RESUMEN
Antiretroviral drugs that target various stages of the Human Immunodeficiency Virus (HIV) life cycle have been effective in curbing the AIDS epidemic. However, drug resistance, off-target effects of antiretroviral therapy (ART), and varying efficacy in prevention underscore the need to develop novel and alternative therapeutics. In this study, we investigated whether targeting the signaling molecule Sphingosine-1-phosphate (S1P) would inhibit HIV-1 infection and generation of the latent reservoir in primary CD4 T cells. We show that FTY720 (Fingolimod), an FDA-approved functional antagonist of S1P receptors, blocks cell-free and cell-to-cell transmission of HIV and consequently reduces detectable latent virus. Mechanistically, FTY720 impacts the HIV-1 life cycle at two levels. Firstly, FTY720 reduces the surface density of CD4, thereby inhibiting viral binding and fusion. Secondly, FTY720 decreases the phosphorylation of the innate HIV restriction factor SAMHD1 which is associated with reduced levels of total and integrated HIV, while reducing the expression of Cyclin D3. In conclusion, targeting the S1P pathway with FTY720 could be a novel strategy to inhibit HIV replication and reduce the seeding of the latent reservoir.
Asunto(s)
Clorhidrato de Fingolimod/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/crecimiento & desarrollo , Proteína 1 que Contiene Dominios SAM y HD/antagonistas & inhibidores , Moduladores de los Receptores de fosfatos y esfingosina 1/farmacología , Linfocitos T/inmunología , Replicación Viral , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/inmunología , Humanos , Lisofosfolípidos/metabolismo , Fosforilación , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Linfocitos T/efectos de los fármacos , Latencia del VirusRESUMEN
The HIV reservoir, which comprises diverse proviruses integrated into the genomes of infected, primarily CD4+ T cells, is the main barrier to developing an effective HIV cure. Our understanding of the genetics and dynamics of proviruses persisting within distinct CD4+ T cell subsets, however, remains incomplete. Using single-genome amplification, we characterized subgenomic proviral sequences (nef region) from naive, central memory, transitional memory, and effector memory CD4+ T cells from five HIV-infected individuals on long-term combination antiretroviral therapy (cART) and compared these to HIV RNA sequences isolated longitudinally from archived plasma collected prior to cART initiation, yielding HIV data sets spanning a median of 19.5 years (range, 10 to 20 years) per participant. We inferred a distribution of within-host phylogenies for each participant, from which we characterized proviral ages, phylogenetic diversity, and genetic compartmentalization between CD4+ T cell subsets. While three of five participants exhibited some degree of proviral compartmentalization between CD4+ T cell subsets, combined analyses revealed no evidence that any particular CD4+ T cell subset harbored the longest persisting, most genetically diverse, and/or most genetically distinctive HIV reservoir. In one participant, diverse proviruses archived within naive T cells were significantly younger than those in memory subsets, while for three other participants we observed no significant differences in proviral ages between subsets. In one participant, "old" proviruses were recovered from all subsets, and included one sequence, estimated to be 21.5 years old, that dominated (>93%) their effector memory subset. HIV eradication strategies will need to overcome within- and between-host genetic complexity of proviral landscapes, possibly via personalized approaches.IMPORTANCE The main barrier to HIV cure is the ability of a genetically diverse pool of proviruses, integrated into the genomes of infected CD4+ T cells, to persist despite long-term suppressive combination antiretroviral therapy (cART). CD4+ T cells, however, constitute a heterogeneous population due to their maturation across a developmental continuum, and the genetic "landscapes" of latent proviruses archived within them remains incompletely understood. We applied phylogenetic techniques, largely novel to HIV persistence research, to reconstruct within-host HIV evolutionary history and characterize proviral diversity in CD4+ T cell subsets in five individuals on long-term cART. Participants varied widely in terms of proviral burden, genetic diversity, and age distribution between CD4+ T cell subsets, revealing that proviral landscapes can differ between individuals and between infected cell types within an individual. Our findings expose each within-host latent reservoir as unique in its genetic complexity and support personalized strategies for HIV eradication.
Asunto(s)
Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos/virología , Variación Genética , VIH-1/genética , Provirus/genética , Adolescente , Secuencia de Bases , Niño , ADN Viral/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Filogenia , Subgrupos de Linfocitos T/virología , Carga Viral , Adulto JovenRESUMEN
Although chronic viral infections have evolved mechanisms to interfere with aspects of pathogen recognition by dendritic cells (DCs), the role that these APCs play in virus-specific T cell exhaustion is unclear. Herein we report that NS3-dependent suppression of Toll/IL-1 domain-containing adapter-inducing IFN-beta- and IFN-beta promoter stimulator-1- but not MyD88-coupled pathogen-recognition receptor-induced synthesis of proinflammatory cytokines (IL-12 and TNF-alpha) from DCs by hepatitis C virus (HCV) is a distinctive feature of a subgroup of chronically infected patients. The result is decreased CD8(+) T cell polyfunctional capacities (production of IFN-gamma, IL-2, TNF-alpha, and CD107a mobilization) that is confined to HCV specificities and that relates to the extent to which HCV inhibits DC responses in infected subjects, despite comparable plasma viral load, helper T cell environments, and inhibitory programmed death 1 receptor/ligand signals. Thus, subjects in whom pathogen-recognition receptor signaling in DCs was intact exhibited enhanced polyfunctionality (i.e., IL-2-secretion and CD107a). In addition, differences between HCV-infected patients in the ability of CD8(+) T cells to activate multiple functions in response to HCV did not apply to CD8(+) T cells specific for other immune-controlled viruses (CMV, EBV, and influenza). Our findings identify reversible virus evasion of DC-mediated innate immunity as an additional important factor that impacts the severity of polyfunctional CD8(+) T cell exhaustion during a chronic viral infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Células Dendríticas/inmunología , Células Dendríticas/virología , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Terapia de Inmunosupresión , Antígenos de Superficie/fisiología , Proteínas Reguladoras de la Apoptosis/fisiología , Linfocitos T CD8-positivos/patología , Degranulación de la Célula/inmunología , Células Dendríticas/patología , Femenino , Hepatitis C Crónica/patología , Humanos , Interleucina-2/biosíntesis , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Células Mieloides/inmunología , Células Mieloides/patología , Células Mieloides/virología , Receptor de Muerte Celular Programada 1 , Receptores de Reconocimiento de Patrones/antagonistas & inhibidores , Receptores de Reconocimiento de Patrones/fisiología , Carga Viral/inmunología , Activación Viral/inmunologíaRESUMEN
Although studies have addressed the exhaustion of the host's immune response to HCV and its role in treatment, there is little information about the possible contribution of innate immunity to treatment-induced clearance. We hypothesized that because intact myeloid dendritic cell (MDC) pathogen sensing functions are associated with improved HCV-specific CD8+ T cell functionality in some chronically infected patients, it might enhance HCV clearance rate under standard interferon therapy. To investigate this hypothesis, TLR-induced MDC activation and HCV-specific CD8+ T cell response quality were monitored longitudinally at the single-cell level using polychromatic flow cytometry in chronically infected patients undergoing interferon therapy. We correlated the immunological, biochemical and virological data with response to treatment. We demonstrate that the clinical efficacy of interferon-induced viral clearance is influenced by the extent to which HCV inhibits MDC functions before treatment, rather than solely on a breakdown of the extrinsic T cell immunosuppressive environment. Thus, viral inhibition of MDC functions before treatment emerges as a co-determining factor in the clinical efficacy of interferon therapy during chronic HCV infection.