Progress 05/15/13 to 01/14/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project itself was challenging to the research team on several fronts, all of which provided ample opportunities for training and development. The project presented several technical challenges, including those that pertained togrowth/culture of a number of Campylobacter isolates. The ability toculture these isolates wasmandatory for isolation of genomic DNA for construction of Elibraries, which required the research team to learn new microbiological skills and techniques.Furthermore, theexectuion of the project, which invovled cutting-edge science and new approaches, including novel recombinant DNA methodologies,immunochemistry procedures, and proteomics,have added to professional skill sets ofresearch personnel. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? In regards to this particular SBIR phase I project, we have accomplished the stated goals. In the next phase, funded by aSBIR phase II grant, our goal will beto sytematically examine each protein and evaluate its vaccine potenial in vivo using either a surrogate animal model ornatural hosts, chickens.An experimental vaccine comprising of the most promising proteins will bethen be evaluated for efficacy in chickens both in a controlled environment and in field trials in collaboration with a majordeveloper of veterinary vaccines.
Impacts
What was accomplished under these goals?
The overall goal of this SBIR phase I project was to define a panel of proteins that were highly conserved (immunologically cross reactive proteins/epitopes) across spatially and temporally diverse members of Campylobacter species, representative of those infecting chicken gastrointestinal tracts and with potential to cause human disease. The stated goal was to be accomplished using a novel proteome mining tool, called PELS,in anapproach that involved "walking" across the proteomes of 20 Campylobacter jejuni and 10 Campylobacter coli strains/isolates immunologically (called "proteome walking,"), and then apply specialized bioinformatics-based algorithms to arrive at the panel ofconservedproteins. The experiemental strategy was to use high affinity antibodies ("bait" antibodies) generated experimentally againstthe proteome of C. jejuni RM1221to define proteins that wereshared by the all of the remaining strains. All of these were accomplished. Specifically in line with this goal, we first constructed Elibraries of each of the strains/isolates. We expressed these Elibraries and then harvested recombinant proteins expressed by each of the Elibraries. Using "bait" antibodies, weappliedPELS to each strain/isolateto first define proteins that crossreacted with the "bait" antibodies , andthenusedbioinformatics toarrive at a panel of 88 proteins that contained antigenic determinants (linear/conformational) shared by all 30 strains. Identified proteinsincudedthose that were both identical/near-identical, as well as those that were dissimilarbut shared only short regions (epitopes/determinants). A subset of the identified proteins included thosehad been previously identified by other studies, whichserved to validateour proposed approach and experimental strategy. In addition, further bioinformatics-based analysis revealed thata subpopulation of the PELS-identified proteins had adhesin potential (suggesting that they couldcontribute to binding of Campylobacters toepithelial cells of the chicken gastrointestinal tract), and another groupthat includedorthologs of those that contribute topathogenesisin related pathogens. Severalnovel proteins were also identified.
Publications
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Progress 05/15/12 to 01/14/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided several opportunties for professional development of personnel, including the Principal Investigator. The cross-disciplinary nature of the project, necessitated learning of new skills to grow and handle these pathogens.Cutting-edge bioinformatics-based skills were acquiredto be able to analyze the mass spectral data and to derive the required information. The specialized bioinformatics-based algorithms were also essential to compile the panel of conserved proteins, which was the objective of this SBIR phase I proposal. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The long-term goal of thisstudy isdevelopment of an efficacious protein subunit vaccine thatbroadly protects against infection/colonization of the chicken gastrointestinal tract by diverse C. jejuni and C.coli strains/isolates. An efficacious vaccinewill reducethe burden of human Campylobacteriosis globally,since broiler chickens areprimary sources of human disease. As a preliminary step toward this goal,theobjectivewas to define a panel of immunologically cross reactive (conserved proteins or CPs) proteins that were shared bydiverse C. jejuni (n = 20), and C. coli (n=10), with the rationalethat a subset of suchCPs were likely to becandidates fora universal Campylobacter chicken vaccine. The proposed strategywas to employ aproprietary proteomics-based proteome mining tool for antigen discovery called Proteomics-based Expression Library Screening (PELS; described briefly under Task iii), coupled with bioinformatics to arrive at the panel of CPs. The realization of the stated technical objective was dependent on sequential completion of the following three tasks, all of which were successfully accomplished: (i) Construction of “optimized” inducible genomic DNA expression libraries.Genomic DNA was isolated from each of the 30 Camyplobacter strains/isolates, and fragments in the range of 0.5- 1.5-kbp were cloned into the pET (30) abc expression vectors (Novagen) and then first transformed into E. coli DH5 alpha (primary Elibrary), followed by transformation intoE. coli BL21 (DE3; general high-level expression; Invitrogen), the recommended expression host strain,E. coliOrigami 2 [DE3] (EMD Biosciences), and C41[DE3] (Lucigen; for optimal expression of disulfide bonded and membrane proteins), respectively, to generate optimized secondary Elibraries.Each Elibrary was cultured with shaking at 250 rpm at room temperature to an OD600 of 0.6,induced with 0.5 mM IPTG overnight andrecombinant proteins harvested were from pellet and lysate fractions following 3 cycles of freeze-thaw and 3 cycles of sonication, and stored at -70oC in PBS-0.2 % NOG until further use. (ii) Generation of C. jejuni strain RM 1221 (RM 1221) anti-proteome PAbs in mice. Recombinant proteins from lysate and pellet fractions of the three Elibraries made from genomic DNA of C. jejuni strain RM1221 were pooled, purified via nickel affinity chromatgrapy (by exploting a vector-derived hexa histidine tag added to theamino terminus of recombinant proteins),formulated with a potent adjuvant (alum), and used to generate anti-proteome PAbs in mice for use as “bait” for defining a panel of conserved proteins. (iii) Definition of a panel of conserved proteins. ThePELStechnologyfor antigen discovery wasusedto define CPs. The principle of PELSinvolves the use of covalently immobilized “bait” antibodies from any source (body fluids of acute/convalescent susceptible hosts/reservoirs host or experimental hosts) to immunoaffinity capture proteins, including recombinant proteins expressed from genomic DNA expression libraries ofcognate hosts or cDNA libraries fromcorresponding hosts/tissues.Interrogation of relevant databases with the spectral data, obtained following one dimensional SDS-PAGE and tandem mass spectrometry (LC-MS/MS) of proteins specifically bound to the immobilized “bait” antibodies then results in protein identification. Anti-proteome PAbs generated against RM 1221 were used as “bait” in PELS to sequentially immunoaffinity captured proteins expressed by Elibraries of each of the remaining 29 representative Campylobacter strains. Following identification of proteins of each strain/isolate,specialized bioinformatics-based algorithms to compile the panel of conserved proteins as follows: First, we identified identical/near-identical proteins using the standard alignment software program, LALIGN. We then defined a group of disparate proteins that shared short linear epitopes using the multiple sequence alignment algorithm called M-Coffee (www.tcoffee.org), and followed this up by examining all disparate proteins using a structure-based multiple alignment algorithm called 3D-Coffee/Expresso (www.tcoffee.org) for identification of possible discontinuous/assembled epitopes, and confirmed using the PROMALS 3D web server at http://prodata.swmed.edu/promals3d/ Results of the phase I study. We identified a panel of 88 immunologically cross reactive proteins (conserved proteins or CPs). Bioinformatics revealed that identified CPs (i) were encoded by genes that localized all over the genome; (ii) were of diverse functional classes ( transporters, lipoproteins, sensors of signal transduction systems, extracellular matrix binding proteins, putative adhesins, those involved in chemotaxis, iron regulation, invasion, respiration, periplasmic proteases, chaperones, outer membrane proteins), including unknown proteins; and (iii) localized to all cellular compartments. The major functional categories (COGS) of identified proteins, and the percentage of identified proteins in each functional category were as follows:(i) Energy production &Conversion (5%); (ii) DNA replication, recombination & Repair (2%); (iii) Post translational modification, protein turnover & Chaperones (6%); (iv) Denfense mechanisms (1%); (v) Signal transduction mechanims (1%); (vi) Transcription (1%); (viii) Necleotide transport & Metabolism (1%); (ix) ABC-type dipeptide/oligopeptide nickel transport systems (1%); Coenzyme metabolism (2%); Cell division & Chromosomal partioning (3%); Carbohydrate transport & Metabolism (2%); Intracellular trafficking & Metabolism (2%); ABC-type amino acid transporters/signal transduction systems (5%); Translation, ribosomal structure & Biogenesis (5%); Cell envelope biogenesis and outer membrane (6%); Amino acid transport & Metabolism (6%); Cell motility & Secretion (7%); Inorganic ion transport & Metabolism (11%); Unknown function (33%). Identified CPs included those that had been identified previously by other studies: These proteins, includingFlaA (invovled in virulence); FlaC (role in invasion);CadF (adhesin); PglB (Oligosaccharide transferase); FlpA (adhesin); and Cj0977 (Invasin),have beenreported to be encoded by genes present across diverse Campylobacters. This fact served as an internal control and both validated our experimental approach and lent credibility to our results. This study demonstrated the power ofusing anti-proteome “bait” PAbs, since both identical/ near identical proteins, as well as disparate proteins with only short regions of homology (putative cross reactive epitopes) wereidentified resulting in a large panel of CPs for futher study. The 82 remaining CPs (out of a total of 88 CPs identified in this phase I proposal) include those not yet implicated in infection / colonization of the avian GIT (including numerous conserved domain proteins/hypothetical proteins), and therefore merit further evaluation. The evaluation of vaccine potential of the 82 CPs in relevant animal models, and determination of promisingvaccine candidates will be the subject of the phase II proposal. Additional anticipated outputs, following experimental evalution in animal modelsinclude (i) presentation of results of this study at local/national conferences either in the form of oral/poster presentation; (ii) a patent application to protect intellectual property, given that this study is likely to yield a novel vaccine components;and (iii) dissemination of results of this study to the scientific community via a publication in a peer-reviewed journal. A partnership with a major player in the area of veterinary biologics for further evaluationof the experimental vaccine in chickens, and for development and marketing of the final product will also be sought.
Publications
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Progress 05/15/12 to 05/14/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided opportunities for both professional development and training opportunities. The project presented several challenges to the research team, including those pertaining to culture of certain Campylobacter strains/isolates, which was imperative for isolation of genomic DNAfor expression library construction. The resolution of suchchallenges viaexperimentationadded to the scientific knowledge of the research team, including the principal investigator. In addition,the cutting-edge science, including novel recombinant DNA methodologies, protein and immunochemistry, and proteomicsemployed to attain the major goals of this project and imparted on a one-to-one basis hasprovidedample opportunities for adding to the professional skill setof the bench scientist. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? To accomplish the major goals of the project, we plan to continue along the lines described above, namely, to complete mining of individual proteomes of the remaining three Campylobacter strains/isolates, and tocompile a panel of proteins conserved in all of the 30 Campylobacer strains/isolates using bioinformatics-based algorithms. We anticipate to attain all of the stated major goals by the project end date.
Impacts
What was accomplished under these goals?
The stated major goals were todefine a panel of immunologically cross reactive (conserved)proteins shared among 20 Campylobacter jejuni and 10 Campylobacter coli strains/isolates (total of 30 Campylobacter strains/isolates),representative of those infecting chicken gastrointestinal tracts/causing human disease globally, using a novel proprietary proteome mining tool called Proteomics-based Expression Library Screening (PELS) coupled with bioinformatics. In accordance with these goals, we have completed mining of individual proteomes of 19 C. jejuni and 8 C. coli strains/isolates(total of 27 strains) thus far. We anticipatethe completion of mining individual proteomes of the remaining one C. jejuni and two C. coli strains/isolates, andbioinformatics-based compilation of a panel of conserved Campylobacter proteinsby the project end date (January 14, 2014). Additional anticipated outputsinclude (i)presentation of resultsof this study at local/nationalconferences either in the form of oral/poster presentation; (ii) apatent application to protect intellectual property, given thatthis studyis likely to yield a novel novel vaccine components; and (iii)dissemination ofresults of this study to the scientific community via a publication in a peer-reviewed journal.
Publications
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