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

Area of Science:

• Immunology
• Molecular Biology
• Infectious Diseases

Background:

• Interferon-gamma (IFN-γ) is a key cytokine in cell-mediated immunity.
• Accurate measurement of antigen-specific IFN-γ is crucial for diagnosing and monitoring infectious diseases.
• Existing assays may have limitations in sensitivity or applicability to certain patient populations.

Purpose of the Study:

• To develop and validate a highly sensitive assay for measuring antigen-specific IFN-γ production.
• To apply the assay for detecting immune responses to Cytomegalovirus (CMV), Human Immunodeficiency Virus (HIV), and Mycobacterium tuberculosis (MTB).
• To compare the sensitivity of the new assay with the established IFN-γ Elispot assay.

Main Methods:

• Development and validation of a real-time quantitative PCR (qPCR) assay.
• Utilizing monokine-induced by IFN-γ (MIG) and IFN-γ inducible protein-10 (IP10) as reporters.
• Application to whole blood samples from HIV co-infected patients.

Main Results:

• The qPCR assay demonstrated high sensitivity for detecting MTB-specific T-cell responses.
• A significant quantitative correlation was observed between the qPCR assay and the IFN-γ Elispot assay (P<0.001).
• The assay successfully detected CMV, HIV, and MTB-specific responses and was not affected by HIV-mediated immunosuppression.

Conclusions:

• The developed qPCR assay is a sensitive tool for detecting antigen-specific IFN-γ production.
• The assay can be performed on small whole blood volumes (50 µL) obtained via fingerprick.
• This assay platform holds potential utility for diagnosing infections in various clinical settings, including those with HIV co-infection.