C. Abou-Zeid

A simple new method for using antigens separated by polyacrylamide gel electrophoresis to stimulate lymphocytes in vitro after converting bands cut from Western blots into antigen-bearing particles

The individual antigenic components present in microgram quantities of complex mixtures can be separated reproducibly by polyacrylamide gel electrophoresis and transferred onto nitrocellulose blots. We report that the ng quantities of antigen present in single lines cut from such Western blots can be used to induce maximal lymphoproliferative responses in 30-60 microtitre wells. In order to achieve this the excised lines of antigen-bearing nitrocellulose sheet must be converted into antigen-bearing particles small enough to be engulfed by macrophages. We describe optimal conditions and discuss the applications of this technique.

Transformation of mycobacterial species using hygromycin resistance as selectable marker

Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials (Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [10(3)-10(5) transformants (micrograms DNA)-1] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG. The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed.

The secreted antigens of Mycobacterium tuberculosis and their relationship to those recognized by the available antibodies.

Proteins secreted by strains of Mycobacterium tuberculosis during short-term, zinc-sufficient batch culture were identified in order to define antigens likely to be relevant to the pathogenesis of human disease. [35S]Methionine-labelled proteins in supernatants of 4-7 d cultures were separated by PAGE under both denaturing and non-denaturing conditions, and the position of labelled material was determined. Secreted protein patterns of M. tuberculosis were quite similar to those of Bacillus Calmette-Guérin (BCG) but differed by the absence of the 46 kDa dimeric protein specific to BCG and by the presence in large amounts of a 23 kDa protein which, when denatured, gave 13 kDa subunits. This 13 kDa subunit protein constituted up to 20% of secreted proteins in classical strains of M. tuberculosis of phage type B but was not detected in phage type I strains from South India. This may be relevant to the different pathogenicity of these strains. Western blot analysis showed that antigens defined in supernatants of short-term (3 d) cultures of M. tuberculosis constituted a small subset of those seen in supernatants of organisms cultured for longer periods. One of the secreted proteins has the interesting property of binding to fibronectin. The available monoclonal antibodies and antisera have been used to identify lines on immunoblots corresponding to the secreted/released antigens of M. tuberculosis. The present findings suggest that there are major secreted antigens to which antibodies do not yet appear to have been produced experimentally.

Does antibody to mycobacterial antigens, including lipoarabinomannan, limit dissemination in childhood tuberculosis?

Serum immunoglobulin (Ig) G responses to a variety of mycobacterial antigens were measured in children from the UK, in children with tuberculosis from Hyderabad, India and Dhaka, Bangladesh, classified according to whether the disease was disseminated or localized, and in non-tuberculous controls. Anti-lipoarabinomannan (LAM) IgG responses in UK children showed a marked trough between 6 months and 3 years coincident with the reported peak incidence of disseminated tuberculosis. Geometric mean IgG responses to sonicates of slow-growing mycobacteria (rich in LAM) in 36 children with disseminated tuberculosis were markedly lower than in 99 children with localized tuberculous lesions (for Mycobacterium scrofulaceum P < 0.01, for M. tuberculosis P < 0.01, and for M. vaccae P < 0.01). Responses to purified LAM were also lower in the disseminated tuberculosis group (P < 0.05) but there was no difference between the groups in their response to mycobacterial 65 kDa protein. Multiple regression analysis showed that the reduced response to sonicated mycobacterial antigens and to LAM in children with disseminated disease was independent of age, nutritional status, skin test reactivity, duration of previous symptoms, and city of origin. There was no evidence for sequestration of antibody to immune complexes. These findings are compatible with the hypothesis that children with low levels of antibody to sonicated mycobacterial antigen and to LAM, or those who cannot mount an antibody response, are predisposed to dissemination. A role for antibody in preventing disseminated forms of tuberculosis in childhood has implications for the development of improved vaccines and for the optimum timing of vaccination with bacille Calmette-Guérin.

Attachment of Mycobacteria to Fibronectin-coated Surfaces

This report investigates the extent of the expression of fibronectin (FN) binding properties among the mycobacteria and provides preliminary characteristics of the bacterial molecule(s) mediating attachment. Eight BCG substrains, three Mycobacterium tuberculosis strains and four other mycobacterial species all expressed FN-binding capacity. Treatment of organisms with detergent prior to the binding assay destroyed the FN-binding capacity of BCG but not that of Staphylococcus aureus. Trypsin pretreatment eliminated the FN-binding capacity of both BCG and S. aureus. [35S]Methionine-labelled material in supernatants from BCG and M. tuberculosis cultures attached to FN-coated surfaces. These culture supernatants inhibited the attachment of BCG but not S. aureus to FN-coated surfaces. This inhibitory activity of the supernatants was removed by affinity chromatography on FN-Sepharose but was not affected by similar passage over a control column (human serum albumin attached to Sepharose). These results demonstrate that the ability to bind FN is present in all mycobacterial species tested and suggest that attachment is mediated by trypsin-sensitive cell-surface component(s).

Molecular and immunological analysis of a fibronectin‐binding protein antigen secreted by Mycobacterium leprae

By screening a Mycobacterium lepraeλgt11 genomic DNA library with leprosy‐patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin‐binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327‐amino‐acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75–85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55–266 and 265–327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55–266 and 265–327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin‐binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.

The 19-kD antigen and protective immunity in a murine model of tuberculosis.

The 19‐kD antigen is a cell wall‐associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette–Guérin (BCG) vaccine strains. Expression of the 19‐kD antigen as a recombinant protein in two saprophytic mycobacteria—M. vaccae and M. smegmatis—resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross‐reactive mycobacterial antigens. Induction of an immune response to the 19‐kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19‐kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.

SUBDIVISION OF DAUGHTER STRAINS OF BACILLE CALMETTE-GUERIN (BCG) ACCORDING TO SECRETED PROTEIN-PATTERNS

In order to identify proteins secreted by live organisms, daughter strains of the Bacillus Calmette-Guérin (BCG) were grown for 4-7 d in a defined medium containing [35S]methionine. Secreted components were then separated by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions, and analysed by autoradiography and in an Ambis beta-scanner. The results indicate that BCG daughter strains can be subdivided into two groups according to their secretion of a 46 kDa protein dimer consisting of two similar 23 kDa subunits. High-producer strains (Japanese, Brazilian and Russian) secrete very large quantities of this material, which constitutes approximately 23% of all secreted protein. These findings correlate with earlier studies in which degradation products of the protein dimer may have been identified, and with the data from patterns of cell wall lipids.