A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals

Background: Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging. Methods: We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days. Findings: In suppressed individuals on ART, we found the median frequency of latently infected CD4+ T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6–27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4+ cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART. Interpretations: Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation. Research in context: In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir

CD32 expression is associated to T-cell activation and is not a marker of the HIV-1 reservoir

CD32 has been shown to be preferentially expressed in latently HIV-1-infected cells in an in vitro model of quiescent CD4 T cells. Here we show that stimulation of CD4+ T cells with IL-2, IL-7, PHA, and anti-CD3/CD28 antibodies induces T-cell proliferation, co-expression of CD32 and the activation of the markers HLA-DR and CD69. HIV-1 infection increases CD32 expression. 79.2% of the CD32+/CD4+ T cells from HIV+ individuals under antiretroviral treatment were HLA-DR+. Resting CD4+ T cells infected in vitro generally results in higher integration of provirus. We observe no difference in provirus integration or replication-competent inducible latent HIV-1 in CD32+ or CD32− CD4+ T cells from HIV+ individuals. Our results demonstrate that CD32 expression is a marker of CD4+ T cell activation in HIV+ individuals and raises questions regarding the immune resting status of CD32+ cells harboring HIV-1 proviruses. CD32 has been previously shown to be expressed preferentially by CD4 T cells latently harbouring HIV-1. Here the authors show that CD32 expression in CD4 T cells is associated with T cell activation, is up-regulated by HIV-1 infection and importantly does not appear to represent an enriched cellular niche for latent HIV-1

The Different Modes of Resistance to AIDS: Lessons from HIV/SIV Controllers and SIV Natural Hosts.

International audienceHuman immunodeficiency virus (HIV) infection in humans does not always lead to AIDS. Rare HIV-1-infected individuals known as HIV controllers do not show signs of disease progression in the long term and spontaneously control HIV replication to very low levels in the absence of therapy. Simian species that are naturally infected by Simian immunodeficiency virus (SIV) in the wild provide an apparently radically different model of resistance to AIDS, as the plasma viral load remains persistently high in these species without obvious pathogenic consequences. Infections in HIV controllers and SIV natural hosts differ in several major points, such as the strength and avidity of the antiviral T-cell response, which are remarkably high in controllers. However, close examination of the human and simian models also reveals common threads, highlighting key elements involved in resisting AIDS: these include contained levels of chronic immune activation, a downregulation of the interferon response, a low viral load in lymph nodes, and a relative protection from infection of certain CD4+ T-cell subsets, including central memory cells and possibly follicular helper T cells. Studies of nonpathogenic HIV and SIV infections thus point to the importance of protecting the lymphoid environment from massive infection to avoid the triggering of a deleterious chronic immune activation, a notion that may inform the development of immunotherapeutic and vaccine approaches against HIV