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The leading bacterial cause of human gastrointestinal disease worldwide is Campylobacter. Bacterial gastroenteritis continues to pose a significant threat to the general public here in the United States and abroad for the foreseeable future. Campylobacter jejuni has also been associated with the neuropathological disease Guillain-Barre Syndrome. Increasing pressure to limit Campylobacter infection antemortem in Europe and increasing regulatory interest for this agent in the United States may signal future pressures for the U.S. industry. Vaccination against Campylobacter has had limited success using killed whole-cell or protein based vaccines because of the development of Guillain-Barre syndrome. There is a real need to find an effective and inexpensive Campylobacter vaccine to protect, decrease, or eliminate Campylobacter contamination in poultry. This project utilizes a novel approach in the development of vaccines by inserting multiple copies of linear epitopes, to be expressed on the cell surface of recombinant live attenuated bacterial vaccine vectors. <P>HYPOTHESIS: Oral live attenuated Salmonella or Lactobacillus vaccine vectors expressing Campylobacter epitope(s) will stimulate systemic, mucosal, humoral, and cell-mediated immune responses against multiple serovars of Campylobacter, thereby reducing poultry sources of this food-borne organism. <P>Specific objectives of this project are to: <ol> <LI> identify candidate cell surface polypeptides associated with Campylobacter infection using proteomics<LI> construct several live attenuated Salmonella strains that express Campylobacter epitopes and evaluate the immune response in chickens<LI> develop and evaluate Lactobacillus vaccine vectors expressing Campylobacter epitopes and their ability to elicit immune responses chickens<LI> evaluate the vaccine strains for protection against Campylobacter challenge. </OL><P>An effective vectored vaccine developed in chickens is expected to provide a possible means to greatly reduce the frequency of poultry-mediated food borne illness in the United States. With a bacterial-vectored vaccine, it may not be necessary to stockpile large quantities of vaccine because large amounts of orally-effective bacterially-vectored vaccine could be amplified very quickly and at low cost. Importantly for potential application to poultry, this vectored vaccine approach is expected to be effective by oral administration, an essential attribute for potential commercial adoption due to the cost of handling and individually administering vaccines to poultry.
NON-TECHNICAL SUMMARY: Food-borne bacterial pathogens causes significant public health problems. The bacteria species Campylobacter is a leading cause of food-borne diarrhea, with the primary source being contaminated poultry. In addition, Campylobacter infections have been associated with chronic diseases such as the neurological disease Guillain-Barr? Syndrome. Presently, we are quite limited with regard to our repertoire of safe and cost-effective vaccines against Campylobacter. Therefore, we have a real need to find an effective and inexpensive Campylobacter vaccine to protect, decrease, or eliminate Campylobacter contamination in poultry. The long-term goal of this research is development of safe, rapidly produced, orally effective, and low cost vaccines to protect humans against food-borne bacteria such as Campylobacter. Vaccine vectors that are able to elicit a good immune responses against Campylobacter will offer a promising alternative to existing vaccine strategies. This project utilizes a novel approach in the development of Campylobacter vaccines by inserting multiple copies of selected potent Campylobacter antigens, in combination with the helper molecules, to be obligatorily expressed on the cell surface of a recombinant live harmless bacterial carrier. Specific objectives of this project are to: 1) identify candidate cell surface antigens associated with Campylobacter infection using proteomics; 2) construct several live safe Salmonella strains that express Campylobacter antigens and evaluate the immune response in chickens; 3) develop and evaluate other even safer bacteria expressing Campylobacter antigens and their ability to elicit immune responses in chickens; 4) evaluate the vaccine strains for protection against Campylobacter challenge. <P>APPROACH: In specific objective 1, candidate cell surface polypeptides associated with Campylobacter infection will be identified using proteomics or selected from the literature [Omp18/cjaD (cj0113), cjaA (cj0982c), and ACE393 (cj0420)]. We will identify candidate epitopes that are cell surface proteins that elicit an immune response to Campylobacter on the same blot. Using an aqueous biotinylation reagent to label outer membrane proteins then using two-dimensional electrophoresis to identify both cell surface molecules and immunogenic epitopes in a Western blot. Cell surface proteins that are immunogenic and outer membrane proteins will be identified using Mass Spectrometry. Samples will be run using the standard configuration of the Bruker Reflex III or Ultraflex II MALDI TOF (or TOF/TOF)<P> In specific objective 2, we will construct several live attenuated Salmonella strains that express codon-optimized Campylobacter epitopes that were identified from the literature or specific objective 1. These constructs will be evaluated for their ability to invade, colonize, and persist in tissues and elicit immune responses against the Campylobacter epitopes in chickens. Attenuation of wild-type Salmonella strains will be achieved by deletion mutation of one or more virulence genes. Attenuated Salmonella strains will be transformed by using an overlapping and extension PCR product and the red recombinase system to chromosomally insert the chosen Campylobactor epitopes; a counter selection marker will be utilized to select for transformed clones. Cell surface expression of the Campylobacter epitopes and CD154 inserts will be confirmed with a simple antibody/antigen precipitation reaction and confirmed using DNA sequencing. <P>Specific objective 3, we will develop and evaluate Lactobacillus vaccine vectors that present linear Campylobacter epitopes for their ability to elicit immune responses chickens. The most promising Campylobacter epitopes identified in specific objectives 1 and 2 will be chromosomally inserted into selected LAB candidates in the same fashion as for the Salmonella; slpS gene. The recombinant LAB vaccine strains will be utilized for in vivo challenge studies.<P> In the final specific objective (objective 4), we will evaluate and compare Salmonella and Lactobacillus vector systems with selected epitope expression for the ability to protect chickens against infection from challenge with wild-type Campylobacter isolates. Chickens are highly susceptible to Campylobacter infections and the majority of commercial poultry are infected. In direct challenge studies, we will be able to directly compare the efficacy of these candidates for actually preventing infection in this highly susceptible model. Attenuated Salmonella or Lactobacillus candidate vaccine strains that have demonstrated the ability to elicit sufficient humoral and cell-mediated immune responses in specific objectives 2 and/or 3 will be selected for evaluation in challenge experiments to ascertain their ability to protect chickens against direct Campylobacter challenge.