A molecular assay for sensitive detection of pathogen-specific T-cells

Victoria O. Kasprowicz; Jessica E. Mitchell; Shivan Chetty; Pamla Govender; Kuan Hsiang Gary Huang; Helen A. Fletcher; Daniel P. Webster; Sebastian Brown; Anne Kasmar; Kerry Millington; Cheryl L. Day; Nompumelelo Mkhwanazi; Cheryl McClurg; Fundisiwe Chonco; Ajit Lalvani; Bruce D. Walker; Thumbi Ndung'u; Paul Klenerman

doi:10.1371/journal.pone.0020606

A molecular assay for sensitive detection of pathogen-specific T-cells

Victoria O. Kasprowicz, Jessica E. Mitchell, Shivan Chetty, Pamla Govender, Kuan Hsiang Gary Huang, Helen A. Fletcher, Daniel P. Webster, Sebastian Brown, Anne Kasmar, Kerry Millington, Cheryl L. Day, Nompumelelo Mkhwanazi, Cheryl McClurg, Fundisiwe Chonco, Ajit Lalvani, Bruce D. Walker, Thumbi Ndung'u, Paul Klenerman

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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.

Original language	English
Article number	e20606
Journal	PLoS ONE
Volume	6
Issue number	8
DOIs	https://doi.org/10.1371/journal.pone.0020606
Publication status	Published - 1 Jan 2011
Externally published	Yes

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Kasprowicz, V. O., Mitchell, J. E., Chetty, S., Govender, P., Huang, K. H. G., Fletcher, H. A., Webster, D. P., Brown, S., Kasmar, A., Millington, K., Day, C. L., Mkhwanazi, N., McClurg, C., Chonco, F., Lalvani, A., Walker, B. D., Ndung'u, T., & Klenerman, P. (2011). A molecular assay for sensitive detection of pathogen-specific T-cells. PLoS ONE, 6(8), Article e20606. https://doi.org/10.1371/journal.pone.0020606

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title = "A molecular assay for sensitive detection of pathogen-specific T-cells",

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.",

author = "Kasprowicz, \{Victoria O.\} and Mitchell, \{Jessica E.\} and Shivan Chetty and Pamla Govender and Huang, \{Kuan Hsiang Gary\} and Fletcher, \{Helen A.\} and Webster, \{Daniel P.\} and Sebastian Brown and Anne Kasmar and Kerry Millington and Day, \{Cheryl L.\} and Nompumelelo Mkhwanazi and Cheryl McClurg and Fundisiwe Chonco and Ajit Lalvani and Walker, \{Bruce D.\} and Thumbi Ndung'u and Paul Klenerman",

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Kasprowicz, VO, Mitchell, JE, Chetty, S, Govender, P, Huang, KHG, Fletcher, HA, Webster, DP, Brown, S, Kasmar, A, Millington, K, Day, CL, Mkhwanazi, N, McClurg, C, Chonco, F, Lalvani, A, Walker, BD, Ndung'u, T & Klenerman, P 2011, 'A molecular assay for sensitive detection of pathogen-specific T-cells', PLoS ONE, vol. 6, no. 8, e20606. https://doi.org/10.1371/journal.pone.0020606

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AU - Kasprowicz, Victoria O.

AU - Mitchell, Jessica E.

AU - Chetty, Shivan

AU - Govender, Pamla

AU - Huang, Kuan Hsiang Gary

AU - Fletcher, Helen A.

AU - Webster, Daniel P.

AU - Brown, Sebastian

AU - Kasmar, Anne

AU - Millington, Kerry

AU - Day, Cheryl L.

AU - Mkhwanazi, Nompumelelo

AU - McClurg, Cheryl

AU - Chonco, Fundisiwe

AU - Lalvani, Ajit

AU - Walker, Bruce D.

AU - Ndung'u, Thumbi

AU - Klenerman, Paul

PY - 2011/1/1

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AB - 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.

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DO - 10.1371/journal.pone.0020606

M3 - Article

VL - 6

JO - PLoS ONE

JF - PLoS ONE

IS - 8

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ER -

A molecular assay for sensitive detection of pathogen-specific T-cells

Abstract

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