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The objective of this application is to use quantitative proteomic<P> protein profiling and an animal model of ETEC disease to discover and characterize novel antigens that are protective against ETEC. The primary need that motivates this research is that previous ETEC vaccine trials have not been effective and most have focused on proteins known as colonization factor antigens (CFs). Vaccination approaches focusing on CFs will not be effective, as the CFs do not exist in ETEC strains of current significance. An alternative strategy is demanded. <P>We will utilize our proven experience in ETEC, proteomics, and animal research to characterize novel ETEC vaccine candidates through discovery of novel surface-exposed ETEC proteins likely to be recognized by the host immune system. The proposed research is significant and innovative because it introduces a novel strategy for protective antigen discovery. <P>We have already discovered 26 surface-exposed antigens that are potential components of a new vaccine formulation. We are thus well positioned to test their efficacy and potentially commercialize antigens found to be protective against ETEC.
Non-Technical Summary: The primary need for this research is that previous ETEC vaccine trials have not been effective because they have focused on antigens that are unlikely to be highly conserved. An alternative strategy is demanded. The objective of this application is to use quantitative proteomic protein profiling and an animal model of ETEC disease to discover and characterize novel antigens protective against ETEC. <P> Approach: PCR products will be generated from chromosomal ETEC H10407 DNA and introduced into plasmid expression vectors to generate recombinant proteins fused to a hexahistidine (His) epitope. Proteins will be purified by passage over nickel-agarose matrices with high affinity for hexahistidine-containing proteins. Affinity-purified proteins (2 doses, 20 mg/dose, every 2 weeks) will be used to immunize adult BALB/c mice. After completion of their immunization regimen, mice will be challenged via dropwise administration of ETEC to their external nares. To quantify mouse survival as a function of ETEC challenge and immunization, we will observe mice every 4 h after challenge and record the % mortality as a function of time post-challenge. To quantify ETEC clearance, 1 mouse/day will be euthanized and the lungs will be aseptically removed, homogenized, serially diluted and plated for bacterial quantification. Antibodies from serum collected from mice 2, 4, and 6 weeks post-immunization will be measured with an enzyme-linked immunosorbent assay (ELISA).