Single-Cell Characterization of Viral Translation-Competent Reservoirs in HIV-Infected Individuals

Abstract

SUMMARY HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5–1 gag-pol mRNA+/Gag protein+ infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia, and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally-suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART

“…The method described here, CARD-SGS, can assess the genetics of HIV RNA in single cells collected from both viremic and virologically suppressed individuals; in combination with proviral quantification (9) and end-point dilution of proviruses, it can also be used to estimate the fraction of all proviruses that express HIV RNA. Although other methods, such as florescence in situ hybridization, are capable of detecting HIV RNA in single infected cells (30,31), such techniques cannot be used to assess the genetics of the expressed proviruses (or the latent ones), and therefore cannot be used determine the fraction of expressed proviruses that are in expanded clones or that are constitutively expressed (i.e., without ex vivo stimulation). The essential features of CARD-SGS are dilution of cells to the end point, or near the end point, for HIV RNA expression before nucleic acid extraction and use of very efficient methods for the extraction of cellassociated HIV DNA and HIV RNA, for cDNA synthesis, and for PCR of single DNA or cDNA templates (less than 30% of PCR reactions positive for 1.3-kb p6-PR-RT amplicon and informatic removal of the few remaining mixtures).…”

Immunologic characteristics of HIV‐infected individuals who make broadly neutralizing antibodies

“…Furthermore, Tfh cell populations in both LNs and blood constitute a major (although not the only) site of SIV/HIV latency in macaques/humans receiving antiretroviral therapy 171, 176, 177, 178, 179. GC Tfh cells are not only highly activated cells that are good targets for HIV‐1 infection but are also located in close proximity to FDCs, which are an important reservoir of infectious virus and can readily transmit infection to Tfh cells 180, 181, 182, 183.…”

Relationship between Measures of HIV Reactivation and Decline of the Latent Reservoir under Latency-Reversing Agents

“…The other challenge is identifying all the effects of these drugs in the context of HIV (32). New tools that detect the frequency of HIV RNA-producing cells could, for example, clarify if they facilitate expansion of infected cells (34). Above all, we need a better understanding of the mechanisms of maintenance and decay of the reservoir and the role that activation plays in the decay in order to decide if virus reactivation is the best approach or if interventions that, for example, enhance cellular apoptosis in infected cells (35) would yield better results.…”