(588j) Nucleic Acid Cytometry of Rare Intact HIV Reservoir Cells

In the U.S., approximately 1.2 million individuals are living with HIV, with 38,100 new diagnoses reported in 2022 [1]. Managing HIV requires lifelong antiretroviral therapy (ART), creating a substantial economic burden that averages $420,000 per patient and totals approximately $2.25 billion annually nationwide [2]. Lifelong ART is required to prevent HIV rebound, which occurs within weeks of treatment interruption from blood and tissue CD4+ T cell reservoirs. Although HIV-infected cells constitute less than 1% of the total CD4+ T cell population, only approximately 0.01% (about 100 per million CD4⁺ T cells) harbor intact, replication-competent proviruses capable of producing infectious virion [3]. This reservoir is considered the primary barrier to a cure, but the molecular mechanisms that control virus persistence remain incompletely understood, primarily due to the difficulty in isolating and studying these exceedingly rare reservoir cells ex vivo.

We developed FIND-seq (Focused Interrogation of Cells by Nucleic Acid Detection and Sequencing), a droplet-based microfluidics platform designed to selectively isolate and RNA-sequence rare HIV-infected cells using nucleic acid biomarkers [4]. In this approach, single cells are encapsulated into 50-micron agarose hydrogel beads, and cellular mRNA is reverse-transcribed to cDNA using agarose functionalized oligo dT primers. The cDNA-containing beads are reinjected into droplets containing TaqMan reagents and subjected to thermocycling. Finally, fluorescent droplets are sorted and RNA-sequenced [5].

In its original implementation, FIND-seq targeted a single region of the HIV genome (gag), which was insufficient to differentiate intact from defective proviruses, limiting the application of this technology for reservoir studies. To address this, we implemented the intact proviral DNA assay (IPDA) [3], which uses multiplexed detection of two distinct HIV genomic regions (psi, env). We validated the FIND-seq IPDA assay using a J.Lat 6.3 cell line model of HIV latency that harbors a near-intact provirus, achieving over 90% psi+ env+ detection. To assess the sensitivity of FIND-seq, we successfully recovered the transcriptomes of HIV+ J.Lat cells diluted into HIV- Jurkat cells at frequencies as low as 0.1%. These results demonstrate that FIND-seq can accurately identify and isolate rare HIV-infected cells with intact proviruses, providing a powerful platform for understanding the biology of HIV persistence.

References

[1] Centers for Disease Control and Prevention. National HIV Progress Report, 2024;(No. 1).

[2] Colson, Amy, et al. "Health care resource utilization and costs for treatment-experienced people with HIV switching or restarting antiretroviral regimens since 2018." Journal of Managed Care & Specialty Pharmacy 30.8 (2024): 817-824.

[3] Bruner, Katherine M., et al. "A quantitative approach for measuring the reservoir of latent HIV-1 proviruses." Nature 566.7742 (2019): 120-125.

[4] Clark, Iain C., et al. "HIV silencing and cell survival signatures in infected T cell reservoirs." Nature 614.7947 (2023): 318-325.

[5] Shin, Seung Won, et al. "FIND-seq: high-throughput nucleic acid cytometry for rare single-cell transcriptomics." Nature Protocols 19.11 (2024): 3191-3218.