Exploring diagnostic opportunities in active and latent TB: Stratifying transmission risk using PCR, and identification of immunogenic CD8+ T-cell epitopes

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling

Background: Tuberculosis remains a major health issue worldwide. One of several obstacles for effective handling of in-hospital patients with TB is the need for long-term in-hospital isolation, also for patients who subsequently turn out not to have (contagious) TB. Another obstacle to TB control is the vast number of latently infected individuals, and the lack of immunodiagnostic tests with the power to discriminate latent from active TB, or to predict subsequent development of TB. Detection of M. tuberculosis (MTB) specific CD8+ T-cells may be a key to improved immunologic tests. Aims: 1. To evaluate the potential of shortening isolation of patients with suspected TB by relying on a single sputum-sample polymerase chain reaction (PCR)-analysis for MTB DNA 2. To establish and validate a panel of HLA A*0201-restricted viral and endogenous epitopes that are recognized by CD8+ T-cells in the blood from >95% healthy and MTB infected persons 3. To establish a panel of MTB-derived epitopes readily recognized by CD8+ T-cells in MTB-infected individuals, by evaluation of CD8+ T-cell recognition of previously described and newly predicted MTB epitopes, restricted by commonly expressed HLA A genotypes. Materials and methods: Study I: We evaluated all culture-confirmed TB cases in Denmark from 2002-2011 with ≥3 samples evaluated within 14 days before or after the initial culture-positive sample Hereof, we identified the occurrence of smear-positive, first-sample PCR negative patients. We repeated the process identifying persons with ≥2 initial samples. Study II: We identified a set of HLA A*0201 restricted peptides from viral antigens and melanoma targets through literature search and in-silico prediction. MHC Dextramers presenting these epitopes were analyzed on PBMC from healthy blood donors, using flow cytometry. The best performing epitopes were tested on PBMC from patients undergoing testing for M. Tuberculosis (MTB) infection to assess the coverage of the epitope panel as a positive control. Study III: MTB-derived, HLA A*0101- HLA A*0201- or HLA A*2402-restricted epitopes from 12 previously described immunogenic antigens were identified from literature search and computer prediction. We analyzed the presence of antigen-specific T-cells against these epitopes in the blood of healthy, MTB-exposed persons, and in patients with latent MTB infection (LTBI) and active TB, using flow cytometry and MHC Dextramer staining. Results: Study I: A total of 1636 sputum culture confirmed TB cases were identified. 482 had min. 3 samples analyzed within 28 days, and at least one PCR result. Nine patients (2.5% of smear-positive patients) were smear-positive and PCR negative. Of 722 patients with 2 samples within 28 days, 7 (1.3% of smear-positive patients) were smear positive/PCR negative. Most smear positive, PCR negative patients had only one low-grade positive smear. Study II: Twenty-one candidate epitopes were identified and tested in PBMC from 18 healthy blood donors. Antigen-specific T-cells could be detected against 12 (57,1%) of the epitopes. We selected the 6 best-performing epitopes and demonstrated positive response in 42 (97.7%) of 43 patient samples (healthy, latent and active MTB infection) Study III: A total of 44 MHC Dextramers presenting MTB epitopes were successfully constructed. Among 18 previously described epitopes, only a single HLA A*0201-restricted Ag85B-epitope revealed specific CD8+ T-cells in our study. Furthermore, we identified five new epitopes, representing the antigens Ag85B, rv1490, rv3615, rv2626c; but each epitope yielded response in only few patient samples. Conclusions: Study I: As a single sputum-sample analyzed with PCR for MTB identifies >97% of smear-positive TB patients, and as the majority of missed smear-positive TB patients have only one low-grade smear, de-isolation of patients with a single negative sputum PCR-result is safe. Study II: Six HLA A*0201-restricted epitopes from five common vira and one endogenous target yield antigen-specific CD8+ T-cells in >97% of healthy indiviuals and patients in different stages of MTB-infection. Thus, this panel of epitopes suffices for use as a positive control in ex vivo MHC Dextramer-detection of HLA A*0201-restricted antigen-specific CD8+ T-cells. Study III: The CD8+ T-cell response to MTB is highly variable and unpredictable, targeting a wide panel of differently expressed antigens. However, the novel epitopes described here could play a role in future immunodiagnostic tools as well as in vaccine development, if confirmed in different studies and settings.
StatusUdgivet - 21 feb. 2018

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