A Proteome-Scale Identification of Novel Antigenic Proteins inMycobacterium tuberculosistoward Diagnostic and Vaccine Development

Abstract

Tuberculosis (TB) remains to be a major infectious disease throughout the world. However, the current vaccine for TB has variable protective efficacy, and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use. One of the potential strategies in developing a new diagnostic method and in improving the TB vaccine involves the identification of novel antigenic candidates. This paper aims to identify systematically the novel antigenic proteins with the greatest potential as protective or diagnostic antigens by using the differential response of Mycobacterium tuberculosis proteins to serum from TB patients and healthy individuals. Approximately 87% of the open reading frames of M. tuberculosis were successfully cloned into IPTG-inducible expression vectors. The clone sets were expressed in Escherichia coli, purified under denatured conditions, and tested for antigenicity using a mixture of sera from 15 TB patients. Out of the 3480 proteins screened, 249 proteins had significant reactions with the serum samples. Among the 249 proteins, 20 proteins were identified as most reactive. Compared with the commercial test kits, 3 novel antigens from the top 20 proteins, namely, Rv1987, Rv3807c, and Rv3887c, provided better sensitivity and accuracy. These newly identified antigenic proteins may be used as candidates for serodiagnostic application and vaccine development. Overall, this study's findings may serve as an essential reference for developing new TB diagnostic methods and more effective tuberculosis vaccines.

Exploration of Novel Cellular and Serological Antigen Biomarkers in the ORFeome of Mycobacterium tuberculosis

“…S4). According to the results of first-round screening, these two studies found that 29 out of 249 and 484 proteins were positive as determined by ELISA assay and protein array, respectively (26,27). Other antigenic proteins were also identified in our study (31 proteins were identified in addition to those reported by Li et al (26), and another 55 proteins in addition to those reported by Kunnath-Velayudhan et al (27)).…”

“…A commercially used recombinant 38-kDa protein has been reported to have a specificity of 94% in smear-positive TB patients, supporting the use of tests for serological reactivity (37). In previous studies, two methods, protein array and ELISA, have been used to detect antibody responses when screening for M. tuberculosis antigens (26,27). In our study, 1,250 M. tuberculosis proteins were screened for antibody response using a semi-quantitative Western blot assay.…”

“…The sequences of protein candidates were further confirmed by mass spectrometry. sistent data from different groups using various methods (26,27). In the current study, a semi-quantitative Western blot assay was used to detect M. tuberculosis antigen-specific serum IgG antibody for the following reasons: (1) all positive reactions could be judged by reading visible bands, which guaranteed an accurate and specific reaction; (2) it is easy to calculate the reaction degree relative to the well-studied and commercialized M. tuberculosis 38-kDa protein, which is the most used M. tuberculosis protein for serum antibody detection at present (28,29); and (3) the results are not influenced by denatured proteins.…”

Surface Proteome of “Mycobacterium avium subsp. hominissuis” during the Early Stages of Macrophage Infection

“…Effective cellular and humoral immune responses depend on targeting accessible molecules, which tend to be on the surface of a pathogen (87). With respect to mycobacteria, putative and known surface-exposed proteins comprise a substantial proportion of antigens observed in comprehensive screens using sera from mycobacterium-infected hosts (37,42,45,85). The characterization of these proteins improves our understanding of bacterial pathogenesis and host immunity, providing insights that can lead to new diagnostic tools and vaccines (38,87).…”

Proteomic progress in studying tuberculosis from 2010 to 2011

“…These antigens might offer new targets for vaccine development. Nearly, an immunoproteome microarray study of M. tuberculosis-proteins with sera from patients was developed [34], in which, 3 novel antigens, namely, Rv1987, Rv3807c, and Rv3887c, provided better sensitivity and accuracy for TB detection.…”