The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation.

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

Immunodominant B-cell epitope in the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis cross-reacting with glutathione S-transferase.

The TB1-5 76C monoclonal antibody raised against a synthetic 60-mer peptide in the N-terminal part of the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis has previously been shown to react with a linear epitope in the KRRITPKD region, residues 131-138 in Mce1A, and to cross-react with Mce1F. Six additional monoclonal antibodies raised against the same peptide were also shown to cross-react with Mce1F. Four of them reacted with a linear epitope in the same area, indicating that this area is immunodominant but showed distinct differrences in fine specificity. Two monoclonal antibodies did not react with synthetic peptides from this region on the solid phase in enzyme-linked immunosorbent assay, indicating greater influence of conformation on reactivity. None of the monoclonal antibodies reacted with 14-mer synthetic peptides from the corresponding area in Mce2A, Mce3A, Mce4A, M. avium, M. smegmatis or M. leprae. The reaction pattern of the monoclonal antibodies was analysed in relation to our model of the Mce1A molecule (AK Das et al. Biochem Biophys Res Commun 2003;302:442-7). The epitope is located on the surface of Mce1A, at the distal beta-strand-loop region in the beta-domain supporting its potential role in promoting uptake of M. tuberculosis in host cells. Monoclonal antibody TB1-5 19C cross-reacted with glutathione S-transferase of Schistosoma japonicum containing a PKE triplet. Monoclonal antibody TB1-5 76C gave a major band at about 44 kDa in Western blotting of M. tuberculosis sonicate, whereas polyclonal rabbit anti-Mce1A peptide antibodies reacting with the extended TTPKNPTKRRITPKDVI area of Mce1A showed a distinct band above the 160 kDa molecular mass standard.

Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis.

In addition to the previously cloned Mce1A and Mce1E genes of the Mce1 operon of Mycobacterium tuberculosis (Ahmad et al. Scand J Immunol 1999;50:510-8), Mce1B, Mce1D and Mce1F were cloned and expressed as glutathione-S-transferase (GST) fusion proteins in recombinant Escherichia coli. Polyclonal antibodies against a predicted B-cell epitope of each of the Mce1 proteins of M. tuberculosis were produced by immunizing rabbits with synthetic peptides coupled to keyhole limpet haemocyanin. These antibodies reacted specifically with the corresponding fusion protein, except for GST-Mce1F. A mouse monoclonal antibody, TB1-5 76C, raised against a synthetic 60-mer peptide corresponding to the residues 106-165 in the N-terminal part of Mce1A, reacted strongly with GST-Mce1A. The antibody cross-reacted with GST-Mce1F, but not with the other recombinant GST-Mce1 fusion proteins or free GST. Bioinformatic analysis revealed only slight homology between Mce1A and Mce1F, along the length of the polypeptide chains. Higher homology was found between the residues 106-165 of Mce1A and the residues 347-406, further into the mature Mce1F polypeptide chain. There was a striking, localized homology, indicating that the epitope reacting with the monoclonal antibody TB1-5 76C may be narrowed to the KRRITPKD region, the residues 131-138 in Mce1A corresponding to the residues 372-379 in Mce1F. This was confirmed in enzyme-linked immunosorbent assay, showing binding of TB1-5 76C to a 17-mer synthetic peptide containing the KRRITPKD sequence.

Generation of antibodies to the signal peptide of the MPT83 lipoprotein of Mycobacterium tuberculosis.

The mpt83 gene (Rv2873) encodes the exported MPT83 lipoprotein of Mycobacterium tuberculosis. The corresponding identical mpb83 gene of Mycobacterium bovis is expressed to varying extents in different substrains of M. bovis Bacille Calmette Guerin (BCG), BCG Tokyo and BCG Moreau being high producers and BCG Danish 1331, a low producer of the MPB83 protein. Immunization with the 13-mer N-terminal part of the signal peptide of MPT83, MINVQAKPAAAASC, coupled to keyhole limpet haemocyanin (KLH) through the added C-terminal cysteine resulted in rapid antibody formation monitored by enzyme-linked immunosorbent assay (ELISA) with free immunizing peptide on the solid phase. In ELISA, with four 20-mer overlapping peptides covering the N-terminal part of the MPT83 sequence, three polyclonal rabbit antisera reacted only with the N-terminal peptide. Antigenic signal peptide could not be detected in sonicates of BCG Tokyo and BCG Moreau. After SDS-PAGE and blotting, the antibodies reacted with sonicates of recombinant Escherichia coli containing the entire mpt83 gene including the signal sequence, but not with the 22 kDa form of native MPB83 purified from BCG culture filtrate. In partition chromatography the recMPT83 partitioned in the water phase while 26 kDa MPB83 in BCG culture filtrate partitioned in the lipid phase confirming that lipidation at the N-terminal cysteine residue occurs after the splitting of the polypeptide chain by signal peptidase II.

Demonstration of expression of six proteins of the mammalian cell entry (mce1) operon of Mycobacterium tuberculosis by anti-peptide antibodies, enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction.

Polyclonal rabbit antibodies were generated to synthetic peptides corresponding to predicted B-cell epitopes of six proteins of the mce1 operon of Mycobacterium tuberculosis. Antipeptide antibodies reacted with Mce1A and Mce1E fusion proteins in sonicates of recombinant Escherichia coli as well as with distinct bands in sonicates, but not in culture fluids of M. tuberculosis and M. bovis bacillus Calmette-Guérin (BCG). Polyvalent rabbit antibodies generated by immunization with sonicates of BCG bacilli reacted with synthetic peptides from the six Mce1 proteins on the solid phase in enzyme-linked immunosorbent assay (ELISA), albeit with different frequencies. The Mce1A peptide (p124-140) reacted most frequently, with seven of the nine antibodies tested, while the Mce1F peptide (p329-343) reacted with two. Used as a control, 20 polyclonal rabbit antibodies to 12 isolated proteins of M. tuberculosis and M. bovis BCG did not react with any of the six synthetic peptides, except in one case. mRNA expression of the six mce1A-mce1F genes of M. tuberculosis was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR). These data indicate that all Mce1A-Mce1F proteins of the mce1 operon are expressed by in vitro-grown M. tuberculosis and M. bovis BCG.

B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.

MPB70 and MPB83 as indicators of protein localization in mycobacterial cells.

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.