An Immunocytochemistry Method to Investigate the Translationally Active HIV Reservoir

Area of Science:

• Virology
• Immunology
• Biotechnology

Background:

• Combination antiretroviral therapy (cART) suppresses HIV replication but does not eliminate the virus.
• HIV persists in a long-lived reservoir, leading to viral rebound upon cART cessation.
• Active HIV reservoir cells contribute to chronic immune activation in people living with HIV (PLWH).

Purpose of the Study:

• To develop and validate a novel automated immunocytochemistry (ICC) assay for detecting and quantifying intracellular HIV Gag capsid protein (CA).
• To establish a method for measuring the translationally active HIV reservoir.

Main Methods:

• Developed an automated immunocytochemistry assay (CA-ICC) using anti-CA antibodies and computational image analysis.
• Validated CA-ICC against digital ELISA, qPCR, and flow cytometry using cell lines and primary cells.
• Assessed CA-ICC sensitivity by limiting dilution and specificity using HIV-seronegative and infected cells.
• Applied CA-ICC to detect HIV CA and SIV p27 in PBMCs from animal models and clinical specimens.

Main Results:

• CA-ICC demonstrated high specificity and sensitivity, detecting as few as 10 CA-positive cells per million.
• The assay's sensitivity surpassed that of flow cytometry.
• Quantified intracellular CA in animal models correlated with plasma viral loads and cell-associated CA.
• CA-ICC was successfully applied to evaluate therapeutic interventions in clinical specimens.

Conclusions:

• CA-ICC is a simple, specific, and sensitive imaging method for detecting CA-positive cells in bulk preparations.
• This assay provides a valuable tool for quantifying the active HIV reservoir.
• CA-ICC has potential applications in pre-clinical research and evaluating therapeutic strategies for HIV.