Juraj Ivanyi

38 000 MW antigen-specific major histocompatibility complex class I restricted interferon-gamma-secreting CD8+ T cells in healthy contacts of tuberculosis.

CD8+ T lymphocytes are required to protect mice against Mycobacterium tuberculosis, although in early infection the mechanism appears not to be via perforin or granzyme‐mediated lysis of the infected target, and may be via interferon‐γ (IFN‐γ) production. We therefore investigated whether CD8+ T cells specific for the immunoprotective 38u2003000u2003MW antigen of M. tuberculosis could be detected in infected humans. Using a recombinant vaccinia virus expressing the 38u2003000u2003MW antigen of M. tuberculosis (rV38) and a control vaccinia virus (rVras) we demonstrated that both viruses stimulated IFN‐γ production from freshly isolated peripheral blood mononuclear cells (PBMC) in a 36‐hr enzyme‐linked immunospot assay. Cell depletion and antibody blockade established that the bulk of the 38u2003000u2003MW antigen‐specific IFN‐γ response was mediated by CD8+, major histocompatibility complex class I‐restricted T cells, whereas the anti‐vaccinia virus response was predominantly mediated by CD4+ T cells. In further evaluations PBMC from all seven healthy tuberculosis‐exposed contacts had a 38u2003000u2003MW antigen‐specific IFN‐γ response, whereas seven patients with untreated sputum‐positive pulmonary tuberculosis had very low levels of 38u2003000 antigen‐specific IFN‐γ‐producing cells. These preliminary observations demonstrate the utility of recombinant vaccinia viruses in restimulating freshly isolated CD4+ and CD8+ T cells. The bias towards a higher frequency of IFN‐γ‐producing CD8+ T cells in contacts rather than patients may indicate a protective role for CD8+ cells in human tuberculosis.

Enhancement of the T cell response to a mycobacterial peptide by conjugation to synthetic branched polypeptide

A peptide‐based approach towards improving the immunodiagnosis of, and vaccination against, tuberculosis faces the problems of MHC restriction of T cell recognition and the poor immunogenicity of peptides in the absence of adjuvant. We sought to compensate this by the use of synthetic branched polypeptides of the poly[Lys‐(Xi‐DL‐Alam)]u2009type, containing a glutamic acid residue (EAK), and further modified either by succinylation (SucEAK) or acetylation (AcEAK). These carriers were conjugated to two permissively recognized peptides of Mycobacterium tuberculosis. The 38p350u2009–u2009369‐SucEAK conjugate enhanced IFN‐γ production more than 13‐fold (from 22.6 to 294 pgu2009/u2009ml, pu2004= 0.001) in peripheral blood mononuclear cells from healthy subjects, and 8.7‐fold (pu2004= 0.012) in cells from tuberculosis patients. The effect was dependent on the carrier used and on covalent linkage of SucEAK to 38p350u2009–u2009369. An increased response occurred best in cells from subjects bearing at least one HLA‐DR allele for which 38p350u2009–u2009369 had high binding affinity and required cellular processing of the conjugate as inhibitors (chloroquine and wortmannin) blocked the IFN‐γ response. SucEAK conjugation of peptide 16p91u2009–u2009110 did not significantly increase IFN‐γ production, indicating that the ability of conjugation to enhance the response was peptide structure dependent. These data indicate that the use of SucEAK polymer coupled with permissively recognized peptides could contribute to the development of an improved immunodiagnostic or vaccine reagent for tuberculosis.