engleski

Study of the MHC/peptide interaction-application in diagnosis of tuberculosis infection

The diversity of immunodominant sequences recognized in the context of the widely polymorphic HLA molecules in man represents inherent obstacle for the development of synthetic peptides as potential diagnostic tools or vaccine subunits. Therefore, a special interest is attached to promiscuous, immunodominant peptides which are recognized in context of multiple HLA alleles. For this study I have selected the immunodominant epitope p38G (amino acids 350-369) from the 38 kDa anti gen of Mycobacterium tuberculosis. This peptide stimulates proliferation of peripheral blood mononuclear cells (PBMC) in the majority of tuberculin-positive individuals. Its HLA binding affinities were found to be high for DR 1 (IC50 <10 mM), moderate for DR2, DR? and DR8 (IC50 10-100 mM), low for DR4, DR5, DR6 and DR9 (IC50 100-1000 mM), and below detection for DR3 (IC50 >1000 mM). Peptide binding to DR2 was attributed to DRB5*0101 and not toDRB1*1501 gene product. Substitution analysis of the amino acid residues involved in binding to DR I and DRB5*0101 identified F-354 as the common primary contact residue (Pl), while allele specific differences were found in positions P4, P6 and the most notably in the C- terminal anchor residue (valine at P9 for DRl or lysine at PIO for DRB5*0l.01). Computer assisted evaluation of these empirical data produced a molecular model, suggesting that the peptide binds to DR 1 in an elongated conformation, similar to that of other peptide MHC class II comp1exes. In contrast, the DRB5*0101 bound peptide is likely to be kinked, which so far was considered characteristic only for peptides within MHC class I complexes. Furthermore, structural flexibility of p38G peptide and its ability to adopt different conformations imposed by distinct HLA alleles may represent an important feature of promiscuous peptides. However, HLA promiscuity of p38G and other immunodominant peptides is relative as they often fail to bind certain alleles. Therefore I have selected eight M. tuberculosis peptides from sequences of 1.6, 1.9 and 38 kDa antigens (MTBmix-8) on the basis of their complementary binding to nine HLA-DR molecules (ORl to DR9). MTBmix-8 at 6.25 and 50 mg/ml gave rise to significant stimulation (P<0.05) of PBMC from healthy tuberculin-positive and both untreated and treated diseased subjects, but not in any of a control group of healthy tuberculin-negative subjects. MTBmix-8 . stimulated proliferation of PBMC from healthy tuberculin-positive individuals at lower concentrations than the individual component peptides. However, the maximal stimulation achieved was only slightly higher than that achieved with individual peptides. MTBmix-8 also stimulated the production of IFN-g in vitro. Using the mean + 2SD of the values for IFN- g production in the tuberculin-negative population as a cut-off, MTBmix-8 at 6.25 mg/ml was able to detect infection with a sensitivity of 1.00% in untreated patients, 87% in treated patients, and 82% in tuberculin-positive controls, whereas the corresponding figures for the most patent single peptide (16p91-1. 00) were: 66%, 71% and 42%. Thus, using the IFN- g based assay, the MTBmix-8 is more sensitive than single peptides in diagnosing infection.

HLA; M. tubercuosis; paptide; diagnosis

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