Antibody Biomarkers of Mycobacterium tuberculosis infection.
In collaboration with Jacqueline Achkar at Albert Einstein College of Medicine in New York, Mitch Magee's lab is studying Mycobacterium tuberculosis (Mtb) and the humoral immune response. M. tuberculosis is a major pathogen in human history and infects over one-third of the current world's population. Together, the pair are investigating the role of antibodies to discover potential biomarkers for disease diagnosis. To analyze the humoral response to the various proteins of tuberculosis, Magee and Achkar are utilizing the process called Nucleic Acid Programmable Protein Arrays (NAPPA) (1).
With this process, they clone all of the genes of the microorganism into an expression vector so that it will encode for a protein with a tag. Then, they print the plasmid in a printing mix that contains an agent to capture the tag to display the protein of interest. The proteins are produced and displayed by treating the plasmid microarray with a coupled in vitro transcription/translation cell lysate. The protein array is queried with antisera containing potential antibodies to the microbial proteins. The lab detects patient sample binding with flourescently labeled secondary antibody followed by image acquisition and quantitation. With this technique, Magee and Achkar were the first to publish on antibody profiling of tuberculosis patients co-infected HIV in a comparison of patients from South Africa or the United States (2).
An example of patient sera binding to the Mtb-HD-NAPPA protein array is shown in the figure to the right. The lab has detected 40 proteins out of 4,500 tested that could be used as potential biomarkers of tuberculosis infection. These arrays were also used to test protective immune responses in an animal model of tuberculosis (3).
Microarray image of patient sera binding to the M. tuberculosis-HD-NAPPA array. The color of the spot ranges from blue (no patient sampling binding) to yellow and read (high patient sample binding).
In Vivo Detection of Microbial Antigens.
In a collaborative project with David Aucoin at the University of Nevada in Reno, Nevada, Mitch's Magee's lab is funded by the Defense Threat Reduction Agency (DTRA) to investigate detection methodologies to find microbial antigens in patient fluids (blood, saliva, or urine) during the infection process. The technique called In vivo Microbial Antigen Detection (inMAD) was pioneered by Aucoin and the lab is coupling the InMAD technique with NAPPA.
In this process, human or primate samples from infected individuals, which contain bacterial antigens, are used to immunize mice. The microbial antigens in the sample will then elicit an antibody response during the immunization process in the mice. Magee and Aucoin then take the mouse immune sera to determine which of the microbial antigens are detected on the NAPPA protein array. If the mouse makes an antibody response to a protein, then that microbial protein, is in the patient sample. These can be further used to develop diagnostic tests to find the microbial proteins in the patient sample.
The prototyped coupling of the InMAD process and NAPPA in a model of Lyme disease caused by the bacterium Borrelia burgdorferi (4). In this study, primates were infected by the bite of a Borrelia-infected tick to initiate the infection. The researchers tested the primate sera on the array to identify the antibodies made during the infection and the data are shown in the figure below. They can see the rise and fall of antibody binding to multiple Borrelia proteins. The results of these studies are now being applied to the potential biothreat agents of Francisella tularensis, Burkholderia pseudomallei, and Bacillus anthracis.
The image in Panel A is a heatmap of longitudinally-collected, normalized antibody responses. The pink to red color indicates high level patient binding to the microarray. The image in panel B is representative NAPPA microarray images of responses to Borrelia burgdorferi proteins in tick-infected primates.
Yu X, Song L, Petritis B, Bian X, Wang H, Viloria J, Park J, Bui H, Li H, Wang J, Liu L, Yang L, Duan H, McMurray DN, Achkar JM, Magee M, Qiu J, LaBaer J. Multiplexed Nucleic Acid Programmable Protein Arrays. Theranostics. 2017;7(16):4057-70. PubMed PMID: 29109798.
Song L, Wallstrom G, Yu X, Hopper M, Van Duine J, Steel J, Park J, Wiktor P, Kahn P, Brunner A, Wilson D, Jenny-Avital ER, Qiu J, Labaer J, Magee DM, Achkar JM. Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays. Mol Cell Proteomics. 2017;16(4 suppl 1):S277-S89. PubMed PMID: 28223349.
Prados-Rosales R, Carreno LJ, Batista-Gonzalez A, Baena A, Venkataswamy MM, Xu J, Yu X, Wallstrom G, Magee DM, LaBaer J, Achkar JM, Jacobs WR, Jr., Chan J, Porcelli SA, Casadevall A. Mycobacterial membrane vesicles administered systemically in mice induce a protective immune response to surface compartments of Mycobacterium tuberculosis. mBio. 2014;5(5):e01921-14. PubMed PMID: 25271291.
Pflughoeft KJ, Mash M, Hasenkampf NR, Jacobs MB, Tardo AC, Magee DM, Song L, LaBaer J, Philipp MT, Embers ME, AuCoin DP. Multi-platform Approach for Microbial Biomarker Identification Using Borrelia burgdorferi as a Model. Front Cell Infect Microbiol. 2019;9:179. Epub 2019/06/28. doi: 10.3389/fcimb.2019.00179. PubMed PMID: 31245298.