Cell mediated immunity assays identify proteins of diagnostic and vaccine potential from genomic regions of difference of mycobacterium tuberculosis

Tuberculosis [TB] is a health problem of global concern. To control TB, tuberculin and BCG have been used as diagnostic reagent and vaccine, respectively. However, BCG has failed to provide consistent protection in different parts of the world, and tuberculin has poor specificity due to the presence of crossreactive antigens. Therefore, there is an urgent need to identify M. tuberculosis-specific antigens as new vaccines and diagnostic reagents to control the global epidemic of TB. The advances in comparative genomics have identified 11 genomic regions of My cobacterium tuberculosis, which are deleted/absent in all vaccine strains of BCG currently used to immunize against TB. By using overlapping synthetic peptides corresponding to 89 predicted proteins of these regions of difference [RDs] in cell mediated immunity [CMI] assays, attempts have been made to identify the RDs and antigens with potential in specific diagnosis and development of new vaccines against TB. The most promising regions [antigens] identified for diagnostic applications are RD1 [ESAT-6 and CFP10], and for vaccine applications are RD1 [PPE68], RD7 [Rv2346c and Rv2347c] and RD9 [Rv3619 and Rv3620]. The promise of ESAT-6 and CFP10 in the diagnosis of latent and active TB has been demonstrated in industrialized and low-burden countries, but more work is needed in poor and high-burden countries to establish the utility of these high cost tests in specific diagnosis and epidemiology of TB. Furthermore, the vaccine application of RD antigen requires additional experiments to confirm their protective efficacy in animal models of TB, before they are evaluated in humans

In silico binding predictions for identification of HLA-DR-promiscuous regions and epitopes of mycobacterium tuberculosis protein MPT64 [Rv1980c] and their recognition by human Th1 cells

To identify HLA-promiscuous regions and epitopes of MPT64 [Rv1980c], a major secreted antigen of Mycobacterium tuberculosis, by in silico analysis for binding to HLA-DR molecules. The sequence of mature MPT64 protein [aa 1-205] was analyzed in silico for HLA-DR binding regions and T cell epitopes using ProPred, a web-based prediction server. The prediction results were experimentally verified by testing 20-mer synthetic peptides corresponding to the predicted HLA-DR binding regions and epitopes with T cell lines established from peripheral blood mononuclear cells of PPD-positive and HLA-heterogeneous healthy subjects in Th1 cell assays [antigen-induced proliferation and IFN-gamma secretion]. The in silico analysis for binding of the mature MPT64 sequence to HLA-DR molecules suggested that it could bind to molecules expressed from all HLA-DR alleles [n=51] included in ProPred. Furthermore, ProPred identified 26 epitopes and 8 nonoverlapping HLA-DR binding regions [9-35 aa in length] in the Rv1980c sequence, with 5 regions [aa 20-44, aa 68-102, aa 132-146, aa 164-186 and aa 194-202] being HLA-DR-promiscuous. By using synthetic peptides and T cell lines in Th1 cell assays, 4 peptides of MPT64 [aa 21-40, aa 81-100, aa 171-190 and aa 191-20], from 4 of the 5 HLA-DR-promiscuous regions predicted by ProPred, were experimentally verified to be HLA-DR-promiscuous and to have immunodominant epitopes. The in silico method [ProPred] suggested promiscuous HLA-DR-binding of mature MPT64 and identified HLA-promiscuous and immunodominant regions and epitopes of this protein

Mapping of TH1 -cell epitope regions of mycobacterium tuberculosis protein MPT64 [Rv1980c] using synthetic peptides and t-cell lines from M. tuberculosis-infected healthy humans

To identify T helper 1 [Th1]-cell stimulating and HLA-promiscuous peptides of MPT64 [Rv1980c], a major secreted antigen of Mycobacterium tuberculosis. Peripheral blood mononuclear cells [PBMCs] were obtained from 35 healthy subjects and typed for HLA-DR molecules using genomic methods. To identify subjects infected with M. tuberculosis, PBMCs were tested in antigen-induced proliferation assays with whole cells and culture filtrate antigens of M. tuberculosis, M. tuberculosis-specific antigens ESAT-6 and CFP10, and MPT64. Culture filtrate-induced T-cell lines were established in vitro from 12 M. tuberculosis-infected and HLA-heterogeneous healthy subjects, and tested with 20 overlapping synthetic peptides covering the sequence of MPT64 in Th1-cell assays, i.e. antigen-induced proliferation and/or IFN-Gamma secretion. In addition, T-cell lines from three HLA-heterogeneous subjects were tested for cytotoxic activity against peptide-pulsed antigen-presenting cells. PBMCs from 12 of 35 subjects responded to M. tuberculosis-specific antigens ESAT-6 and CEP10 as well as to MPT64, which suggested that they were infected with M. tuberculosis. Ten of twelve T-cell lines established from these donors responded to MPT64, and nine T-cell lines responded to 1 or more of the peptides of MPT64 in antigen-induced proliferation assays. Furthermore, 18 of the 20 peptides of MPT64 were recognized by the T-cell lines in 1 or more assay systems, and at least 5 peptides were recognized by T-cell lines from HLA-DR-heterogeneous subjects. Th1-cell-reactive epitopes are scattered throughout the sequence of MPT64, and at least 5 of its peptides are presented to Th1-cells in a HLA-promiscuous manner

Detection and monitoring of cytomegalovirus infection in renal transplant patients by quantitative reAl time PCR

To establish a sensitive and specific real time PCR for quantitation of cytomegalovirus [CMV] DNA in clinical specimens. In a prospective study, CMV DNA was quantified in blood samples of 255 kidney recipients with and without CMV-related symptoms between the years 2000 and 2005 in Kuwait. In a selected group of patients, the effect of anti-CMV chemotherapy was monitored by quantitative real time PCR [qRT-PCR]. The established qRT-PCR assay had a sensitivity to detect 30 CMV DNA copies. CMV DNA was detected in 54/255 [24%] patients; of these, 17 [31.5%] were asymptomatic, and 37 patients [68.5%] had symptomatic CMV infection. Sequential blood specimens were collected from all CMV-positive patients and tested by CMV pp65 antigenemia and qRT-PCR assays. There was a moderate positive correlation between the two assays [Pearson's correlation=0.52]. The median CMV viral load measured by qRT-PCR was higher in symptomatic [6.5 +/- 10.4 copies/ml] than in asymptomatic [185 copies/ml] patients [p=0.001]. The estimated cut-off value of CMV DNA for CMV symptoms/disease was 6 800 copies/ml of blood. Testing of sequential samples from patients treated with symptomatic CMV infection showed that the viral load was significantly reduced after 3 weeks of anti-CMV chemotherapy [p=0.001]. The reported qRT-PCR is a sensitive method for quantitation of CMV DNA in the blood of kidney recipients and can be useful in monitoring the efficacy of anti-CMV therapy