Immune responses against Mycobacterium tuberculosis-specific proteins PE35 and CFP10 in mice immunized with recombinant Mycobacterium vaccae

To clone and express Mycobacterium tuberculosis [M. tuberculosis] proteins PE35 and culture filtrate protein [CFP]10 in Mycobacterium vaccae [M. vaccae], and subsequently, evaluate the humoral and cellular immunity responses against these recombinant constructs in mice. The DNA of PE35 and CFP 10 genes were cloned into the shuttle plasmid pDE22, and the recombinant plasmids were electroporated into M. vaccae. The recombinant constructs were then tested for expression of PE35 and CFP10 by Western immunoblotting using rabbit anti-sera. Furthermore, splenocytes and sera from groups of 5 mice immunized with recombinant M. vaccae [rVaccae] were tested for cellular and humoral responses in proliferation, and antibody assays. Experiments were carried out in the laboratory of the Faculty of Medicine, Kuwait University, Safat, Kuwait between 2009 and 2011. The results of Western immunoblot suggested the expression of only PE35. However, splenocyte assays showed positive proliferation in response to peptide pools, and 4 and 5 of the 6 overlapping synthetic peptides covering the sequence of PE35 and CFP10. In addition, positive antibody reactivity was detected with PE35 peptide pool and a single peptide, namely, P2. The expression of PE35 and CFP10 proteins in rVaccae constructs led to the induction of cellular immune responses to multiple epitopes

Cell-mediated immune responses to complex and single mycobacterial antigens in tuberculosis patients with diabetes

To evaluate cell-mediated immune [CMI] response in diabetic and non-diabetic tuberculosis [TB] patients and healthy subjects in response to complex, fractionated and single antigens of Mycobacterium tuberculosis. Peripheral blood mononuclear cells [PBMC] were obtained from patients suffering from pulmonary TB and type II diabetes [n = 7], pulmonary TB without diabetes [n = 10] and healthy subjects without TB and diabetes [n = 10]. PBMC were assessed for CMI responses in antigen-induced proliferation assays in response to complex mycobacterial antigens [whole cells, cell walls and culture filtrate of M. tuberculosis], a battery of naturally purified or recombinant produced secreted [ESAT6, MPT59, MPT64 and MTB38] and cytosolic [MTB10, MTB70, ML10, ML28, ML36, ML65 and MB65] mycobacterial antigens and fractionated culture filtrate proteins [fractions F1-F10] of M. tuberculosis. The majority [>70%] of diabetic and non-diabetic TB patients and healthy subjects responded to the complex antigens of M. tuberculosis. However, among the single antigens, ESAT6 was most frequently recognized by TB patients with and without diabetes, but least recognized by healthy subjects. The secreted antigens MPT59 and MPT64 were recognized by all the groups, whereas the cytosolic antigens were recognized best by healthy subjects. When tested with fractionated secreted proteins present in the culture filtrate of M. tuberculosis, the best responses in both diabetic and non-diabetic TB patients were obtained with fractions containing low-molecular-weight proteins. Diabetic and non-diabetic TB patients respond frequently to secreted low-molecular-weight ESAT6 antigen of M. tuberculosis, indicating that this antigen may be useful in the diagnosis of TB in both the groups

Amplification of six putative RDI genes of Mycobacterium tuberculosis for cloning and expression in Escherichia coli and purifications of expressed proteins

To amplify, clone and express in Escherichia coli six open reading frames [ORFs] predicted in the RD1 DNA segment of Mycobacterium tuberculosis and purify the expressed proteins to homogeneity. DNA corresponding to the coding regions of six RD1 ORFs, i.e. ORF10 to ORF15, was amplified from genomic DNA of M. tuberculosis, cloned in the plasmid vector pPCR-Script and subcloned in expression plasmid vectors pET29a and/or pGEX-4T for expression in E. coli as fusion proteins. The recombinant fusion proteins were identified by sodium dodecyl polyacrylamide gel electrophoresis and Western immunoblotting. Attempts were made to obtain purified proteins, free of the fusion partner, using affinity and fast protein liquid chromatography. DNA corresponding to all six targeted RD1 ORFs was amplified from the genomic DNA of M. tuberculosis and five of the six ORFs, with the exception of ORF13, were cloned in the plasmid vectors and expressed in E. coli. Because of extensive degradation of ORF10 and ORF12 fusion proteins or nonbinding to the affinity columns of ORF15 fusion proteins, only ORF11 and ORF14 proteins were purified, free of the fusion partner, to homogeneity. All of the six targeted RD1 genes were amplified and five expressed using E. coli hosts, but only two of the expressed proteins were purified to homogeneity. Alternative expression systems are required to obtain all RD1 proteins for functional characterization

Whole blood assays to identify Th1 cell antigens and peptides encoded by mycobacterium tuberculosis-specific RD1 genes

To identify Th1 cell-stimulating antigens/peptides encoded by the genes predicted in the Mycobacterium tuberculosis-specific genomic region of difference [RD]1, deleted in Mycobacterium bovis Bacille Calmette-Guerin[BCG], by using synthetic peptides and whole blood from tuberculosis [TB] patients. Heparinized peripheral blood was obtained from culture-proven pulmonary TB patients [n = 16] attending the Chest Disease Hospital, Kuwait. Whole blood was diluted with tissue culture medium RPMI-1640 and tested for Th1 cell stimulation using antigen-induced proliferation and interferon- [IFN-] secretion assays. The antigens included a peptide pool of 220 peptides covering the sequence of 12 open reading frames [ORFs] of RD1 [RD1mix], peptide pools of RD1 ORF5 [ORF5mix], ORF6 [ORF6mix] and ORF7 [ORF7mix], and individual peptides of ORF6 [P6.1-P6.6] and ORF7 [P7.1-P7.6]. M. tuberculosis culture filtrate, cell walls and whole-cell M. bovis BCG were used as complex mycobacterial antigens. The results obtained with different antigens and peptides were statistically analyzed for significant differences using Z test. The complex mycobacterial antigens [culture filtrate, cell walls and M.bovis BCG] and RD1mix induced comparable [p > 0.05] positive antigen-induced proliferation and IFN- responses with whole blood from TB patients. However, the positive IFN- responses induced by ORF6mix and ORF7mix were higher than ORF5mix. Among the individual peptides, P6.4 and P7.1 of ORF6 and ORF7, respectively, induced the highest IFN- responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested. The whole blood assays with synthetic peptides are useful to identify Th1 cell antigens/peptides encoded by genes located in M. tuberculosis-specific genomic regions

Identification of transcriptionally active open reading frames within the RD1 genomic segment of Mycobacterium tuberculosis

To identify transcriptionally active open reading frames [ORFs], predicted by bioinformatics, within RD1 genomic segment of Mycobacterium tuberculosis using reverse transcription-polymerase chain reaction [RT-PCR]. M. tuberculosis H37Rv was grown in Middlebrook 7H9 medium for 8 weeks and total RNA was isolated using standard procedures. The cDNA was synthesized using first-strand cDNA synthesis kit and general primers provided in the kit [pd [N][6], and/or Not I-d[T][18]] as well as forward primers specific for each predicted RD1 ORF. Specific forward and reverse primers in PCR were used to amplify ORF-specific cDNA. The amplified products were identified on the basis of size using agarose gel electrophoresis, and their identity was confirmed by DNA sequencing. RT-PCR demonstrated expression of 13 of the 14 bioinformatics-predicted ORFs within RD1 genomic segment of M. tuberculosis. However, cDNA synthesis and PCR amplifications of specific products varied with respect to primer requirement and reaction conditions, respectively. All ORFs of <1.5 kb were amplified in standard RT-PCR, whereas several large-size ORFs [>1.5 kb] required internal primers for amplification in semi-nested RT-PCR. The sequencing of RT-PCR-amplified products of ORFs confirmed their identity. Bioinformatics analysis of DNA can accurately predict ORFs within M. tuberculosis-specific genomic regions, and RT-PCR is a suitable technique to confirm their expression in bacteria