Pattern Recognition in Pulmonary Tuberculosis Defined by High Content Peptide Microarray Chip Analysis Representing 61 Proteins from M. tuberculosis

Abstract

BackgroundSerum antibody-based target identification has been used to identify tumor-associated antigens (TAAs) for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M.tuberculosis (MTB) infection for diagnosis or TB vaccine development in clinically well defined populations.MethodWe constructed a high-content peptide microarray with 61 M.tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/−.FindingsThree distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals) and are highly predictive of the division into TB+ and TB−, other targets were exclusively recognized in all patients with TB (e.g. sigmaF) but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35) but no in TB+ patients. The segregation between TB+ and TB− does not cluster into specific recognition of distinct MTB proteins, but into specific peptide epitope ‘hotspots’ at different locations within the same protein. Antigen recognition pattern profiles in serum from TB+ patients from Armenia vs. patients recruited in Sweden showed that IgG-defined MTB epitopes are very similar in individuals with different genetic background.ConclusionsA uniform target MTB IgG-epitope recognition pattern exists in pulmonary tuberculosis. Unbiased, high-content peptide microarray chip-based testing of clinically well-defined populations allows to visualize biologically relevant targets useful for development of novel TB diagnostics and vaccines.

Evaluation of Lionex TB kits and mycobacterial antigens for IgG and IgA detection in cerebrospinal fluid from tuberculosis meningitis patients

“…For antigen-specific IgG in CSF of TBM patients, 16 kDa and 38 kDa performed better (31.6% and 26.3%) than MPT-64 (21%), but in contrast to IgA, combinations of all antigens led to a slight increase in sensitivity (36.8%) but lowered the specificity. These data corroborate findings that most TB patients develop an antibodymediated immune response to mycobacterial antigens, but the predominant isotype elicited may be dependent not only on the antigen but also on the site of infection (Chandramuki et al 2002, Gaseitsiwe et al 2008. Nevertheless, some of the patient samples were not recognized by any of the recombinant proteins tested, which indicates that single antigens, even those of immune dominant response, must be combined with other molecules that may be recognized by less responsive patients (Raja et al 2008).…”

Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401

“…Sixty-one M. tuberculosis proteins were printed as overlapping peptide (15-amino-acid) stretches on microarray slides, as reported previously (10). Most of these proteins have not been mapped for MHC class II binding, except for antigen 85B, heat shock protein HSPX, and MPT63 (Rv1926c) (20).…”

“…These data were therefore available for comparative analysis. The biological functions of the 61 proteins in the M. tuberculosis life cycle have been addressed in detail previously (10), and an overview is provided in Table 1.…”

Optimization of Peptide Density on Microarray Surface for Quantitative Phosphoproteomics

“…1 These microarrays are a powerful tool not only for the high throughput screening of substrate peptides of enzymes including protein kinases (PKs) 2 and proteases, 3 but also for drug screening 4,5 and epitope mapping. 6 Recently, peptide microarrays have been extensively used for the analysis of intracellular PKs, which are enzymes used to catalyze the phosphorylation of amino acid residues (Ser, Thr, and Tyr) of specific proteins to regulate their activity to control intracellular signal transduction processes. 7,8 The analysis of intracellular PKs is an attractive application of peptide microarrays because it enables cell-based drug discovery, 5 the biochemical study to elucidate signal transduction pathways, 9,10 and diagnosis using tissue lysates.…”