TILDA: Tat/Rev Induced Limiting Dilution Assay

Area of Science:

• Virology
• Immunology
• Molecular Biology

Background:

• Quantifying the latent HIV-1 reservoir is crucial for developing effective cure strategies.
• Current methods like HIV-1 DNA measurements may overestimate reservoir size due to including defective viral genomes.
• A more precise method is needed to accurately assess the burden of HIV-1 infection.

Purpose of the Study:

• To describe the execution of the Tat/rev Induced Limiting Dilution Assay (TILDA) for quantifying the HIV-1 reservoir.
• To highlight the advantages of TILDA, including reduced overestimation and suitability for clinical settings.
• To provide detailed guidance on cell preparation and limiting dilution schemes for TILDA implementation.

Main Methods:

• TILDA utilizes a limiting dilution format to probe for tat and rev multiply spliced HIV-1 RNA.
• This method estimates the frequency of latently infected cells based on the presence or inducibility of specific viral RNA.
• Emphasis is placed on optimized cell preparation and the limiting dilution strategy.

Main Results:

• TILDA reduces overestimation of the HIV-1 reservoir size compared to traditional HIV-1 DNA measurements.
• Multiply spliced HIV-1 RNA is less likely to be transcribed from replication-defective genomes, improving accuracy.
• The assay is easy to perform, requires minimal cell input, and offers a rapid turnaround time.

Conclusions:

• TILDA presents a viable and accurate alternative for quantifying the latent HIV-1 reservoir.
• Its efficiency and accuracy make it ideal for clinical research and monitoring treatment efficacy.
• Standardized protocols for cell preparation and limiting dilution are key to successful TILDA execution.