The discussion sessions in the workshop focused on the biological significance of the information in the epitope database. One topic of particular interest was the potential variation in structure, sequence, and expression of genes encoding epitope-bearing antigens.
Recent characterization of M. tuberculosis
strains by analysis of large sequence polymorphisms (insertions and deletions) and single-nucleotide polymorphisms has revealed more diversity between strains than previously believed, although strains can be grouped by phylogeography13–16
. This recently-recognized diversity raises concerns that distinct strains of M. tuberculosis
may differ in epitope sequences that epitope-based diagnostic assays or subunit vaccines may not take into account. David Alland (New Jersey Medical School, UMDNJ) presented data on sequence polymorphisms in drug target genes that cannot be accounted for by selection for drug resistance, indicating that selection pressures for genetic variation in M. tuberculosis
are still not well understood. While an earlier publication revealed limited sequence diversity in 24 antigen-coding genes in 16 strains of M. tuberculosis
from geographically-distant sources17
, the availability of information on epitopes in the IEDB will facilitate extension of that study to a greater number of antigenic proteins. In turn, there is a need for characterization of epitope sequence variation in diverse strains, in order to better understand mycobacterial evolution, and to apply this information to the design and interpretation of new diagnostic assays and vaccine candidates. Data on these variations (or the lack of them) should be included as data elements in the IEDB.
Michael Brenner (Harvard Medical School) presented data on the structures of the CD1-restricted non-peptide epitopes identified in M. tuberculosis: lipoglycans, lipopeptides, mycolic acids. Five of these are restricted by CD1b, and one each by CD1a and CD1c. An existing hindrance to further characterization of the roles of these epitopes in the immune response is lack of an optimal animal model for their investigation.
David Lewinsohn (Oregon Health & Sciences University) provided an overview of the human CD8+ response to M. tuberculosis. These responses appear to be evenly divided between classically (HLA-Ia), and nonclassically (such as HLA-E) restricted responses. Ex vivo characterization of the classically-restricted responses reveals diverse and distinct patterns of immunodominance. Furthermore, these responses are frequently restricted by HLA-B, and consist of 10- to -11-amino acid peptides.
A second topic discussed was the expression of epitope-containing genes. Maria L. Gennaro (Public Health Research Institute) noted studies revealing that expression of several well-characterized epitope genes (fbpA
, and fbpC
) is downregulated during the chronic phase of M. tuberculosis
infection in mice18, 19
. Shreemanta Parida (Max Planck Institute for Infection Biology) discussed efforts to define global gene expression profiles of M. tuberculosis
in vivo, including in human tissues during distinct states of infection. Joel Ernst (NYU School of Medicine) showed data indicating that downregulation of expression of the fbpB
gene (encoding antigen 85B) in vivo is accompanied by attenuation of CD4+
T lymphocyte responses to that antigen, using adoptive transfer of cells from mice with a T cell antigen receptor transgene that is specific for an epitope of antigen 85B. Juraj Ivanyi (King’s College) described results of a published study that found that distinct epitopes of the same M. tuberculosis
protein (GroES) are recognized with different frequencies by cells of subjects with active or latent tuberculosis20
. Hardy Kornfeld (University of Massachusetts) presented evidence for heterologous immunity between mycobacteria and certain viruses that influences host susceptibility and immunopathology upon sequential infection. The underlying mechanisms may differ from those reported for heterologous immunity between unrelated viruses, but the data suggest that T cells specific for particular viral epitopes may cross-react with mycobacterial epitopes (and vice versa) with functionally significant consequences.
Additional discussion focused on the utility of reference sets of epitopes for studies of CD4+
T cell and antibody responses in humans, mice, and other experimental animals. Reference sets of epitopes will facilitate comparison of the results of studies in distinct systems and clinical contexts, and will also facilitate development of additional reagents for studies of immune responses to infection and immunization. Sam Behar (Harvard Medical School) advocated the use of define peptide epitopes to evaluate vaccine efficacy. It is important to understand why vaccines fail, whether because of a vaccine failure (no immune response was generated), or because of immunological failure (the immune response that resulted from vaccination does not mediate protection). This is an important role of defined peptide epitopes, as there are several examples in which varying the vaccination strategy for the same antigen led to recognition of different epitopes in that antigen21
. Whether an epitope is protective or not is determined at least in part by whether it is presented by M. tuberculosis
infected cells, and vaccination often elicits T cells recognizing a broader epitope repertoire than elicited following infection22
. Data was presented showing that a CFP10 DNA vaccine elicits CFP10-specific CD8+
T cells in C3H mice that recognize the same epitope (CFP1032–39
) that is recognized following M. tuberculosis
infection. Furthermore, this response protected mice from M. tuberculosis
, demonstrating that immunological success was linked to protective efficacy. A panel of epitopes recognized by both CD4+
T cells in different mouse strains (C57BL/6, BALB/c, and C3H (e.g., H-2b
), was presented which can be used for measuring the T cell response in different mouse strains during various experimental situations (vaccination, genetic studies, and differential susceptibility).
Taken together, these findings indicate that the establishment of reference sets of epitopes, and design and development of new vaccines and diagnostic assays, need to take into account the expression, as well as the sequences, of antigenic epitope-coding genes, as it is possible that immune responses to epitopes expressed only early in infection provide little protection during the chronic phase of infection. They also suggest that additional investigation of the functions of immune responses to non-peptide mycobacterial epitopes should be strongly encouraged.