Sara Svensson Akusjärvi [1], Wang Zhang[1,2], Anders Sönnerborg[1,3], Ujjwal Neogi[1]

Affiliates

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden[1], Science for Life Laboratory, Division of Proteomics and Nanobiotechnology, KTH Royal Institute of Technology, Stockholm, Sweden[2], Department of Medicine Huddinge, Unit of Infectious Diseases, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden[3]

Abstract

Background:
There are currently no reliable markers for quantification of latently HIV-1-infected cells in vivo nor in vitro. Also, available techniques exhibit large discrepancies in detected viral RNA (vRNA) and viral DNA (vDNA) rendering a hindrance in characterization of latent HIV-1 reservoir. Understanding the source and characteristics of latency on a single cell level is a necessity for overcoming the barriers to an HIV cure. Previously, we have shown that the proportion of latently HIV-1-infected cells can efficiently be quantified on a single-cell level in vitro using RNAscope ISH technology. Herein we employed this technique to assess its sensitivity towards various HIV-1 subtypes and ex vivo usage in clinical samples after combinatory latency reversal agents (cLRA) stimulation.

Methods:
The RNAscope technology was earlier evaluated and optimized by our group (as shown last year by Wang Zhang) to retain maximal cell coverage. Here, the HIV subtype B (HIV-1B) probe set, consisting of 20 zz probes targeting the gag-pol region, was evaluated for subtype sensitivity on patient-derived subtypes HIV-1A1, HIV-1B, HIV-1C and CRF01_AE recombinant plasmids. Plasmids were transfected into HEK293T cells and vRNA detection was complemented with a p24 antibody staining for validation of viral reactivation in each cell. Further, the RNAscope technique was employed on PBMCs from two HIV-1 therapy naïve progressors. PBMCs were treated with or without cLRA stimulation using Prostatin (6μM) and hTNFα (10ng/μL). Wide-field analyses allowed for automated signal detection to quantify proportion of transcriptional inducible, replication competent HIV in each sample.

Results:
The probe set targeting HIV-1B was capable of detecting patient-derived subtypes HIV-1A1, HIV-1B, HIV-1C and CRF01_AE indicating that these probes target a seemingly conserved region within Gag. Detected vRNA that coincided with positive p24 staining validated transcriptional activation of replication competent HIV-1 in the infected cells. Importantly, we could quantify the proportion of transcriptional inducible, replication competent HIV-1 in two therapy naïve progressors. In the absence of cLRA, we detected 319 and 54 HIVvRNA+/protein+ cells per million PBMCs whereas after cLRA stimulation we detected 710 and 184 HIVvRNA+/protein+ cells per million PBMCs, respectively. This increase in transcriptional activation could reflect the proportion of latently HIV-1-infected cells that became reactivated by cLRA stimuli or induction of viral replication. These results show the translational potential of dual RNAscope-protein detection from in vitro model to ex vivo application.

Conclusion:
Herein the RNAscope technology showed the capacity of one probe set to detect several HIV-1 M group subtypes together at single-cell resolution with a translational capacity to quantify latently HIV-1 infected cells ex vivo.