Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the ...distribution of latent replication-competent HIV-1 within memory CD4+ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.
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•FLIPS utilizes NGS to sequence and genetically characterize HIV-1 proviruses•FLIPS identifies genetically intact and likely replication-competent HIV-1 proviruses•Identical HIV-1 proviruses suggest maintenance of reservoir by cellular proliferation•Demonstrate the advantages of FLIPS over other common HIV-1 sequencing assays
Latent, replication-competent HIV-1 proviruses pose a significant barrier to an HIV-1 cure. Hiener et al. present the Full-Length Individual Proviral Sequencing (FLIPS) assay to reveal the distribution of genetically intact and potentially replication-competent HIV-1 proviruses in different T cell subsets isolated from individuals on long-term antiretroviral therapy.
Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural ...virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.
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•In HIV controllers, both TFH and non-TFH lymph node CD4 T cells contain HIV•Lymph node viruses in both TFH and non-TFH have attributes of active replication•Rare, recently infected cells that produce virus upon stimulation circulate in blood•Archival proviruses predominant in clonally expanded blood cells can be inducible
HIV persistence in people who can spontaneously control the infection involves different mechanisms within distinct anatomic and functional compartments.
Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use ...of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH2-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of ...viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominantmechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.
Despite the advent of effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) continues to pose major challenges, with extensive pathogenesis during acute and chronic infection ...prior to ART initiation and continued persistence in a reservoir of infected CD4 T cells during long-term ART. CD101 has recently been characterized to play an important role in CD4 Treg potency. Using the simian immunodeficiency virus (SIV) model of HIV infection in rhesus macaques, we characterized the role and kinetics of CD101+ CD4 T cells in longitudinal SIV infection. Phenotypic analyses and single-cell RNAseq profiling revealed that CD101 marked CD4 Tregs with high immunosuppressive potential, distinct from CD101- Tregs, and these cells also were ideal target cells for HIV/SIV infection, with higher expression of CCR5 and α4β7 in the gut mucosa. Notably, during acute SIV infection, CD101+ CD4 T cells were preferentially depleted across all CD4 subsets when compared with their CD101- counterpart, with a pronounced reduction within the Treg compartment, as well as significant depletion in mucosal tissue. Depletion of CD101+ CD4 was associated with increased viral burden in plasma and gut and elevated levels of inflammatory cytokines. While restored during long-term ART, the reconstituted CD101+ CD4 T cells display a phenotypic profile with high expression of inhibitory receptors (including PD-1 and CTLA-4), immunsuppressive cytokine production, and high levels of Ki-67, consistent with potential for homeostatic proliferation. Both the depletion of CD101+ cells and phenotypic profile of these cells found in the SIV model were confirmed in people with HIV on ART. Overall, these data suggest an important role for CD101-expressing CD4 T cells at all stages of HIV/SIV infection and a potential rationale for targeting CD101 to limit HIV pathogenesis and persistence, particularly at mucosal sites.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The uneven anatomic distribution of cell subsets that harbor human immunodeficiency virus (HIV) during antiretroviral therapy (ART) complicates investigation of the barriers to HIV cure. Here we ...propose that while previous studies done largely in blood cells have led to important investigations into HIV latency, other important mechanisms of HIV persistence during ART may not be readily apparent in the bloodstream. We specifically consider as an example the question of ongoing HIV replication during ART. We discuss how growing understanding of key anatomic sanctuaries for the virus can inform future experiments aimed at further clarifying this issue.
Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure
, but the infeasibility of isolating and characterizing these cells in their natural state ...has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA
memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA
cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA
cells, positive regulators of HIV transcription that were lower in HIV-DNA
cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.
OBJECTIVE:The contribution of HLA-DR+ memory CD4 T cells to the HIV reservoir during prolonged antiretroviral therapy is unclear as these cells are commonly excluded when assessing for ...replication-competent HIV. To address this issue, we examined the distribution of genetically intact HIV DNA within HLA-DR− and HLA-DR+ memory CD4 T cells and the RNA transcriptional profile of these cells during antiretroviral therapy.
DESIGN/METHODS:Full-length DNA sequencing was used to examine the HIV DNA landscape within HLA-DR+ and HLA-DR− memory CD4 T cells. RNA quantification and sequencing was used to interrogate the relationship between HLA-DR status and HIV RNA transcription.
RESULTS:HLA-DR+ CD4 T cells contained a high frequency of genetically intact HIV genomes, contributing over half of the genetically intact viral sequences to the reservoir. Expansions of genetically identical sequences were identified in all T-cell subsets, indicating that cellular proliferation maintains genetically intact and defective viral DNA during therapy. Intracellular HIV RNA levels in HLA-DR+ and HLA-DR− T cells were not statistically different by either long terminal repeat quantitative PCR quantification or single-genome RNA sequencing of the p6-RT region.
CONCLUSION:The high proportion of intact viral DNA sequences in the proliferative HLA-DR+ subset suggests they are critical in maintaining HIV infection during effective therapy. As such, these cells should be included in any immune intervention targeting HIV during effective therapy.
The human immunodeficiency virus (HIV) integrates its genome into that of infected cells and may enter an inactive state of reversible latency that cannot be targeted using antiretroviral therapy. ...Sequencing such a provirus and the adjacent host junctions in individual cells may elucidate the mechanisms of the persistence of infected cells, but this is difficult owing to the 150-million-fold higher amount of background human DNA. Here we show that full-length proviruses connected to their contiguous HIV-host DNA junctions can be assembled via a high-throughput microfluidic assay where droplet-based whole-genome amplification of HIV DNA in its native context is followed by a polymerase chain reaction (PCR) to tag droplets containing proviruses for sequencing. We assayed infected cells from people with HIV receiving suppressive antiretroviral therapy, resulting in the detection and sequencing of paired proviral genomes and integration sites, 90% of which were not recovered by commonly used nested-PCR methods. The sequencing of individual proviral genomes with their integration sites could improve the genetic analysis of persistent HIV-infected cell reservoirs.
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