Identification of novel seroreactive antigens in Johne’s disease cattle using the Mycobacterium tuberculosis protein array

Authors: Bannantine, John; CAMPO, JOSEPH - Antigen Discovery, Inc; RANDALL, ARLO - Antigen Discovery, Inc; PABLO, JOZELYN - Antigen Discovery, Inc; PRAUL, CRAIG - Pennsylvania State University; RAYGOZA GARAY, JUAN - Pennsylvania State University; LI, LINGLING - Pennsylvania State University; Stabel, Judith; KAPUR, VIVEK - Pennsylvania State University

Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/10/2017
Publication Date: 5/17/2017
Citation: Bannantine, J.P., Campo, J., Randall, A., Pablo, J., Praul, C.A., Raygoza Garay, J.A., Li, L., Stabel, J.R., Kapur, V. 2017. Identification of novel seroreactive antigens in Johne’s disease cattle using the Mycobacterium tuberculosis protein array. Clinical and Vaccine Immunology. 24(7). https://doi.org/10.1128.CVI.00081-17.
DOI: https://doi.org/10.1128.CVI.00081-17

Interpretive Summary: Evaluating proteins singly and independently for use as an antigen in a diagnostic test is too cumbersome and would take years to analyze thousands of proteins. So our laboratory built and used a protein microarray. This protein microarray (also called a protein chip) is a high-throughput method used to track the reactions of proteins with antibodies, and to determine their function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The problem is that despite years of effort, we only had approximately 850 purified proteins to put on the array, but the bacterium produces about 4,300 proteins total. Thus this study was conducted to explore the possibility of using a previously developed protein array from a related bacterium, Mycobacterium tuberculosis (TB). Through bioinformatic approaches, we showed that by using the TB array, we could quickly add many testable proteins for antigen discovery experiments. This conclusion was further tested and confirmed by comparing the TB array with the Map array using serum from Johne's disease cattle. We identified a number of strongly reacting proteins and incorporated them into an ELISA test. We concluded the study by validating the ELISA test using an expanded serum set. This study is of primary interest to other researchers working in the field, veterinarians, and stakeholders.

Technical Abstract: Johne’s disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis (Map), is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between Map and the human pathogen, Mycobacterium tuberculosis (Mtb), we here applied a whole proteome Mtb protein array to help identify seroreactive and diagnostic Map antigens. A genome-scale pairwise analysis of amino acid identity between orthologous proteins in Map and Mtb showed an average of 62% identity, with more than half the orthologous proteins sharing > 75% identity. Analysis of the Mtb protein array probed with sera from Map-infected cattle showed antibody binding to 729 Mtb proteins, 58% of these having = 70% identity to Map orthologs. The results show only four of the top 40 seroreactive Mtb antigens were orthologs of previously reported Map antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. ELISA testing of 20 Map recombinant proteins, representing reactive and non-reactive Mtb orthologs, further confirmed that the Mtb array has utility as a screening tool for identifying candidate antigens for Johne’s disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed MAP2942c as having the strongest seroreactivity with Johne’s positive samples. Collectively, our studies have considerably expanded the number of candidate Map proteins with potential utility in the next generation of rationally designed Johne’s disease diagnostic assays.