Identification of B cell antigenome in Mycobacterium bovis by immunoproteomic analysis

Bovine tuberculosis (bTB) is a common zoonosis prevalent in many countries with grave economic consequences. Most developed and developing countries have implemented the test-and-slaughter policy to protect public health and reduce economic losses in the cattle industry. The official diagnosis of bTB is based on assays dependent on cell-mediated immunity (CMI). CMI-based diagnosis demonstrates diagnostic incapability at late stages of infection, which could be overcome by diagnosis based on humoral immunity (HI). Therefore, there is an urgent need to identify and define the B cell antigenome of Mycobacterium bovis. In this study, the B cell antigenome of culture filtrate proteins (CFP) was defined by mass spectrometry-based proteomics technology. Four spots were detected on 2-dimensional gel electrophoresis (2-DE) against M. bovis-positive serum in an immunoblotting experiment. Twenty-one proteins were identified in four spots by proteomic tools, such as Mb2900, Mb2898, Mb0448, Mb3834c, Mb1918c, Mb0134c, Mb0358 and Mb1868c, which are known B cell antigens, including 13 new proteins, i.e. Mb3751, Mb2006c, Mb3276c, Mb2244, Mb1164c, Mb2553c, Mb2946c, Mb1849c, Mb1511c, Mb1034c, Mb2616c, Mb0854c and Mb2267. These new proteins identified by 2-DE and immunoblotting were the B cell antigens used in developing serological diagnostic methods based on HI to bTB.

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications

ABSTRACTDirect pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity forMycobacterium tuberculosisproteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to nativeM. tuberculosisproteins was confirmed by usingM. tuberculosisculture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P< 0.0001) differences in the medianM. tuberculosissignals and in specific pathogen markers, such as antigen 85B. Samples where manyM. tuberculosisaptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P< 0.0276), particularly in TB patients with HIV coinfection. In conclusion, directM. tuberculosisantigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation ofM. tuberculosisSOMAmers using other platforms and sample types is warranted.

Immunogenicity Studies with Microbial Fractions of M. tuberculosis H37Rv Total Culture Filtrate

<p class="1Body">Current study investigates the whole secretory proteome of <em>Mycobacterium tuberculosis</em> as culture filtrate fractions to identify immunoprotective protein antigens on the basis of protection studies in animal (mouse and guinea pig) models. Secretory culture filtrate proteins (CFPs) of <em>M. tuberculosis</em> H₃₇Rv were fractionated into fifteen narrow molecular mass fractions in the order of increasing molecular size (F1-F15) by electroelution. Immunization studies revealed proteins in the molecular weight range of 20-24kDa (F7), 25-30kDa (F8) and 37-42kDa (F11) as key protective fractions against experimental tuberculosis in both the animal (mice and guinea pig) models. Amongst these fractions, F7 imparted even better protection as compared to BCG. Immunological studies with all the fractions demonstrated that although selected three protective fractions were able to induce significant immune responses in both short term culture filtrate (STCF) immunized and Mtb infected animals, there were number of other non-protective fractions also that were inducing higher immune responses either in immunized animals (e.g.F12-F15) or in Mtb challenged animals (e.g.F1-F6). These results demonstrate that only those mycobacterial proteins that are recognized by the host immune system both during immunization and infection can induce significant protection against experimental tuberculosis, however there is no direct correlation between the level of immune responses and degree of protective efficacy.</p>