This is a proof-of-concept project designed to evaluate a novel strategy to develop vaccines to protect against intracellular pathogens, taking advantage of a peptide that high a highly efficient cell delivery function. The peptide called Inv3 was derived from a sequence based on the most active domain of a protein cloned from Mycobacterium tuberculosis called mycobacterium cell entry protein or Mcep. In this project, this peptide sequence will be used to identify an oral vaccine candidate to protect against bacteremia in a murine model of Salmonella enteritidis infection. The ultimate goal of the project is to identify a set of protective polypeptides that can be used to prevent ovarian infection in chickens, in order to interrupt transmission of this very important human pathogen to the human population by eggs and egg products. The two specific aims are to 1) demonstrate that chimeric Salmonella polypeptides containing the Inv3 sequence at the N-terminus will enter mammalian cells in vitro and in vivo, and 2) identify a set of fusion proteins cloned from Salmonella enteritidis phage type 8 that is broadly cross-protective against bacteremia induced by a wide variety of S. enteritidis stains in a murine model of salmonellosis. An expression plasmid pInv3 designed to express any polypeptide with an N- terminus polyhistidine tag (6xHis) followed by Inv3, a 22-amino acid peptide, was constructed. In a preliminary study, the E. coli beta- galactosidase gene was cloned into pInv3 and its product was shown to efficient associate with HeLa cells, while the enzyme beta-galactosidase without the Inv3 at its N-terminus failed to associate with HeLa cells. The enzymatic activity was preserved. The observation demonstrated that it is possible to express any coding sequence downstream of Inv3 in the expression plasmid pInv3. Thus, in this project, the entire coding sequences of genome of S. enteritidis will be cloned in fragments of approximately 1000bp into pINV3 and expressed. These Inv3-fusion proteins will be pooled and used to impregnate biodegradable poly(lactice-co- glycolipid))PLG) microspheres, which will be delivered orally or subcutaneously to mice. These animals will be challenged with an oral dose of S. enteritidis, and evaluated for bacteremia. Sub-pools of polypeptides most protective against bacteremia will be divided into smaller pools, and used again to immunize mice. This will be repeated until a final pool of 5-10 polypeptides most protective for mice is identified. This set of polypeptides will then be used as a vaccine candidate for further cross- protection studies. This study is designed to test the feasibility of using this expression system to develop a new oral vaccine that may be used in poultry farms to reduce infection of eggs which are the major source of human S. enteritidis infections. The successful outcome of this project will not only identify such a vaccine candidate, but will serve as a proof-of- concept for a completely novel approach to vaccine development. This ability to deliver any polypeptide into cells enables one to screen the entire coding capacity of a genome of a pathogen for immunodominant or immunoprotective peptide sequences. The ability to identify such domains may open a completely new direction in identifying peptides that may be able to elicit protective CTL response against not just intracellular pathogens, but against tumor cells. S. enteritidis infection was selected as the initial mode to evaluate this strategy because of the ready availability of an animal model, knowledge of its genetics, and its importance in human and veterinary public health.
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