Background: Traveler's diarrhea is a leading cause of disease and temporary incapacitation in deployed U.S. forces. Enterotoxigenic Escherichia coli (ETEC) is the principal etiology causing secretory diarrhea by expression of fimbrial colonization factors and enterotoxins. In volunteer challenge-protection studies, high level protection against disease was achieved by passive, oral supplementation with bovine milk immunoglobulins (BIgG) made by immunizing cows with the archetypal ETEC colonization factor antigen I (CFA/I). However, commercial development of a traveler's diarrhea BIgG product was discontinued several years ago when efforts to broaden coverage to include structurally divergent ETEC colonization factors were unsuccessful. Our recent insights into the structure and function of the large CFA/I class of ETEC fimbriae and the highly conserved nature of the class-specific minor fimbrial tip adhesin suggest an innovative approach for formulating a BIgG product with widened protective coverage for ETEC diarrhea.

Objectives/Hypothesis: We hypothesize that ETEC expressing CS17, an epidemiologically prevalent fimbriae belonging to the CFA/I class, will likewise be preventable by passive administration of BIgG with specific activity against CS17 fimbriae. Furthermore, we hypothesize that BIgG with specific activity against the minor tip-associated adhesive proteins of CFA/I and CS17 will confer passive protection against not only wild-type ETEC that express the homologous CF, but also against heterologous CFs with structurally related adhesins. These hypotheses are founded on two main bases: (1) that anti-adhesion is the operative protective mechanism of passively administered BIgG, and (2) that the minor adhesins of the CFA/I fimbrial class are more highly conserved than the immunodominant major fimbrial protein that makes up the fimbrial fiber.

Study Design: To test these hypotheses, we will first develop the processes for production of CS17 fimbriae, following existing procedures for production of CFA/I, and of the CFA/I and CS17 tip adhesins, using stabilization and expression technology developed by the Navy. These antigens will be used to immunize parturient cows, from which BIgG will be purified under food 'good manufacturing practice' (GMP) conditions and characterized. We will develop potency assays for these products that is based on either ELISA or on functional adhesion neutralization titers. Simultaneous with product manufacturing, we will expand the human ETEC diarrhea challenge model to include challenge with ETEC that express three other CFA/I class fimbriae (CS14, CS17, and CS19) that are needed to test the project hypotheses. These trials will be designed based on precedent studies in which the CFA/I-ETEC challenge strain has been firmly established. In parallel, we will expand development of a nonhuman primate disease model in Aotus nancymae to include challenge with CS14, CS17, and CS19, and validation, for the already established CFA/I-ETEC Aotus challenge model, that CFA/I is necessary for disease production. Lastly, in both the human and nonhuman primate models, we will assess the efficacy of each BIgG product in passive protection studies first against ETEC that express the homologous CF and then a heterologous CF.

Relevance: These studies will build upon the proven concept that passive administration of anti-CFA/I BIgG is highly protective against disease. The results are expected to provide the scientific basis and common pathway for development of a commercially viable, multivalent, food-based, anti-diarrheal supplement. Based on a successful outcome, we would expect to attract commercial support for a final multivalent BIgG formulation suitable for field use and expanded efficacy evaluation in pivotal studies for support of licensure. An anti-ETEC BIgG product will fill an important military need and have dual use in the civilian sector.