Anbarasu Deenadayalan

Immunoproteomic Identification of Human T Cell Antigens of Mycobacterium tuberculosis That Differentiate Healthy Contacts from Tuberculosis Patients

Identification of Mycobacterium tuberculosis antigens inducing cellular immune responses is required to improve the diagnosis of and vaccine development against tuberculosis. To identify the antigens of M. tuberculosis that differentiated between tuberculosis (TB) patients and healthy contacts based on T cell reactivity, the culture filtrate of in vitro grown M. tuberculosis was fractionated by two-dimensional liquid phase electrophoresis and tested for the ability to stimulate T cells in a whole blood assay. This approach separated the culture filtrate into 350 fractions with sufficient protein quantity (at least 200 μg of protein) for mass spectrometry and immunological analyses. High levels of interferon-γ (IFN-γ) secretion were induced by 105 fractions in healthy contacts compared with TB patients (p < 0.05). Most interesting was the identification of 10 fractions that specifically induced strong IFN-γ production in the healthy contact population but not in TB patients. Other immunological measurements showed 42 fractions that induced significant lymphocyte proliferative responses in the healthy contact group compared with the TB patients. The tumor necrosis factor-α response for most of the fractions did not significantly differ in the tested groups, and the interleukin-4 response was below the detectable range for all fractions and both study groups. Proteomic characterization of the 105 fractions that induced a significant IFN-γ response in the healthy contacts compared with the TB patients led to the identification of 59 proteins of which 24 represented potentially novel T cell antigens. Likewise, the protein identification in the 10 healthy “contact-specific fractions” revealed 16 proteins that are key candidates as vaccine or diagnostic targets.

Comparison of whole blood and PBMC assays for T-cell functional analysis

BackgroundTuberculosis remains the foremost cause of morbidity and mortality, more than any other single infectious disease in the world. Cell mediated immune response plays a crucial role in the control of tuberculosis. Therefore, measuring cell mediated immune response against the antigens is having a vital role in understanding the pathogenesis of tuberculosis, which will also help in the diagnosis of and vaccination for tuberculosis.FindingsThe aim of the present study was to compare and optimize the assay conditions to measure the cell mediated immune response against M. tuberculosis specific antigens. Because the conventional PBMC assays (due to requirement of large volume of blood sample) are unable to screen more number of antigens within the same blood sample. So, here we have compared 6 days culture supernatants of 1:5 and 1:10 diluted blood and PBMCs from healthy laboratory volunteers, to assess the proliferative response of T lymphocytes and secreted IFN-γ levels against purified recombinant antigen of M. tuberculosis (MPT51, Rv3803c), crude antigens of M. tuberculosis (PPD) and mitogen (PHA).ConclusionsWe have observed good correlation between each assay and also the mean difference of these assays did not reach the statistical significance (p > 0.05). From these results, we conclude that 1:10 diluted whole-blood cultures can be well-suited as an alternative assay to measure cytokine production and lymphocyte proliferation in comparison to the conventional PBMC assays. Moreover, 1:10 diluted blood assays require less volume of blood when compared to PBMC assays which will be useful particularly in paediatric and field studies in endemic countries, where blood volume is a limiting factor.

In silico analysis of potential human T Cell antigens from Mycobacterium tuberculosis for the development of subunit vaccines against tuberculosis

In silico analysis was used to predict MHC class I and class II promiscuous epitopes and potential antigens, from 24 novel T cell antigens of Mycobacterium tuberculosis. Majority of the antigens (16/24) had high affinity peptides to both MHC class I and class II alleles and higher population coverage compared to well-proven T cell antigens ESAT-6, CFP-10 and Ag85B. Among these, highest population coverage were calculated for three novel T cell antigens Rv0733 (97.24%), Rv0462 (96.9%) and Rv2251 (96.3%). The prediction results were experimentally tested by in vitro stimulation of these novel T cell antigens with blood drawn from QuantiFERON-TB Gold In-Tube (QFT-IT) positive healthy household contacts of tuberculosis patients and pulmonary TB patients. Significantly higher level interferon-γ (IFN-γ) was observed, with these novel T cell antigens, in healthy household contacts compared to pulmonary TB subjects (p = 0.0001). In silico analysis also resulted in prediction of 36 promiscuous epitopes from the novel 24 T cell antigens. Population coverage for 4 out of the 36 promiscuous epitopes was >90% [67 VVLLWSPRS (Rv1324), 42 VVGVTTNPS (Rv1448c), 178 MRFLLSAKS (Rv0242c) and 842 IRLMALVEY (Rv3800c)]. Our results shows that these novel antigens and promiscuous epitopes identified from our analysis can further be investigated for their usefulness for subunit vaccine development.

Immunological and proteomic analysis of preparative isoelectric focusing separated culture filtrate antigens of Mycobacterium tuberculosis.

Isolation of the secreted proteins and studying the immune response they induce is an essential prerequisite for understanding the pathogenesis of M. tuberculosis. In this study, preparative liquid-phase isoelectric focusing was used for the separation of culture filtrate protein (CFP) of M. tuberculosis. This procedure resolved culture filtrate proteins into 20 fractions with a pI range of 2.59 to 12.9. These 20 fractions were subjected to immunological analysis in healthy laboratory volunteers from our endemic area. Eleven fractions (Fractions 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 19) showed increased interferon gamma (IFN-gamma) secretion and 5 fractions induced increased proliferative response, when compared to unfractionated CFP. In the 11 fractions which showed increased IFN-gamma secretion, mass spectrometric analysis identified 19 different proteins. Apart from the already reported immunodominant antigens like FbpB, CFP-10 and ESAT-6, two new T cell antigens (AcpM and PpiA) were also identified in the immunologically active fractions. Immunoinformatic analysis showed that PpiA was predicted to bind more number of class I and class II HLA alleles compared with the immunodominant ESAT-6 and CFP-10. Population coverage calculations also showed that PpiA protein (85%) had a higher population coverage compared with ESAT-6 (79%) and CFP-10 (77%). This result shows that the PpiA protein has a potential to be a novel T cell antigen.

In vitro QuantiFERON-TB gold antigen specific interleukin-1beta to diagnose TB among HIV-positive subjects.

BACKGROUND The recently introduced IFN-γ release assay (IGRA) has been reported to improve the diagnosis of TB. However, IGRA has suboptimal sensitivity to diagnose TB among HIV co-infected subjects. Apart from IFN-γ, the pro inflammatory cytokines such as Interleukin-1beta (IL-1β), Tumor necrosis factor-alpha (TNF-α), IL-2, IL-6, IL-8 and IL-12 are also play a major role in mycobacterial infections. This study aimed to analyze these cytokines for detecting active TB among HIV sero positive subjects. MATERIALS AND METHODS We had prospectively enrolled 53 HIV positive subjects and 55 HIV-TB co-infected patients from India. IGRA was performed by using QuantiFERON TB-Gold In tube (QFT-GIT) method. TB antigen specific IL-1β, TNF-α, IL-2, IL-6, IL-8 and IL-12 levels were evaluated by ELISA in plasma harvested from QFT-GIT tubes. RESULTS AND CONCLUSION The TB antigen specific IL-1β levels were significantly elevated in HIV-TB co-infected patients compared to HIV positive subjects (p = 0.0004). The specificity of both IL-1β (50.94%) and QFT-GIT (52.83%) remained similar in HIV positive subjects (p = 0.24). However, IL-1β had shown higher sensitivity (72.73%) than QFT-GIT (54.55%) to diagnose TB among HIV co-infected patients. Moreover, in culture test positive HIV-TB patients, antigen specific IL-1β exhibited sensitivity of 84.21%; whereas QFT-GIT exhibited only 57.89% sensitivity. Unlike IFN-γ (the read out marker of QFT-GIT), antigen specific IL-1β levels were not influenced by low CD4 counts. The other cytokine levels were not significantly differ between the 2 groups.From this study we concluded that TB antigen specific IL-1β may be an additional biomarker for active TB diagnosis among HIV positive subjects.