separation-fractionation-culture-filtrate-proteins-cfps-from

Separation and Fractionation of Culture Filtrate Proteins (CFPs) from <em>Mycobacterium tuberculosis</em>

Secreted proteins of Mycobacterium tuberculosis (M. tuberculosis) play a crucial role in tuberculosis pathogenesis, immune modulation, and disease ...

separation-fractionation-cell-wall-cell-membrane-proteins-from

Separation and Fractionation of Cell Wall and Cell Membrane Proteins from <em>Mycobacterium tuberculosis</em> for Downstream Protein Analysis

Mycobacterium cell wall and membrane proteins, which play a central role in tuberculosis pathogenesis, were successfully separated using preparative ...

National Institute for Research in Tuberculosis

Tuberculosis 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.

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

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.

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

The main objective of the study was to evaluate whether in vitro QuantiFERON-TB Gold In-Tube (QFT-GIT) assay antigen-specific IL-1β, TNF-α, IL-2, IL-6, IL-8 and IL-12 (p40) production is associated with active TB. In a cohort of 77 pulmonary TB patients (PTB), 67 healthy household contacts (HHC) and 83 healthy control subjects (HCS), the antigen-specific cytokines levels were determined in supernatants generated from QFT-GIT tubes. Antigen-specific IL-1β levels were significantly higher in PTB than HHC and HCS. At a fixed cutoff point (1,108 pg/ml), IL-1β showed positivity of 62.33% in PTB, 22.38% in HHC and 22.89% in HCS. Moreover, antigen-specific IL-1β assay can differentiate PTB and HHC (believed to be latently infected) (p < 0.0001). Like IL-1β, significantly higher levels of antigen-specific TNF-α were associated with PTB and displayed 43.63% positivity in PTB. The antigen-specific IL-2 levels were associated both with PTB (54.54%) and HHC (48.14%). Other cytokines levels did not differ among the groups. Our results suggest that antigen-specific IL-1β can be used as a biomarker for active TB diagnosis as well as for differential diagnosis of PTB and LTBI.

Role of QuantiFERON-TB Gold antigen-specific IL-1β in diagnosis of active tuberculosis.

One third of the world's population is estimated to harbour latent tuberculosis infection (LTBI). Around 10% of them have the life time risk of developing active tuberculosis (PTB). Currently there is no gold standard test for identifying LTBI. Therefore identification of specific markers for LTBI will help as to develop a test specific for LTBI. Earlier, in our immunoproteomic analysis, we found that peptidyl-prolyl cis-trans isomerase A (PpiA) protein-containing fractions induced significantly higher interferon-gamma (IFN-γ) response in LTBI than in PTB. Immunological characterisation of recombinant PpiA protein was carried out in the current study. We have studied 10 cytokines and 2 chemokine responses against PpiA and standard antigens such as early secretory antigenic target-6 (ESAT-6) and culture filtrate antigen-10 (CFP-10). In healthy household contacts (HHC), all the tested antigens induced significantly higher levels of IFN-γ and Interlukin-8 (IL-8) compared with those in PTB. PpiA-specific IL-12p40 response was significantly increased in HHC compared with that in PTB. PpiA antigen-specific IFN-γ and IL-12p40 both showed 86% positivity in HHC, whereas in PTB, they showed 20% and 38% positivity, respectively. In terms of IFN-γ/TNF-α ratio, PpiA displayed 86% (30/35) positivity in HHC and 18% (7/39) positivity in PTB. In summary we found that PpiA-specific IFN-γ and IFN-γ/TNF-α ratio response were specific biomarkers for LTBI identification.

PpiA antigen specific immune response is a potential biomarker for latent tuberculosis infection.

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.

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

Even though various techniques have been developed for rapid diagnosis of tuberculosis (TB), still there is an immense need for a simple, cost effective, highly sensitive and specific test. Hence, one of the possibilities is identification of Mycobacterium tuberculosis specific antibodies in infected serum by using specific antigens.

Dynamic IgG antibody response to immunodominant antigens of M. tuberculosis for active TB diagnosis in high endemic settings.

, which causes tuberculosis, is one of the leading infectious agents worldwide with a high rate of mortality. Following aerosol inhalation,  primarily infects the alveolar macrophages, which results in a host immune response that gradually activates various antimicrobial mechanisms, including the production of reactive oxygen species (ROS), within the phagocytes to neutralize the bacteria.  is the master regulator of oxidative stress response in several bacterial species. However, due to the absence of a functional  locus in , the peroxidase stress is controlled by alkylhydroperoxidases.  expresses alkylhydroperoxide reductase to counteract the toxic effects of ROS. In the current study, we report the functional characterization of an orthologue of alkylhydroperoxidase family member, Rv2159c, a conserved protein with putative peroxidase activity, during stress response and virulence of . We generated a gene knockout mutant of  Rv2159c (MtbΔ2159) by specialized transduction. The MtbΔ2159 was sensitive to oxidative stress and exposure to toxic transition metals. In a human monocyte (THP-1) cell infection model, MtbΔ2159 showed reduced uptake and intracellular survival and increased expression of pro-inflammatory molecules, including IL-1β, IP-10, and MIP-1α, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Similarly, in a guinea pig model of pulmonary infection, MtbΔ2159 displayed growth attenuation in the lungs, compared to the wild type  and Rv2159c-complemented MtbΔ2159 strains. Our study suggests that Rv2159c has a significant role in maintaining the cellular homeostasis during stress and virulence of .

ArticleTotal : 2	Year
Publication title	2025
Publication title	2025

Article	Year
Comparison of whole blood and PBMC assays for T-cell functional analysis. BMC research notes\| PubMed ID: 23531281	2013
In silico analysis of potential human T Cell antigens from Mycobacterium tuberculosis for the development of subunit vaccines against tuberculosis. Immunological investigations\| PubMed ID: 24467664	2014
Role of QuantiFERON-TB Gold antigen-specific IL-1β in diagnosis of active tuberculosis. Medical microbiology and immunology\| PubMed ID: 25504009	2015
PpiA antigen specific immune response is a potential biomarker for latent tuberculosis infection. Tuberculosis (Edinburgh, Scotland)\| PubMed ID: 26546406	2015
In vitro QuantiFERON-TB gold antigen specific interleukin-1beta to diagnose TB among HIV-positive subjects. Tuberculosis (Edinburgh, Scotland)\| PubMed ID: 26786651	2016
Dynamic IgG antibody response to immunodominant antigens of M. tuberculosis for active TB diagnosis in high endemic settings. Clinica chimica acta; international journal of clinical chemistry\| PubMed ID: 27370403	2016
Role of a Putative Alkylhydroperoxidase Rv2159c in the Oxidative Stress Response and Virulence of . Pathogens (Basel, Switzerland)\| PubMed ID: 35745538	2022

Anbarasu Deenadayalan

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