Q4ddPCR (May the Fourth Be Precise): A Flexible, 4-Target Assay for High-Resolution HIV Reservoir Profiling.

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Accurate quantification of the intact HIV reservoir is essential for developing curative strategies.
• Existing assays like IPDA face challenges with quantification failures and misclassification of defective HIV genomes due to sequence variability.

Purpose of the Study:

• To introduce and validate Q4ddPCR, a novel droplet digital PCR assay designed for precise and scalable quantification of the intact HIV reservoir.
• To compare the performance of Q4ddPCR against established methods using proviral sequences and clinical samples.

Main Methods:

• Development of Q4ddPCR, a modular assay targeting four conserved HIV genome regions for enhanced specificity and multi-layered readouts.
• Benchmarking Q4ddPCR against 3,650 near full-length proviral sequences and comparing results with IPDA in independent cohorts.

Main Results:

• Q4ddPCR achieved intact reservoir quantification in 95% of samples across three cohorts, closely aligning with sequence-confirmed Q4PCR measurements.
• Multi-probe readouts from Q4ddPCR identified clonal intact reservoir dynamics not detectable by IPDA.
• Longitudinal analysis showed Q4ddPCR reported lower proviral frequencies and a faster decline in intact HIV DNA compared to IPDA.

Conclusions:

• Q4ddPCR offers improved specificity and reduced quantification gaps for intact HIV reservoir measurement.
• The multi-target assay accurately captures the dynamics of the intact HIV reservoir, confirming predictions from mathematical modeling.