H. M. Vordermeier

Increase of tuberculous infection in the organs of B cell-deficient mice.

Protective immunity against infection with Mycobacterium tuberculosis is imparted by T cells rather than antibodies, but B cells can play a role as antigen‐presenting cells and in granuloma formation. We re‐evaluated the role of B cells in the course of tuberculous infection in μ‐chain knock‐out (Ig−) mice. Surprisingly, the organs of M. tuberculosis‐infected Ig− mice were found to have three‐to eight‐fold elevated counts of viable bacilli compared with normal littermates at 3–6 weeks post‐infection. Splenic interferon‐gamma responses to whole antigen were unimpaired, whilst proliferation to certain mycobacterial peptides was found to be diminished. However, bacille Calmette–Guérin (BCG) vaccination significantly reduced the infection in Ig− mice. The mechanisms by which B cells can influence primary tuberculous infection need further study.

INDUCTION OF CD8+ CTL RECOGNIZING MYCOBACTERIAL PEPTIDES

Mycobacterium tuberculosis is the single, most important cause of morbidity attributable to a single infectious organism. CD8+ T cells play an important role in anti‐tuberculous immune responses in both mice and humans. Data concerning the identity of mycobacterial antigens recognized by CD8+ T cells is limited; consequently, few CTL epitopes have been characterized. The authors identified allele‐specific (H‐2b and d) MHC class I binding motifs in six prominent M. tuberculosis protein antigens (the 19 and 38 kDa lipoglycoproteins and the 10, 16, 65 and 70 kDa stress proteins). These predicted epitopes were tested for MHC binding as well as their ability to elicit peptide‐specific CTL following in vivo priming. The authors were able to identify eight previously undescribed mycobacterial CTL epitopes by using spleen cells from peptide‐immunized mice. In addition, CTL specific for at least one of these epitopes also recognized the naturally processed epitope presented on transfected EL4 target cells. These mycobacteria‐derived CTL epitopes could be important for future analysis of the involvement of CD8+ T cells in M. tuberculosis infection, pathogenesis and vaccine development.

M. tuberculosis-Complex Specific T-Cell Stimulation and DTH Reactions Induced With a Peptide from the 38-kDa Protein

An immunodominant T‐cell‐stimulatory epitope located near the carboxy terminus of the 38‐kDa antigen from M. tuberculosis (38.G, residues 350–369) was found to be M. tuberculosis‐complex specific. This was demonstrated by the presence of proliferative and delayed type hypersensitivity (DTH) responses in mice immunized with Mycobacterium tuberculosis and Mycobacterium bovis BCG, whereas mice immunized with M. avium or other non‐tuberculous species of mycobacteria showed no such responses. Peptide 38.G stimulated the proliferation of peripheral blood lymphocytes from healthy purified protein derivative (PPD)‐positive individuals but not from PPD‐negative individuals. It also elicited DTH responses in M. tuberculosis sensitized mice and in PPD‐positive healthy human volunteers. Peptide 38.G could therefore prove to be an important component in any new molecularly defined reagent used in the immunodiagnosis of tuberculous infection.

Immunogenicity of peptides for B cells is not impaired by overlapping T-cell epitope topology

The epitope specificity of T‐cell help to B cells and of surface immunoglobulin‐mediated B‐cell‐binding of antigens usually involves topographically distinct antigenic determinants. The possibility of cross‐recognition of the same peptide sequence by both T cells and antibodies has been a matter of conflicting opinions. We investigated this subject by detailed mapping of T‐ and B‐cell epitopes within four immunogenic mycobacterial peptides. The identified core sequences of T‐ and B‐cell epitopes showed different topology within each peptide: they were partially overlapping or adjacent in two P38‐derived peptides, but entirely overlapping in two P19‐derived peptides. The critically important result using the two truncated peptides (P19/67–78 and P19/146–155) containing only the fully overlapping epitope cores was, that they retained full potency for inducing antibody responses. However, despite this desirable overlap of determinants, anti‐peptide sera failed to block the proliferation of corresponding T‐cell hybridomas. We conclude, that our study, in contrast to previous findings, suggests that overlapping topology of T‐ and B‐cell epitopes within synthetic peptides does not necessarily impair B‐cell immunogenicity.

Predominant recognition of species-specific determinants of the GroES homologues from Mycobacterium leprae and M. tuberculosis

The Mycobacterium leprae and M. tuberculosis 10 000 MW heat‐shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequence identity between these two proteins, T cells recognize prominently the species‐specific determinants localized within amino acid residues 21–40 and 49–72. Analysis of the molecular determinants of species‐specificity for the M. leprae GroES sequence 25–40, using T‐cell hybridomas and major histocompatibility complex (MHC)‐binding assays, led to the identification of epitope cores and critical residues. Interestingly, closely overlapping epitope cores were found to be restricted by either H‐2Ad (24–34) or H‐2Ed (28–34). Furthermore, the site recognized by the M. leprae‐specific monoclonal antibodies ML06 and ML10 was also localized in the overlapping sequences 25–31 and 25–29. In conclusion, we demonstrated that immunodominant species‐specific T‐ and B‐cell epitopes can be found in a mycobacterial heat‐shock protein despite its highly conserved amino acid sequence. This finding suggests the feasibility of identifying a sufficient number of M. leprae‐specific determinants for a composite T‐cell immunodiagnostic reagent for tuberculoid leprosy.

Mutagenesis of an immunodominant T cell epitope can affect recognition of different T and B determinants within the same antigen

The effects of mutagenesis of residues of a major T cell epitope were investigated in order to expand knowledge from synthetic peptides to the naturally processed antigen. The impact of substitutions within the core of the immunodominant p61-80/PT19 mycobacterial epitope was ascertained in respect of this epitope per se, or of a C-terminal (140-159) overlapping T/B epitope and of a conformational B epitope. The core substitution A71L impaired T immunogenicity of the target epitope within the protein, but not in the peptide, whereas the N73A substitution impaired the responses in both instances. Notably, each of these single amino acid mutations abrogated the T but not the B immunogenicity of the C-terminal epitope. Furthermore, mutation of five core residues (71-76) also ablated expression of a monoclonal antibody defined conformational B epitope. In conclusion, immunological analysis of mutated proteins revealed functional associations between topographically distinct antigenic determinants which may account for the previously observed differences in the specificity of immune responses between immunised and infected hosts.