Single-cell characterization and quantification of translation-competent viral reservoirs in treated and untreated HIV infection

Abstract

The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins α4β7 and α4β1. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/106 cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin α4β1. Remarkably, α4β1 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir.

TIGIT is upregulated by HIV-1 infection and marks a highly functional adaptive and mature subset of natural killer cells

“…Early initiation of antiretroviral treatment (ART) in HIV infected individuals does not return TIGIT/CD115 to normal levels on CD8 + T cells [37]. Additionally, the co-expression of TIGIT with immune checkpoint inhibitor PD-1 marks CD4 + T cells harboring latent virus [45][46][47]. These data suggest that the TIGIT/CD155/CD112 pathway in T cells could contribute to HIV pathogenesis.…”

Complementation can maintain a quasispecies of drug sensitive and resistant HIV

“…Other studies did not show multiple infections at a frequency greater than that predicted by the Poisson distribution ( Josefsson et al ( 2011 , 2013)). Given that many proviruses are not expressed ( Bruner et al ( 2016 )) and that cells with multiple infections may be more likely to express HIV and be infectious ( Wodarz and Levy ( 2017 )), it is possible that the multiplicity of infection may be higher than predicted by Poisson in cells where HIV is actively replicating ( Pardons et al ( 2019 )). Other possibilities where localized multiple infection per cell can occur is in cell subsets ( Banga et al ( 2016 )) where HIV infection is particularly efficient in lymph nodes or gut ( Fletcher et al ( 2014 ); Brenchley et al ( 2004 ); Deleage et al ( 2016 )).…”

Reduce and Control: A Combinatorial Strategy for Achieving Sustained HIV Remissions in the Absence of Antiretroviral Therapy

“…All previously described "block and lock" approaches require at least minimal viral transcription and/or translation to be effective. However, recent studies suggest that viral reservoirs are mostly devoid of significant virus replication [174] and HIV gene expression [175,176]. Thus, most latently infected cells would not be affected by treatments targeting either viral transcripts or viral proteins, except during the narrow window of viral reactivation.…”