Human Campylobacter disease caused by the foodborne pathogens, Campylobacter jejuni and Campylobacter coli, is a huge public health problem globally. The economic burden of this disease, which ranges from mild self-limiting diarrhea to devastating neurological complications, in the United States alone is estimated at more than USD 8 billion annually. Broiler chickens harbor these pathogens in their gastrointestinal tracts (GITs) without developing disease, and are primary reservoirs considered to be the principal source of human infection. Hence, efficacious vaccines for elimination of these pathogens from the GITs of broiler chickens will significantly reduce incidence of human Campylobacteriosis; however, despite an urgent need, currently there are no efficacious chicken Campylobacter vaccines commercially available. Thus far, progress toward development of efficacious Campylobacter chicken vaccines has been slow. This is because Campylobacter species that infect broiler chickens and have human disease causing potential are a very diverse group, which dictates that efficacious vaccines comprise multiple components, including proteins that are shared by the vast majority chicken Campylobacter strains/isolates with human disease causing potential (conserved proteins). Also, the lack of innovative discovery tools for identification of conserved pathogen proteins is another contributory factor, since such conserved proteins are "hidden" by the pathogen during natural infection to escape host defenses. PELS is one such innovative tool that has the ability to identify conserved pathogen proteins (antigens). This antigen discovery technology makes clever use of the response of a host infected with a pathogen to identify components (proteins) with potential for vaccines against the same pathogen. In this project, PELS will be applied in an innovative manner, and coupled with a suite of computer-based algorithms for identification of proteins that are shared by diverse Campylobacter chicken and human strains/isolates. It is expected that this study will result in a panel of conserved Campylobacter proteins, a subset of which is likely to have potential for development of broadly protective Campylobacter chicken vaccines. Future studies in broiler chickens will examine if these proteins can function as vaccines. Such vaccines are anticipated to reduce the public health burden, as well as significantly reduce health care costs currently expended toward management of human Campylobacteriosis in the United States. OBJECTIVES: The goal of this project is to identify and define a panel of immunologically cross reactive (conserved) proteins of 20 Campylobacter jejuni and 10 Campylobacter coli strains/isolates, representative of those infecting chicken gastrointestinal tracts/causing human disease globally, using an innovative and powerful proteome mining tool for antigen discovery called Proteomics-based Expression Library Screening (PELS). PELS involves immunoaffinity capture of recombinant proteins expressed from genes on inserts within clones constituting inducible genomic DNA expressible libraries of a pathogen of interest using "bait" polyclonal antibodies (PAbs) from diverse sources. Specifically captured proteins are then identified using tandem mass spectrometry (LC-MS/MS). The specific strategy adopted in this study to accomplish the above objective involves the following tasks: The first task is the construction of highly functional, inducible expression libraries (Elibraries) using genomic DNA isolated from each of the 30 Campylobacter strains/isolates. The second task entails generation of high titer polyclonal antibodies (PAbs) against the recombinant proteome of C. jejuni strain RM1221, a sequenced chicken isolate, in an experimental host. The next task involves PELS-based sequential immunoaffinity capture of recombinant proteins expressed from Elibraries of each of the C. jejuni and C. coli strains/isolates using the anti-RM1221 proteome PAbs as "bait". This task will be followed by identification of specifically captured proteins of each strain/isolate by interrogation of the sequenced Campylobacter genomes in non redundant public databases using mass spectral data. The final task will be the compilation of a core panel of proteins conserved across all 30 Campylobacter strains/isolates using bioinformatics. TARGET DATES (Project duration: 05/01/2012 - 04/30/2013) Construction of Elibraries of 30 Campylobacter strains/isolates: 05/01/2012 - 10/30/2012 Generation of anti-RM1221 proteome PAbs: 05/15/2012 - 07/15/2012 PELS - Harvesting of recombinant proteins, Immunoaffinity capture: 07/15/2012 - 11/30/2012 PELS - Proteomics-based protein identification: 10/15/2012 - 02/28/2013 Bioinformatics for definition of a panel of conserved proteins: 03/01/2013 - 04/30/2013 EXPECTED OUTPUTS Expected outputs from this project include activities such as planning and execution of relevant experiments, strict adherence to established protocols pertaining to use and care of animals in this study, and the use of stringent statistical analysis of data to ensure relevance of experimental results. Also anticipated is the dissemination of data/results of this study at local/national events such as conferences either in the form of oral/poster presentation. Another expected output is a patent application to protect intellectual property, since this study involves the first application of a new technology to develop prophylactic measures against the pathogen in this study, and hence likely to yield a novel product. A final anticipated output is the publication of results in a peer-reviewed journal to disseminate results of this study to the scientific community. APPROACH: An innovative and sensitive proteome mining tool for antigen discovery called Proteomics-based expression library screening (PELS), coupled with bioinformatics-based algorithms will be used to define and compile a panel of proteins that are conserved across geographically diverse C. jejuni (n=20) and C. coli (n=10) strains/isolates of both avian and human origin. PELS involves immunoaffinity capture of recombinant proteins expressed from genes on inserts within clones constituting inducible genomic DNA expressible libraries of a pathogen of interest using "bait" polyclonal antibodies (PAbs) from diverse sources. Specifically captured proteins are then identified by querying relevant non redundant public databases with the mass spectral data obtained using tandem mass spectrometry (LC-MS/MS). The unique approach proposed in this first application of PELS-based mining of Campylobacter proteomes for identification of potential universal vaccine candidates will begin with the construction of inducible, functional expression DNA libraries (Elibraries) using genomic DNA from each of the 30 Campylobacter strains/isolates. Each Elibrary will be induced, and then recombinant proteins expressed by genes on inserts within clones comprising these libraries will be harvested and purified "en masse" using immobilized metal ion chromatography (IMAC). IMAC-purified recombinant proteins expressed from the Elibrary of a prototypical sequenced C. jejuni chicken isolate, strain RM1221 (RM1221 proteome), depleted of "housekeeping" proteins will be used to generate high titer anti-RM1221 proteome polyclonal antibodies (PAbs) in an experimental host. These anti-RM1221 proteome PAbs will be then used as "bait" to sequentially effect PELS-based immunoaffinity capture of IMAC-purified recombinant proteins comprising the proteomes of each of the remaining 29 Campylobacter strains/isolates. Protein identification for each of the Campylobacter strains/isolates will be identified by querying sequenced C. jejuni and C. coli genomes at http://www.ncbi.nlm.nih.gov/sites/genome, and www.ebi.ac.uk/newt/display with the generated mass spectral data. Following rigorous statistical analysis to ensure relevance of the identified proteins, a suite of bioinformatics-based algorithms will be deployed to define a core panel of conserved proteins, which are expected to include both proteins that are identical as well as those that share only domains/epitopes. Efforts to change knowledge /inform target audiences will include dissemination of results of this project at relevant local/national scientific meetings in the form of a poster/oral presentation, and also via a publication in a scientific peer-reviewed journal. The scope of this project is to employ an innovative proteome mining tool and bioinformatics to define a panel of highly conserved C. jejuni and C. coli proteins that have potential for inclusion in broadly protective chicken vaccines. Evaluation of potential of these conserved proteins to prevent C. jejuni and C. coli infection of chicken gastrointestinal tracts will be the focus of future studies
