RESUMEN
Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.
Asunto(s)
COVID-19 , Memoria Epigenética , Síndrome Post Agudo de COVID-19 , Animales , Humanos , Ratones , Diferenciación Celular , COVID-19/inmunología , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas , Inflamación/genética , Inmunidad Entrenada , Monocitos/inmunología , Síndrome Post Agudo de COVID-19/genética , Síndrome Post Agudo de COVID-19/inmunología , Síndrome Post Agudo de COVID-19/patologíaRESUMEN
HIV infection persists during antiretroviral therapy (ART) due to a reservoir of latently infected cells that harbor replication-competent virus and evade immunity. Previous ex vivo studies suggested that CD8+ T cells from people with HIV may suppress HIV expression via non-cytolytic mechanisms, but the mechanisms responsible for this effect remain unclear. Here, we used a primary cell-based in vitro latency model and demonstrated that co-culture of autologous activated CD8+ T cells with HIV-infected memory CD4+ T cells promoted specific changes in metabolic and/or signaling pathways resulting in increased CD4+ T cell survival, quiescence, and stemness. Collectively, these pathways negatively regulated HIV expression and ultimately promoted the establishment of latency. As shown previously, we observed that macrophages, but not B cells, promoted latency in CD4+ T cells. The identification of CD8-specific mechanisms of pro-latency activity may favor the development of approaches to eliminate the viral reservoir in people with HIV.
Asunto(s)
Infecciones por VIH , Humanos , Linfocitos T CD8-positivos , Latencia del Virus , Linfocitos T CD4-Positivos , Replicación ViralRESUMEN
HIV-1 infection greatly alters the NK cell phenotypic and functional repertoire. This is highlighted by the expansion of a rare population of FcRγ- NK cells exhibiting characteristics of traditional immunologic memory in people with HIV (PWH). Although current antiretroviral therapy (ART) effectively controls HIV-1 viremia and disease progression, its impact on HIV-1-associated NK cell abnormalities remains unclear. To address this, we performed a longitudinal analysis detailing conventional and memory-like NK cell characteristics in n = 60 PWH during the first 4 y of ART. Throughout this regimen, a skewed repertoire of cytokine unresponsive FcRγ- memory-like NK cells persisted and accompanied an overall increase in NK surface expression of CD57 and KLRG1, suggestive of progression toward immune senescence. These traits were linked to elevated serum inflammatory biomarkers and increasing Ab titers to human CMV, with human CMV viremia detected in approximately one-third of PWH at years 1-4 of ART. Interestingly, 40% of PWH displayed atypical NK cell subsets, representing intermediate stages of NK-poiesis based on single-cell multiomic trajectory analysis. Our findings indicate that NK cell irregularities persist in PWH despite long-term ART, underscoring the need to better understand the causative mechanisms that prevent full restoration of immune health in PWH.
Asunto(s)
Antígenos CD57 , Infecciones por VIH , VIH-1 , Células Asesinas Naturales , Humanos , Células Asesinas Naturales/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/inmunología , Masculino , Femenino , Antígenos CD57/inmunología , Adulto , Persona de Mediana Edad , Memoria Inmunológica/inmunología , Lectinas Tipo C/inmunología , Receptores Inmunológicos , Viremia/inmunología , Viremia/tratamiento farmacológico , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/tratamiento farmacológico , Receptores de IgG/inmunología , Estudios Longitudinales , Antirretrovirales/uso terapéuticoRESUMEN
Despite antiretroviral therapy (ART), HIV-1 persists as proviruses integrated into the genomic DNA of CD4+ T cells. The mechanisms underlying the persistence and clonal expansion of these cells remain incompletely understood. Cases have been described in which proviral integration can alter host gene expression to drive cellular proliferation. Here, we review observations from other genome-integrating human viruses to propose additional putative modalities by which HIV-1 integration may alter cellular function to favor persistence, such as by altering susceptibility to cytotoxicity in virus-expressing cells. We propose that signals implicating such mechanisms may have been masked thus far by the preponderance of defective and/or nonreactivatable HIV-1 proviruses, but could be revealed by focusing on the integration sites of intact proviruses with expression potential.
Asunto(s)
Infecciones por VIH , VIH-1 , Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos , Infecciones por VIH/tratamiento farmacológico , Humanos , Provirus/genética , Integración ViralRESUMEN
CD8+ T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8+ T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor ß (TCRß) analysis revealed that class II-restricted CD8+ T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8+ T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8+ T cell responses can exist in a chronic human viral infection, and may contribute to immune control.
Asunto(s)
Antivirales/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Antígenos HLA/inmunología , HumanosRESUMEN
Combination antiretroviral therapy (ART) to treat human immunodeficiency virus (HIV) infection has proven remarkably successful - for those who can access and afford it - yet HIV infection persists indefinitely in a reservoir of cells, despite effective ART and despite host antiviral immune responses. An HIV cure is therefore the next aspirational goal and challenge, though approaches differ in their objectives - with 'functional cures' aiming for durable viral control in the absence of ART, and 'sterilizing cures' aiming for the more difficult to realize objective of complete viral eradication. Mechanisms of HIV persistence, including viral latency, anatomical sequestration, suboptimal immune functioning, reservoir replenishment, target cell-intrinsic immune resistance, and, potentially, target cell distraction of immune effectors, likely need to be overcome in order to achieve a cure. A small fraction of people living with HIV (PLWH) naturally control infection via immune-mediated mechanisms, however, providing both sound rationale and optimism that an immunological approach to cure is possible. Herein we review up to date knowledge and emerging evidence on: the mechanisms contributing to HIV persistence, as well as potential strategies to overcome these barriers; promising immunological approaches to achieve viral control and elimination of reservoir-harboring cells, including harnessing adaptive immune responses to HIV and engineered therapies, as well as enhancers of their functions and of complementary innate immune functioning; and combination strategies that are most likely to succeed. Ultimately, a cure must be safe, effective, durable, and, eventually, scalable in order to be widely acceptable and available.
Asunto(s)
Infecciones por VIH , VIH-1 , Antivirales/uso terapéutico , Linfocitos T CD4-Positivos , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Latencia del VirusRESUMEN
Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos , COVID-19 , Antígenos de Histocompatibilidad Clase I , Proteínas Virales , Aminoácidos , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Complejo Mayor de Histocompatibilidad , Péptidos , SARS-CoV-2 , Proteínas Virales/inmunologíaRESUMEN
In this Brief Communications Arising Comment, the first three authors (Osuna, Lim and Kublin) should have been listed as equally contributing authors; this has been corrected online.
RESUMEN
The establishment of HIV-1 latency has hindered an HIV-1 cure. "Shock and Kill" strategies to target this reservoir aim to induce the latent provirus with latency reversing agents (LRAs). However, recent studies have shown that the majority of the intact HIV-1 viral reservoir found in ART-suppressed HIV infected individuals is not inducible. We sought to understand whether this non-inducible reservoir is established, and thus able to be studied, in an in vitro primary TCM model of latency. Furthermore, we wanted to expand this model system to include R5-tropic and non-B subtype viruses. To that end, we generated our TCM model of latency with an R5 subtype B virus, AD8 and an R5 subtype C virus, MJ4. Our results demonstrate that both intact and defective proviruses are generated in this model. Less than 50% of intact proviruses are inducible regardless of viral strain in the context of maximal stimulation through the TCR or with different clinically relevant LRAs including the HDAC inhibitors SAHA and MS-275, the PKC agonist Ingenol 3,20-dibenzoate or the SMAC mimetic AZD-5582. Our findings suggest that current LRA strategies are insufficient to effectively reactivate intact latent HIV-1 proviruses in primary CD4 TCM cells and that the mechanisms involved in the generation of the non-inducible HIV-1 reservoir can be studied using this primary in vitro model.Importance: HIV-1 establishes a latent reservoir that persists under antiretroviral therapy. Antiretroviral therapy is able to stop the spread of the virus and the progression of the disease but does not target this latent reservoir. If antiretroviral therapy is stopped, the virus is able to resume replication and the disease progresses. Recently, it has been demonstrated that most of the latent reservoir capable of generating replication competent virus cannot be induced in the laboratory setting. However, the mechanisms that influence the generation of this intact and non-inducible latent reservoir are still under investigation. Here we demonstrate the generation of defective, intact and intact non-inducible latent HIV-1 in a TCM model of latency using different HIV-1 strains. Thus, the mechanisms which control inducibility can be studied using this primary cell model of latency, which may accelerate our understanding of the latent reservoir and the development of curative strategies.
RESUMEN
HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.
Asunto(s)
Benzotiazoles/farmacología , Brioestatinas/farmacología , Reservorios de Enfermedades/virología , Isoquinolinas/farmacología , Latencia del Virus/efectos de los fármacos , Proteína bcl-X/antagonistas & inhibidores , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Infecciones por VIH/prevención & control , VIH-1/crecimiento & desarrollo , Humanos , Replicación Viral/efectos de los fármacos , Proteína bcl-X/metabolismoRESUMEN
It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/genética , Transcriptoma , Latencia del Virus/genética , Antirretrovirales/uso terapéutico , VIH-1/fisiología , Humanos , ARN Viral/genéticaRESUMEN
The primary reservoir for HIV is within memory CD4+ T cells residing within tissues, yet the features that make some of these cells more susceptible than others to infection by HIV is not well understood. Recent studies demonstrated that CCR5-tropic HIV-1 efficiently enters tissue-derived memory CD4+ T cells expressing CD127, the alpha chain of the IL7 receptor, but rarely completes the replication cycle. We now demonstrate that the inability of HIV to replicate in these CD127-expressing cells is not due to post-entry restriction by SAMHD1. Rather, relative to other memory T cell subsets, these cells are highly prone to undergoing latent infection with HIV, as revealed by the high levels of integrated HIV DNA in these cells. Host gene expression profiling revealed that CD127-expressing memory CD4+ T cells are phenotypically distinct from other tissue memory CD4+ T cells, and are defined by a quiescent state with diminished NFκB, NFAT, and Ox40 signaling. However, latently-infected CD127+ cells harbored unspliced HIV transcripts and stimulation of these cells with anti-CD3/CD28 reversed latency. These findings identify a novel subset of memory CD4+ T cells found in tissue and not in blood that are preferentially targeted for latent infection by HIV, and may serve as an important reservoir to target for HIV eradication efforts.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Subunidad alfa del Receptor de Interleucina-7/inmunología , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Memoria Inmunológica , Subunidad alfa del Receptor de Interleucina-7/genética , Latencia del Virus , Replicación ViralRESUMEN
T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , COVID-19/inmunología , Técnicas de Cultivo de Célula/métodos , Inmunoterapia Adoptiva/métodos , SARS-CoV-2/inmunología , Adulto , Anciano , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Epítopos Inmunodominantes/inmunología , Masculino , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , Proteínas Virales/inmunología , Adulto Joven , Tratamiento Farmacológico de COVID-19RESUMEN
The success of the shock and kill strategy for the HIV cure depends both on the reactivation of the latent reservoir and on the ability of the immune system to eliminate infected cells. As latency reversal alone has not shown any impact in the size of the latent reservoir, ensuring that effector CTLs are able to recognize and kill HIV-infected cells could contribute to reservoir reduction. In this study, we investigated which functional aspects of human CTLs are associated with a better capacity to kill HIV-infected CD4+ T cells. We isolated Gag- and Nef-specific CTL clones with different TCR sequences from the PBMC of donors in acute and chronic infection. High-affinity clonotypes that showed IFN-γ production preserved even when the CD8 coreceptor was blocked, and clones with high Ag sensitivity exhibited higher efficiency at reducing the latent reservoir. Although intrinsic cytotoxic capacity did not differ according to TCR affinity, clonotypes with high TCR affinity showed a better ability to kill HIV-infected CD4+ T cells obtained from in vivo-infected PBMC and subjected to viral reactivation. Strategies aiming to specifically boost and maintain long-living memory CTLs with high TCR affinity in vivo prior to latency-reversing treatment might improve the efficacy of the shock and kill approach to reduce the latent reservoir.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Latencia del Virus/inmunología , Linfocitos T CD4-Positivos/virología , Humanos , Interferón gamma/inmunologíaRESUMEN
Clinical trials investigating histone deacetylase inhibitors (HDACi) to reverse HIV-1 latency aim to expose reservoirs in antiretroviral (ARV)-treated individuals to clearance by immune effectors, yet have not driven measurable reductions in the frequencies of infected cells. We therefore investigated the effects of the class I-selective HDACi nanatinostat and romidepsin on various blocks to latency reversal and elimination, including viral splicing, antigen presentation, and CD8+ T cell function. In ex vivo CD4+ T cells from ARV-suppressed individuals, both HDACi significantly induced viral transcription, but not splicing nor supernatant HIV-1 RNA. In an HIV-1 latency model using autologous CD8+ T cell clones as biosensors of antigen presentation, neither HDACi-treated CD4+ T cell condition induced clone degranulation. Both HDACi also impaired the function of primary CD8+ T cells in viral inhibition assays, with nanatinostat causing less impairment. These findings suggest that spliced or cell-free HIV-1 RNAs are more indicative of antigen expression than unspliced HIV-RNAs and may help to explain the limited abilities of HDACi to generate CD8+ T cell targets in vivoIMPORTANCE Antiretroviral (ARV) drug regimens suppress HIV-1 replication but are unable to cure infection. This leaves people living with HIV-1 burdened by a lifelong commitment to expensive daily medication. Furthermore, it has become clear that ARV therapy does not fully restore health, leaving individuals at elevated risk for cardiovascular disease, certain types of cancers, and neurocognitive disorders, as well as leaving them exposed to stigma. Efforts are therefore under way to develop therapies capable of curing infection. A key focus of these efforts has been on a class of drugs called histone deacetylase inhibitors (HDACi), which have the potential of exposing hidden reservoirs of HIV-1 to elimination by the immune system. Unfortunately, clinical trial results with HDACi have thus far been disappointing. In the current study, we integrate a number of experimental approaches to build a model that provides insights into the limited activity of HDACi in clinical trials and offers direction for future approaches.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Latencia del Virus/efectos de los fármacos , Adulto , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Depsipéptidos/farmacología , Femenino , Infecciones por VIH/inmunología , Seropositividad para VIH/tratamiento farmacológico , VIH-1/metabolismo , VIH-1/patogenicidad , VIH-1/fisiología , Histona Desacetilasas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Latencia del Virus/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Given that HIV evolution and latent reservoir establishment occur continually within-host, and that latently infected cells can persist long-term, the HIV reservoir should comprise a genetically heterogeneous archive recapitulating within-host HIV evolution. However, this has yet to be conclusively demonstrated, in part due to the challenges of reconstructing within-host reservoir establishment dynamics over long timescales. We developed a phylogenetic framework to reconstruct the integration dates of individual latent HIV lineages. The framework first involves inference and rooting of a maximum-likelihood phylogeny relating plasma HIV RNA sequences serially sampled before the initiation of suppressive antiretroviral therapy, along with putative latent sequences sampled thereafter. A linear model relating root-to-tip distances of plasma HIV RNA sequences to their sampling dates is used to convert root-to-tip distances of putative latent lineages to their establishment (integration) dates. Reconstruction of the ages of putative latent sequences sampled from chronically HIV-infected individuals up to 10 y following initiation of suppressive therapy revealed a genetically heterogeneous reservoir that recapitulated HIV's within-host evolutionary history. Reservoir sequences were interspersed throughout multiple within-host lineages, with the oldest dating to >20 y before sampling; historic genetic bottleneck events were also recorded therein. Notably, plasma HIV RNA sequences isolated from a viremia blip in an individual receiving otherwise suppressive therapy were highly genetically diverse and spanned a 20-y age range, suggestive of spontaneous in vivo HIV reactivation from a large latently infected cell pool. Our framework for reservoir dating provides a potentially powerful addition to the HIV persistence research toolkit.
Asunto(s)
Infecciones por VIH/genética , VIH-1/genética , Interacciones Huésped-Patógeno/genética , Filogenia , Latencia del Virus/genética , Conjuntos de Datos como Asunto , Infecciones por VIH/sangre , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Humanos , Modelos Genéticos , Provirus/genética , Provirus/aislamiento & purificación , ARN Viral/genética , ARN Viral/aislamiento & purificación , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Factores de Tiempo , Viremia/sangre , Viremia/genética , Viremia/virología , Integración Viral/genéticaRESUMEN
False-negative severe acute respiratory syndrome coronavirus 2 test results can negatively impact the clinical and public health response to coronavirus disease 2019 (COVID-19). We used droplet digital polymerase chain reaction (ddPCR) to demonstrate that human DNA levels, a stable molecular marker of sampling quality, were significantly lower in samples from 40 confirmed or suspected COVID-19 cases that yielded negative diagnostic test results (ie, suspected false-negative test results) compared with a representative pool of 87 specimens submitted for COVID-19 testing. Our results support suboptimal biological sampling as a contributor to false-negative COVID-19 test results and underscore the importance of proper training and technique in the collection of nasopharyngeal specimens.
Asunto(s)
Betacoronavirus/aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Manejo de Especímenes/métodos , Betacoronavirus/genética , COVID-19 , Prueba de COVID-19 , Reacciones Falso Negativas , Humanos , Nasofaringe/virología , Pandemias , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2 , Carga ViralRESUMEN
"Shock and kill" therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.
Asunto(s)
Infecciones por VIH , VIH-1 , Animales , Linfocitos T CD4-Positivos , Infecciones por VIH/tratamiento farmacológico , Ratones , Relación Estructura-Actividad , Triazinas , Activación Viral , Latencia del VirusRESUMEN
The low frequency of latently HIV-infected cells in vivo limits the testing of potential HIV cure strategies using cells from successfully suppressed individuals. To date, primary cell models of latency use cells infected in vitro Primary CD4+ T cell models carrying an individual's endogenous HIV reservoir that recapitulate in vivo conditions of HIV latency are still outstanding. We developed a primary CD4+ T cell model of HIV latency derived from memory CD4+ T cells isolated from virally suppressed HIV-infected individuals that recapitulates HIV-1 latency and viral reactivation events. This model is based on the expansion of primary CD4+ T cells up to 300-fold in cell number. These cells reestablish a resting state without active virus production after extended culture and maintain a stable number of total HIV proviruses. The ability of these cells to respond to various classes of latency-reversing agents is similar to that of ex vivo CD4+ T cells directly isolated from blood. Importantly, viral outgrowth assays confirmed the ability of these expanded cells to produce replication-competent endogenous virus. In sum, this model recapitulates ex vivo viral reactivation conditions, captures the variability between individuals with different HIV reservoirs, and provides large numbers of cells for testing multiple agents from a single donor. The use of this novel model will allow accurate exploration of novel cure approaches aimed either at promoting viral reactivation or maintaining sustained latency.IMPORTANCE Primary cell models of HIV latency have been very useful to identify mechanisms contributing to HIV latency and to evaluate potential HIV cure strategies. However, the current models utilize in vitro infection with exogenous virus that does not fully recapitulate virus reactivation profiles of endogenous HIV in in vivo-infected CD4+ T cells. In contrast, obtaining sufficient amounts of CD4+ T cells from HIV-infected individuals to interrogate the HIV reservoir in vitro requires leukapheresis. In the model we propose here, in vitro expansion and extended culture of primary CD4+ T cells isolated from virally suppressed HIV-infected individuals enable obtaining large numbers of cells harboring endogenous latent HIV reservoirs without performing leukapheresis. This model captures the variability of HIV reservoirs seeded in different individuals and should be useful to evaluate future HIV cure strategies.
Asunto(s)
Latencia del Virus/fisiología , Replicación Viral/fisiología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Seropositividad para VIH , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Modelos Biológicos , Cultivo Primario de Células , Provirus , Activación ViralRESUMEN
Curing HIV infection has been thwarted by the persistent reservoir of latently infected CD4+ T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir depletion strategies, we developed a novel preclinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, 2 weeks after PBMC injection, was 1,000-fold higher (mean = 9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean = 6,838 HIV copies/ml) or intravenously (mean = 591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo-reactivated primary latently infected cells. The time to viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, compared to treatment with 10-1074-FcRnull, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated, or 10-1074-FcRnull-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in the plasma of 10-1074-treated mice, whereas two new clusters emerged that were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time to viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.