RESUMEN
Pegylated interferon-alpha (pIFN-α) is suggested to lower human immunodeficiency virus type-1 (HIV-1) DNA load in antiretroviral therapy (ART)-treated patients. We studied kinetics of HIV-1 DNA levels in 40 HIV-1/hepatitis C virus (HCV) coinfected patients, treated with pIFN-α for HCV and categorized into 3 groups according to start of ART: chronic HIV-1 infection (n = 22), acute HIV-1 infection (n = 8), no-ART (n = 10). Total HIV-1 DNA levels in 247 peripheral blood mononuclear cell samples were stable before, during, and after pIFN-α treatment in all groups. Our results question the benefit of pIFN-α as an immunotherapeutic agent for reducing the HIV-1 reservoir.
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Coinfección/tratamiento farmacológico , ADN Viral/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Hepatitis C Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Carga Viral/efectos de los fármacos , Adulto , Antivirales/uso terapéutico , Coinfección/virología , Femenino , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/virología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Objectives: To determine the most recent prevalence, transmission patterns and risk factors of transmitted drug-resistance mutations (TDRMs) in Cameroon, we initiated a multicentre study monitoring HIV-1 drug resistance in newly HIV-1-diagnosed individuals using a novel next-generation sequencing (NGS) assay applicable to fingerprick dried blood spot (DBS) samples. Methods: Fingerprick DBS samples and questionnaires were collected from 360 newly HIV-1-diagnosed individuals in four hospitals in urban areas in Cameroon in the years 2015-16. We developed an HIV-1 protease and reverse transcriptase drug resistance genotyping assay applicable to DBS samples and HIV-1 genomes of groups M, N and O. The WHO 2009 list of mutations for surveillance of transmitted drug-resistant HIV strains was used to analyse TDRMs. Results: Applying our 'DBS-NGS-genotypic resistance test', baseline HIV-1 drug resistance data were successfully obtained from 82.8% (298/360) of newly diagnosed individuals. At nucleotide frequencies >15%, TDRMs to NRTIs were observed in 3.0% (9/298), to NNRTIs in 4.0% (12/298) and to PIs in 1.3% (3/240). The NNRTI mutation K103N was most commonly detected (2.7%). Expanding the analysis to low-abundance TDRMs, i.e. 3%-15%, 12 additional individuals (4.0%) harbouring TDRMs were identified. Having unprotected sex with a known HIV-1-positive person was significantly associated with the transmission of DRMs (adjusted OR 9.6; 95% CI 1.79-51.3). Conclusions: The prevalence of transmitted HIV-1 drug resistance is currently low in the study sites in Cameroon. Evidence of some risky sexual behaviours depicts a public health problem with possible implications for the prevention of new HIV-1 infections.
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Pruebas con Sangre Seca , Farmacorresistencia Viral , Infecciones por VIH/epidemiología , VIH-1/efectos de los fármacos , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Adolescente , Adulto , Anciano , Fármacos Anti-VIH/uso terapéutico , Camerún/epidemiología , Femenino , Técnicas de Genotipaje , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , Seropositividad para VIH , Humanos , Masculino , Persona de Mediana Edad , Mutación , Prevalencia , ARN Viral/genética , Factores de Riesgo , Adulto JovenRESUMEN
HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T cells from 12 HIV-1-infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes, genes associated with clonal expansion of latently HIV-1-infected CD4+ T cells, cancer-related genes, and highly expressed genes. The preference for cancer-related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during HIV-1 infection in both resting and activated CD4+ T cells. In summary, characteristic HIV-1 integration site features are preestablished as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T cells.
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Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular/genética , Infecciones por VIH/genética , VIH-1/genética , Interacciones Microbiota-Huesped/genética , Neoplasias/genética , Integración Viral/genética , Latencia del Virus/genética , Terapia Antirretroviral Altamente Activa , Progresión de la Enfermedad , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Humanos , Activación de Linfocitos , Carga ViralRESUMEN
Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.
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Infecciones por VIH , VIH-1 , Receptores CCR5 , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Resistencia a la Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/prevención & control , VIH-1/genética , Humanos , Receptores CCR5/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/farmacología , Efectores Tipo Activadores de la TranscripciónRESUMEN
BACKGROUND: The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). METHODS: We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean-/mCherry+) HIV-1 promoters were characterised. RESULTS: Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. CONCLUSION: Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.
RESUMEN
BACKGROUND: The primary hurdle for the eradication of HIV-1 is the establishment of a latent viral reservoir early after primary infection. Here, we investigated the potential influence of human genetic variation on the HIV-1 reservoir size and its decay rate during suppressive antiretroviral treatment. SETTING: Genome-wide association study and exome sequencing study to look for host genetic determinants of HIV-1 reservoir measurements in patients enrolled in the Swiss HIV Cohort Study, a nation-wide prospective observational study. METHODS: We measured total HIV-1 DNA in peripheral blood mononuclear cells from study participants, as a proxy for the reservoir size at 3 time points over a median of 5.4 years, and searched for associations between human genetic variation and 2 phenotypic readouts: the reservoir size at the first time point and its decay rate over the study period. We assessed the contribution of common genetic variants using genome-wide genotyping data from 797 patients with European ancestry enrolled in the Swiss HIV Cohort Study and searched for a potential impact of rare variants and exonic copy number variants using exome sequencing data generated in a subset of 194 study participants. RESULTS: Genome-wide and exome-wide analyses did not reveal any significant association with the size of the HIV-1 reservoir or its decay rate on suppressive antiretroviral treatment. CONCLUSIONS: Our results point to a limited influence of human genetics on the size of the HIV-1 reservoir and its long-term dynamics in successfully treated individuals.
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Fármacos Anti-VIH/uso terapéutico , Variación Genética , Genoma Humano , Genómica/métodos , Infecciones por VIH/genética , VIH-1 , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Infecciones por VIH/tratamiento farmacológico , Humanos , Factores de TiempoRESUMEN
HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell-mediated immune control.
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Infecciones por VIH/inmunología , Infecciones por VIH/virología , Herpesvirus Humano 4/patogenicidad , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Linfocitos B/virología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Coinfección , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/metabolismo , Susceptibilidad a Enfermedades/virología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por VIH/genética , Seropositividad para VIH , VIH-1/metabolismo , VIH-1/patogenicidad , Células Madre Hematopoyéticas/patología , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Receptores CXCR4/metabolismo , Receptores CXCR4/fisiología , Linfocitos T/inmunologíaRESUMEN
The HIV-1 reservoir is the major hurdle to a cure. We here evaluate viral and host characteristics associated with reservoir size and long-term dynamics in 1,057 individuals on suppressive antiretroviral therapy for a median of 5.4 years. At the population level, the reservoir decreases with diminishing differences over time, but increases in 26.6% of individuals. Viral blips and low-level viremia are significantly associated with slower reservoir decay. Initiation of ART within the first year of infection, pretreatment viral load, and ethnicity affect reservoir size, but less so long-term dynamics. Viral blips and low-level viremia are thus relevant for reservoir and cure studies.
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Fármacos Anti-VIH/uso terapéutico , Reservorios de Enfermedades , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Adulto , Femenino , Infecciones por VIH/sangre , VIH-1/genética , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Modelos Biológicos , ARN Viral/sangre , Carga Viral , Viremia , Latencia del Virus/efectos de los fármacosRESUMEN
Long-lived latently HIV-1-infected cells represent a barrier to cure. We developed a dual-fluorescence HIV-1-based vector containing a pair of genetic insulators flanking a constitutive fluorescent reporter gene to study HIV-1 latency. The protective effects of these genetic insulators are demonstrated through long-term (up to 394 days) stable fluorescence profiles in transduced SUP-T1 cells. Analysis of 1,941 vector integration sites confirmed reproduction of HIV-1 integration patterns. We sorted monoclonal cells representing latent HIV-1 infections and found that both vector integration sites and integrity of the vector genomes influence the reactivation potentials of latent HIV-1 promoters. Interestingly, some latent monoclonal cells exhibited a small cell subpopulation with a spontaneously reactivated HIV-1 promoter. Higher expression levels of genes involved in cell cycle progression are observed in these cell subpopulations compared to their counterparts with HIV-1 promoters that remained latent. Consistently, larger fractions of spontaneously reactivated cells are in the S and G2 phases of the cell cycle. Furthermore, genistein and nocodazole treatments of these cell clones, which halted cells in the G2 phase, resulted in a 1.4-2.9-fold increase in spontaneous reactivation. Taken together, our HIV-1 latency model reveals that the spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle.
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Infecciones por VIH/virología , VIH-1/fisiología , Regiones Promotoras Genéticas , Activación Viral , Ciclo Celular , Línea Celular , Células Clonales/efectos de los fármacos , Células Clonales/virología , Genes Reporteros , Genisteína/farmacología , Infecciones por VIH/genética , Humanos , Nocodazol/farmacología , Transducción Genética , Integración Viral , Latencia del VirusRESUMEN
A single virus is capable of infecting and replicating in a single cell. Recent advances across single-cell omics technologies - genomics, epigenomics, transcriptomics, epitranscriptomics, proteomics, and metabolomics - will offer unprecedented opportunities to gain more insights into the various aspects of the life cycle of viruses and their impact on the host cell. Here, using the human immunodeficiency virus type 1 (HIV-1) as an example, we summarize the current knowledge and the future potential of single-cell omics in the investigation of an important aspect of the life cycle of HIV-1 that represents a major hurdle in achieving viral eradication, HIV-1 latency.
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Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Latencia del Virus/fisiología , Genómica , Interacciones Huésped-Patógeno , Humanos , MetabolómicaRESUMEN
The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells.