. One aspect that has not been sufficiently explored is the role played by flagella. E. coli are serotyoed based on their lipopolysaccharide (O) and flagellar (H) antigens, and enterotoxigenic Escherichia coli flagella have largely been relegated to serotyping with virtually no investigation of a role in pathogenesis (Wolf, 1997). However, flagellar organelles from a number of Gram-negative pathogens play critical roles in pathogenesis by promoting adherence to epithelial cells (Giron et al., 2002; Yao et al., 1994), formation of biofilms (Pratt and Kolter, 1998) and colonization (Wright et al., 2005; Lane et al., 2005; Correa et al., 2000). It has also been recently demonstrated that production of intact flagella and motility are essential for the successful delivery of heat-labile enterotoxin to epithelial cells by enterotoxigenic Escherichia coli (Dorsey et al., 2006).
Flagella are complex extracellular structures each composed of approximately 20,000 molecules of the protein flagellin (FliC) arranged in a cylindrical lattice of 11 helical protofilaments. The FliC monomer is thought to travel down the nascent cylinder in an unfolded state to the distal end (tip) where the flagellar cap protein, FliD (Yonekura et al., 2000), directs assembly of flagellin monomers into the growing flagellum. Flagellin has several major domains: the central variable domain projects outward along the surface of the flagellar shaft and accounts for the antigenic variation on which the “H” serotyping is based; conserved amino and carboxy terminal regions participate in interactions between individual subunits and face the inaccessible inner core of the flagellar shaft once incorporated into the cylinder (Yonekura et al., 2003).
Thus, prior art lacks complete understanding of the pathogenesis of enterotoxigenic Escherichia coli and is deficient in the knowledge of antigens of enterotoxigenic Escherichia coli that can be targeted to prevent enterotoxigenic Escherichia coli infection. The current invention fulfils this long-standing need in the art.