A Molecular Assay for Sensitive Detection of Pathogen-Specific T-Cells

• Type of Material: Article
• Authors: Victoria O. Kasprowicz, Harvard University; Jessica E. Mitchell, Harvard University; Shivan Chetty, Nelson R. Mandela School of Medicine; Pamla Govender, Nelson R. Mandela School of Medicine; Kuan-Hsiang Gary Huang, University of Oxford; Helen A. Fletcher, University of Oxford; Daniel P. Webster, University of Oxford; Sebastian Brown, University of Oxford; Anna Kasmar, Harvard University; Kerry Millington, Imperial College London; Cheryl Day, Emory University; Nompumelelo Mkhwanazi, Nelson R. Mandela School of Medicine; Cheryl McClurg, Harvard University; Fundisiwe Chonco, Nelson R. Mandela School of Medicine; Ajit Lalvani, Imperial College London; Bruce D. Walker, Harvard University; Thumbi Ndung'u, Harvard University; Paul Klenerman, University of Oxford
• Language: English
• Date: 2011-08-11
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2011 Kasprowicz et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0020606
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 6
• Issue: 8
• Start Page: e20606
• End Page: e20606
• Grant/Funding Information: The authors would like to thank the Howard Hughes Medical Institute, the KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH), the Wellcome Trust, the James Martin School for 21st Century (Oxford), the NIHR Biomedical Research Centre Programme (Oxford), the MRC, and the National Institutes of Health (NIH) (National Institute of Allergy and Infectious Diseases [NIAID] 1U19AI082630-01).
• Keywords: DISEASE; Science & Technology - Other Topics; IFN-GAMMA; INTERFERON-GAMMA; IP-10; LATENT TUBERCULOSIS INFECTION; Multidisciplinary Sciences; DIAGNOSIS; TOOLS; Science & Technology; EXPRESSION; MYCOBACTERIUM-TUBERCULOSIS; CHILDREN
• Research Categories: Engineering, Biomedical; Health Sciences, Immunology

Abstract

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters - monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.