360 Mycobacterial infections 25 June 1997 - Oral presentations

MaterIala and Methods: In order to get more precise information, we have analyzed the intramuscular vaccination with two plasmld DNAs encoding either a mature or a secreted form of the Ag85A protein and compared it to the immunogenicity of an intravenously given live M. bovis BCG vaccine.

FteeulB: Significant and comparable Ag85-specific IgG levels of IgGl, IgG2a and IgGPb isotype were detected in sera from mice vaccinated with either ma- ture or secreted Ag85A DNA, whereas BCG-vaccination only induced minimal antibody levels. Vaccination with DNA stimulated comparable levels of IL-2 and IFN-y responses in response to whole BCG culture filtrate and its purt- fied Ag85 complex as vaccination with BCG. On the other hand, Thl cytokine production against PPD, BCG cytoplasmic extract and whole BCG bacilli, was cleatiy lower following vaccination with DNA encoding Ag85A than following BCG. A Thl T cell epitope mapping using synthetic 20mer peptides covering the complete mature sequence of Ag85A, indicated that DNA vaccination stim- ulated a broader IL-2 and IFN-y epitopic repertoire than BCG, albeit that most BCG-triggered epitopes were also recognized following DNA vaccination. The same peptides were recognized following vaccination with DNA encoding the secreted or mature form, and generally responses were strongest against the latter. Moreover, strong CTL activity against Ag85A-transfected P815 cells and peptide-pulsed P815 cells could only be measured following DNA vaccination with ether construct but not after M. bovis BCG infection. Finally, vaccination of guinea pigs with Ag85A encoding DNA induced significant lymphoproliferative responses and antibody production but failed to induce a positive DTH reaction against purified native Ag85, whereas intradermal BCG vaccination did induce a positive DTH reactton but little antibodies or Ag85 specific pmliieration.

Conclualon: These results indicate that vaccination with plasmid DNA en- coding Ag85A may offer several advantages over classical vaccination with M. bovis BCG by the induction of antibodies of IgGl, IgGPa and lgG2b isotype (which may neutralize the enzymatic mycolyl-transferase activity of Ag85), by the stimulation of a stronger, epitope spreadened but antigenically restricted pro- ductton of Thl cytokines, by the absence of DTH induction and by the generation of potent CD8+ CTL responses.

0.4.11.6 Human CD8+ CTL specific for Mycobacterium tlh?rcl4/os/s

R. Bmokes’, A. Lalvani ‘, Ft. Wilkinson 2, A. Malin3, G. Pasvo12, A.V.S. Hill’. ’ Nuffieid Department of Clinical Medicihe, John Radcli& Hospital, Oxford. UK, 2 Unit of kn’ection and Tropical Medicine, Imperial College of Science, Technology and Medicine, Hanow, UK, 3Department of Clinical Sciences, London .&WI of Hygiene and TIspca/ Medicne, London, UK

Introduction: The immune response to Mycobac~erium tubenx/osis (M. tb) involves several components of cell mediated immunity. Mounting evidence implicates a role for CD8+ T cells as a requirement for protection, yet HLA class I restricted responses specific for M. tb have not been de&bed in humans. By using optimal conditions of culture and a particularly sensitive assay system this study sets out to identify CTL effecters and examine their possible involvement in protection.

Materlala and Methods: Using a reverse immunogenetic approach we chose secreted antigens of M. tb and synthesised an array of peptides that were congruent with HLA class I allele-specific peptide motifs. These were used to restimulate lymphocytes from a series of patients with tuberculosis and healthy contacts. Activated T cells were detected using the ELISPOT assay for interferon-gamma (IFN-)I) release and these were charactelized phenotypically by CD4, or CD8 depletion. Cytolytic activity was measured with a standard 4 hour chromium release assay.

Ftesuh We identified HIA-B52 and -A2 restricted CD8+ T cells specific for epitopss in the early secretory antigenic target -8 (ESAT-8) of the M.tb complex. These cells exhibited peptkte-specific IFN-y release as well as strong cytolytic activity for free peptide and endogenously prwessed antigen. Activated CTL were seen ex viw, in the fresh blood of a patient with active disease.

Conclusion: HLA class I restricted CD8+ CTL with specificity for epitopes within a secreted antigen of M. tb were charactettzed. The phenotype and specificity of the T cells suggests they may play an important role in the protective immune response to M. tb in viva.

0.4.11.7 The role of CD80 and CD88 in Mycobacterial antigen induced T cell activation

R. De Jong ‘, A. Janson ‘, W. Faber3. B. Naafs2, T.H.M. Ottenhoff ‘. ‘Dept. lmmunohematol~y & Bloodbank, Univ. Hospital Leiden, The Netherlands, 2 Dept. Dermatd, Univ. Hospital Leiden, The Netherlands, 3 Dept. Dermatol., AC. Med. Center, Univ of Amsterdam, The Netherlands

Introduction: The interaction of CD28 with 87 family molecules (B7-l/CD80 and B7-2/CD88) on APC has been shown to provide an important costimulatory signal for T cell activation. Data that have been obtained on a selective role of CD80 and CD88 in directing selective TH subset responses are not conclusive.

We studied the contribution of costimulation by CD80 and CD88 molecules, respectively, in T cell proliferative responses dtiven by M. lepme.

Materlala and Methods: PBMC of leprosy patients (BT-lT, n = 10) or M. /epme specific Th cell clones were stimulated with M. lepme or the purified protein derivative (PPD) of M.tuberculosis. The role of co-stimulation via CD80 and CD88 molecules in the induction of ag-specific T cell proliferation was tested by the addition of various CD80 mAb, CD88 mAb and by addition of CTLA-4lg fusion protein. Proliferative responses were measured at day 7.

Resuh The results indicate that M. @me induced pmliiemtive responses are inhibited for 25 f 5% (n = 10) by CD80 mAb whereas the responses are inhibited for 82 f 5% by CD88 mAb (n = 10). The combination of CD80 mAb and CD88 mAb have an additive effect resulting in an inhibition of 89 f 3% (n = 10). The critical role of 87 molecules was confirmed in experiments in which CTLA-4lg contruct was used to prevent 87 molecules from interacting with their counter-receptor(s). CTLA4lg was equally potent in blocking M. repme induced proliferation as the combination of CD80 mAb and CD88 mAb. Similar data were obtained following stimulation with PPD. No effects on M. /epma induced proliferation was obsenred by interference of CD4O-CD40 Ligand interaction or CD27-CD70 interaction. In contrast with PBMC, M. lepme dependent prolifera- tion of differentiated Thl call clones was more dependent on CD80 costimulation than on CD88 mediated co-stimulation. These results with Th cell clones were seen irrespective whether monocytes or EBV-B cells were used as APC.

Conclusion: The data show that in vitro primaty responses of PBMC from BT-TT leprosy patients directed against M. lepme are dependent on co-stimula- tion via CD88 rather than CD80 molecules. The proliferative responses of fully differentiated Thl clones on the other hand are more dependent on CD80 than on CD88 molecules, suggesting that the requirements for co-stimulation by 87 molecules is related to the maturation/differentiation stage of the cells. Studies are currently being performed to compare these results on Thl type responses with the involvement of CD80 and CD88 molecules in Th2 type responses.

1 0.4.11.6 1 The secreted M. fubefw/os/s ESAT8 antigen is frequently recognized by human T cells during natural infection

AS. Mustafa’, H. Amoudy I, F. Oftung*, A.T. Aba13, H.G. Weiker4, P. Ravn5, P. Andersen 5. ’ Department of Microbiology Faculty of Medicfne, Kuwait Universih! Kuwait. * Dent of V’inolwv. The National lnstitute of Public Health, &lo. No&y, %?hest Disease~iiospital, Ministry of Public Health, Kuwait, 4 IGRI, The National Hospital Oslo, Norwav. 5 TB Reseat& Unit, Statens Serum Institut, Copenh&en, Denmark .

Introduction: The M. tuberculosis ESATG protein antigen, which is present in culture filtrate, is one of the major antigen fractions recognized by T cells duling recall of a protective immune response against challenge in a mouse model. The objective of this study was to investigate if this antigen was recognized by human T cells during infection with M. tubemukx.is.

Materials and Methods: Peripheral blood mononuclear cells (PBMC) and culture filtrate induced T cell lines from tuberculosis patients in Kuwait were screened for proliferative reactivity by 3H-thymidine incorporation against well defined secreted (ESATG, MPT59, MPT84 and MPB70), somatic (TblO, Tb38, L28, Tb85 and Tb70) and complex antigens like M. tubenx/osis, its culture filtrate and M. bovis BCG. IFNy in culture supematants was measured by an ELISA kit.

Reautta: Both by using PBMC and antigen induced T cell lines, the results demonstrated that the secreted ESATG antigen was frequently recognized by human T cells during infection with M. tubercukxis, but not vaccination with BCG in a nonendemic region. The frequency of ESATG responders, as measured both by antigen specific proliferation and IFN-y secretion, were comparable to the results obtained with complex antigens like culture filtrate, whole M. fubercukxis and M. bovis BCG. Based on the same criteria, the ESATG antigens scores superior to other secreted and nonsecreted cytosolic antigens tested in this study.

Conclusion: We have shown that the M. tubercuhxis ESATG antigen is frequently recognized by human T cells duling natural infection of humans with M. tuberculosis.