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

Abstract

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 g of protein) for mass spectrometry and immunological analyses. High levels of interferon-␥ (IFN-␥) 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-␥ 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-␣ 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-␥ 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. Molecular & Cellular Proteomics 9:538 -549, 2010. Tuberculosis (TB)1 is a major health problem throughout the world. A recent World Health Organization report shows that TB has been increasing at a rate of 1% per year, and an estimated 9.2 million new cases arise each year (1). Although TB is preventable, there has been an increase in its incidence in recent years. Re-emergence of TB is mainly due to its association with human immunodeficiency virus infection (2) and also due to the occurrence of multidrugresistant strains of the causative agent, Mycobacterium tuberculosis (3).Vaccination of general population is cost effective and represents one of the best biological measures for disease control. The current vaccine against tuberculosis, Bacille Calmette-Gué rin (BCG), has been administered to more people than any other vaccine. The side effects of BCG are tolerable, and it prevents miliary and meningeal tuberculosis in young children. In striking contrast, it affords limited and highly variable protection (0 -80%) against pulmonary TB (4). Thus, BCG does not seem to be a satisfactory vaccine (5, 6) and necessitates exploration of newer strategies to improve BCG or to develop a more effective vaccine.One of the potential strategies for the development of an im...

Evaluation of cytokine and chemokine response elicited by Rv2204c and Rv0753c to detect latent tuberculosis infection

“…In order to increase the diagnostic performance of IGRA for identifying LTBI, increasing number of researchers are making efforts to identify the immune-dominant T cell antigens secreted from actively growing M. tb [18]. Earlier, we investigated M. tb culture filtrate separated fractions containing Rv2204c and Rv0753c antigens that stimulated high IFN-c response in HHC than PTB [11]. Thus, we have evaluated cytokine and chemokine response against these 2 selected antigens to check whether addition of these antigens to the existing IGRA will improve the specific detection of LTBI.…”

“…Previously, we found higher IFN-c response in LTBI individuals compared to active TB patients against immuno dominant M. tb secreted protein fractions [11]; Further analyses by mass spectrometry revealed a total of 24 novel T cell antigens in those protein fractions. We have chosen two proteins, Rv2204c and Rv0753c among the 24 for the present study.…”

Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based onPseudomonasexotoxin A

“…3B summarizes the responses of the 50 donors to stimulation by the 22 pools. As previously shown (24,25), individual donors responded variably; to correct for these variations, the values were normalized to the total number of spots for each donor. Pool 3 elicited a positive response in 23 donors, which is extraordinarily strong.…”

Antigens for CD4 and CD8 T Cells in Tuberculosis

“…Several investigations have tried to identify immunodominant antigens through genomics and proteomics (Covert et al 2001;Malen et al 2008;Deenadayalan et al 2010), comparative genomics (Cockle et al 2002), or bioinformatics (Zvi et al 2008), none of which covered the entire genome. The majority of the MTB genome was covered in an investigation for in vivo expression of MTB genes during MTB infection in the lungs of mice (Commandeur et al 2013).…”