Conservation and Immunogenicity of Novel Antigens in Diverse Isolates of Enterotoxigenic Escherichia coli.

Background: Enterotoxigenic Escherichia coli (ETEC) are common causes of diarrheal morbidity and mortality in developing countries for which there is currently no vaccine. Heterogeneity in classical ETEC antigens known as colonization factors (CFs) and poor efficacy of toxoid-based approaches to date have impeded development of a broadly protective ETEC vaccine, prompting searches for novel molecular targets. Methodology: Using a variety of molecular methods, we examined a large collection of ETEC isolates for production of two secreted plasmid-encoded pathotype-specific antigens, the EtpA extracellular adhesin, and EatA, a mucin-degrading serine protease; and two chromosomally-encoded molecules, the YghJ metalloprotease and the EaeH adhesin, that are not specific to the ETEC pathovar, but which have been implicated in ETEC pathogenesis. ELISA assays were also performed on control and convalescent sera to characterize the immune response to these antigens. Finally, mice were immunized with recombinant EtpA (rEtpA), and a protease deficient version of the secreted EatA passenger domain (rEatApH134R) to examine the feasibility of combining these molecules in a subunit vaccine approach. Principal Findings: EtpA and EatA were secreted by more than half of all ETEC, distributed over diverse phylogenetic lineages belonging to multiple CF groups, and exhibited surprisingly little sequence variation. Both chromosomally-encoded molecules were also identified in a wide variety of ETEC strains and YghJ was secreted by 89% of isolates. Antibodies against both the ETEC pathovar-specific and conserved E. coli antigens were present in significantly higher titers in convalescent samples from subjects with ETEC infection than controls suggesting that each of these antigens is produced and recognized during infection. Finally, co-immunization of mice with rEtpA and rEatApH134R offered significant protection against ETEC infection. Conclusions: Collectively, these data suggest that novel antigens could significantly complement current approaches and foster improved strategies for development of broadly protective ETEC vaccines. Author Summary: Infectious diarrhea is one of the leading causes of death among young children in developing countries, and a major cause of morbidity in all age groups. The enterotoxigenic Escherichia coli contribute substantially to this burden of diarrheal illness, and have been a focus of vaccine development efforts for more than forty years following their discovery as a cause of severe diarrheal illness. The heat-labile, and/or heat stable enterotoxins that define ETEC are produced by a diverse population of Escherichia coli. This inherent genetic plasticity of E. coli has made it difficult to identify antigens specific to ETEC that are highly conserved. Therefore, identification of protective antigens shared by many ETEC strains will likely play an essential role in development of the next iteration of vaccines. [ABSTRACT FROM AUTHOR]