Selection of epigenetically privileged HIV-1 proviruses during treatment with panobinostat and interferon-α2a.

CD4+ T cells with latent HIV-1 infection persist despite treatment with antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may expose HIV-1-infected cells to host immune activity, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized-controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-α2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target sites for panobinostat. By contrast, proviruses near H3K27ac marks were actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment can increase the immunological vulnerability of HIV-1 reservoir cells and accelerate the selection of epigenetically privileged HIV-1 proviruses.

IL-15-dependent immune crosstalk between natural killer cells and dendritic cells in HIV-1 elite controllers.

As the principal effector cell population of the innate immune system, natural killer (NK) cells may make critical contributions to natural, immune-mediated control of HIV-1 replication. Using genome-wide assessments of activating and inhibitory chromatin features, we demonstrate here that cytotoxic NK (cNK) cells from elite controllers (ECs) display elevated activating histone modifications at the interleukin 2 (IL-2)/IL-15 receptor ß chain and the BCL2 gene loci. These histone changes translate into increased responsiveness of cNK cells to paracrine IL-15 secretion, which coincides with higher levels of IL-15 transcription by myeloid dendritic cells in ECs. The distinct immune crosstalk between these innate immune cell populations results in improved IL-15-dependent cNK cell survival and cytotoxicity, paired with a metabolic profile biased toward IL-15-mediated glycolytic activities. Together, these results suggest that cNK cells from ECs display a programmed IL-15 response signature and support the emerging role of innate immune pathways in natural, drug-free control of HIV-1.

Persistence of intact HIV-1 proviruses in the brain during antiretroviral therapy.

HIV-1 reservoir cells that circulate in peripheral blood during suppressive antiretroviral therapy (ART) have been well characterized, but little is known about the dissemination of HIV-1-infected cells across multiple anatomical tissues, especially the CNS. Here, we performed single-genome, near full-length HIV-1 next-generation sequencing to evaluate the proviral landscape in distinct anatomical compartments, including multiple CNS tissues, from 3 ART-treated participants at autopsy. While lymph nodes and, to a lesser extent, gastrointestinal and genitourinary tissues represented tissue hotspots for the persistence of intact proviruses, we also observed intact proviruses in CNS tissue sections, particularly in the basal ganglia. Multi-compartment dissemination of clonal intact and defective proviral sequences occurred across multiple anatomical tissues, including the CNS, and evidence for the clonal proliferation of HIV-1-infected cells was found in the basal ganglia, in the frontal lobe, in the thalamus and in periventricular white matter. Deep analysis of HIV-1 reservoirs in distinct tissues will be informative for advancing HIV-1 cure strategies.

Approximately 39 million people in the world live with HIV infection. Currently available treatments can reduce the amount of virus to near undetectable levels. But they do not eliminate the virus. A reservoir of HIV-infected cells persists during treatment. If treatment stops, these cells can cause rebounding virus levels and a return of symptoms. As a result, patients living with HIV must remain on treatment their entire lives. HIV reservoir cells often do not express viral proteins, making them hard for the immune system to find and destroy. Many of these reservoir cells occur in lymph nodes, which makes them difficult for researchers to access for study. Learning more about where these cells hide in the body may enable scientists to develop new treatments to help eliminate them. Sun et al. show that HIV reservoir cells exist in many body tissues, including the brain. In the experiments, Sun et al. used single HIV genome sequencing to identify HIV genetic sequences in the brain and other body tissues from three recently deceased individuals with HIV. The individuals agreed to donate their tissues for postmortem studies before their deaths. All received antiretroviral therapy until death. The experiments identified functional HIV genetic sequences in lymph nodes and gastrointestinal tissues, known hotspots for HIV-infected cells. Sun et al. also found genetically intact HIV in brain tissue from two of the individuals. The HIV genetic sequences were identical to sequences found in other body tissues. This discovery suggests HIV-infected cells had divided into more HIV-infected cells and spread. The results suggest that cells harboring intact HIV invade the brain and persist there for extended periods during antiretroviral therapy. To eradicate the virus, interventions targeting HIV reservoir cells must be able to reach the brain. This new information may help researchers developing HIV-reservoir targeting drugs decide which candidates will likely be the most effective. Future studies may also shed light on how HIV reaches the brain and how the infected cells escape destruction by immune cells, which may suggest more treatment strategies.

Persistence of intact HIV-1 proviruses in the brain during antiretroviral therapy.

HIV-1 reservoir cells that circulate in peripheral blood during suppressive antiretroviral therapy (ART) have been well characterized, but little is known about the dissemination of HIV-1-infected cells across multiple anatomical tissues, especially the central nervous system (CNS). Here, we performed single-genome, near full-length HIV-1 next-generation sequencing to evaluate the proviral landscape in distinct anatomical compartments, including multiple CNS tissues, from 3 ART-treated participants at autopsy. While lymph nodes and, to a lesser extent, gastrointestinal and genitourinary tissues represented tissue hotspots for the persistence of intact proviruses, we also observed intact proviruses in CNS tissue sections, particularly in the basal ganglia. Multi-compartment dissemination of clonal intact and defective proviral sequences occurred across multiple anatomical tissues, including the CNS, and evidence for the clonal proliferation of HIV-1-infected cells was found in the basal ganglia, in the frontal lobe, in the thalamus and in periventricular white matter. Deep analysis of HIV-1 reservoirs in distinct tissues will be informative for advancing HIV-1 cure strategies.

HIV-1 reservoir evolution in infants infected with clade C from Mozambique.

BACKGROUND: The persistence of HIV-1-infected cells during antiretroviral therapy is well documented but may be modulated by early initiation of antiretroviral therapy in infants. METHODS: Here, we longitudinally analyzed the proviral landscape in nine infants with vertical HIV-1 infection from Mozambique over a median period of 24 months, using single-genome, near full-length, next-generation proviral sequencing. RESULTS: We observed a rapid decline in the frequency of intact proviruses, leading to a disproportional under-representation of intact HIV-1 sequences within the total number of HIV-1 DNA sequences after 12-24 months of therapy. In addition, proviral integration site profiling in one infant demonstrated clonal expansion of infected cells harboring intact proviruses and indicated that viral rebound was associated with an integration site profile dominated by intact proviruses integrated into genic and accessible chromatin locations. CONCLUSION: Together, these results permit rare insight into the evolution of the HIV-1 reservoir in infants infected with HIV-1 and suggest that the rapid decline of intact proviruses, relative to defective proviruses, may be attributed to a higher vulnerability of genome-intact proviruses to antiviral immunity. Technologies to analyze combinations of intact proviral sequences and corresponding integration sites permit a high-resolution analysis of HIV-1 reservoir cells after early antiretroviral treatment initiation in infants.

Immune-profiling of SARS-CoV-2 viremic patients reveals dysregulated innate immune responses.

SARS-CoV-2 plasma viremia has been associated with severe disease and death in COVID-19. However, the effects of viremia on immune responses in blood cells remain unclear. The current study comprehensively examined transcriptional signatures of PBMCs involving T cells, B cells, NK cells, monocytes, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) respectively, from three different groups including individuals with moderate (nM), or severe disease with (vS) or without (nS) detectable plasma viral load. Whole transcriptome analysis demonstrated that all seven immune cell subsets were associated with disease severity regardless of cell type. Supervised clustering analysis demonstrated that mDCs and pDCs gene signatures could distinguish disease severity. Notably, transcriptional signatures of the vS group were enriched in pathways related to DNA repair, E2F targets, and G2M checkpoints; in contrast, transcriptional signatures of the nM group were enriched in interferon responses. Moreover, we observed an impaired induction of interferon responses accompanied by imbalanced cell-intrinsic immune sensing and an excessive inflammatory response in patients with severe disease (nS and vS). In sum, our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in seven major immune cells in COVID-19 patients.

Immune correlates of HIV-1 reservoir cell decline in early-treated infants.

Initiation of antiretroviral therapy (ART) in infected neonates within hours after birth limits viral reservoir seeding but does not prevent long-term HIV-1 persistence. Here, we report parallel assessments of HIV-1 reservoir cells and innate antiviral immune responses in a unique cohort of 37 infected neonates from Botswana who started ART extremely early, frequently within hours after birth. Decline of genome-intact HIV-1 proviruses occurs rapidly after initiation of ART and is associated with an increase in natural killer (NK) cell populations expressing the cytotoxicity marker CD57 and with a decrease in NK cell subsets expressing the inhibitory marker NKG2A. Immune perturbations in innate lymphoid cells, myeloid dendritic cells, and monocytes detected at birth normalize after rapid institution of antiretroviral therapy but do not notably influence HIV-1 reservoir cell dynamics. These results suggest that HIV-1 reservoir cell seeding and evolution in early-treated neonates is markedly influenced by antiviral NK cell immune responses.

A Possible Sterilizing Cure of HIV-1 Infection Without Stem Cell Transplantation.

BACKGROUND: A sterilizing cure of HIV-1 infection has been reported in 2 persons living with HIV-1 who underwent allogeneic hematopoietic stem cell transplantations from donors who were homozygous for the CCR5Δ32 gene polymorphism. However, this has been considered elusive during natural infection. OBJECTIVE: To evaluate persistent HIV-1 reservoir cells in an elite controller with undetectable HIV-1 viremia for more than 8 years in the absence of antiretroviral therapy. DESIGN: Detailed investigation of virologic and immunologic characteristics. SETTING: Tertiary care centers in Buenos Aires, Argentina, and Boston, Massachusetts. PATIENT: A patient with HIV-1 infection and durable drug-free suppression of HIV-1 replication. MEASUREMENTS: Analysis of genome-intact and replication-competent HIV-1 using near-full-length individual proviral sequencing and viral outgrowth assays, respectively; analysis of HIV-1 plasma RNA by ultrasensitive HIV-1 viral load testing. RESULTS: No genome-intact HIV-1 proviruses were detected in analysis of a total of 1.188 billion peripheral blood mononuclear cells and 503 million mononuclear cells from placental tissues. Seven defective proviruses, some of them derived from clonally expanded cells, were detected. A viral outgrowth assay failed to retrieve replication-competent HIV-1 from 150 million resting CD4+ T cells. No HIV-1 RNA was detected in 4.5 mL of plasma. LIMITATIONS: Absence of evidence for intact HIV-1 proviruses in large numbers of cells is not evidence of absence of intact HIV-1 proviruses. A sterilizing cure of HIV-1 can never be empirically proved. CONCLUSION: Genome-intact and replication-competent HIV-1 were not detected in an elite controller despite analysis of massive numbers of cells from blood and tissues, suggesting that this patient may have naturally achieved a sterilizing cure of HIV-1 infection. These observations raise the possibility that a sterilizing cure may be an extremely rare but possible outcome of HIV-1 infection. PRIMARY FUNDING SOURCE: National Institutes of Health and Bill & Melinda Gates Foundation.

Single center, open label dose escalating trial evaluating once weekly oral ixazomib in ART-suppressed, HIV positive adults and effects on HIV reservoir size in vivo.

BACKGROUND: Achieving a functional or sterilizing cure for HIV will require identification of therapeutic interventions that reduce HIV reservoir size in infected individuals. Proteasome inhibitors, such as ixazomib, impact multiple aspects of HIV biology including latency, transcription initiation, viral replication, and infected cell killing through the HIV protease - Casp8p41 pathway, resulting in latency reversal and reduced measures of HIV reservoir size ex vivo. METHODS: We conducted a phase 1b/2a dose escalating, open label trial of weekly oral ixazomib for 24 weeks in antiretroviral (ART)-suppressed, HIV positive adults (NCT02946047). The study was conducted from March 2017 to August 2019 at two tertiary referral centers in the United States. The primary outcomes were safety and tolerability of oral ixazomib. Secondary outcomes included changes in immunologic markers and estimates of HIV reservoir size after ixazomib treatment. FINDINGS: Sixteen participants completed the study. Ixazomib up to 4mg weekly was safe and well-tolerated, yielding no treatment-emergent events above grade 1. In exploratory analyses, ixazomib treatment was associated with detectable viremia that was below the lower limit of quantification (LLQ) in 9 participants, and viremia that was above LLQ in 4 of 16 participants. While treatment was associated with reduced CD4 counts [baseline 783 cells/ mm3 vs. week-24 724 cells/ mm3 p=0.003], there were no changes in markers of cellular activation, exhaustion or inflammation. Total HIV DNA and proviral sequencing were not altered by ixazomib treatment. Intact proviral DNA assay (IPDA) identified intact proviruses in 14 patients pre-treatment, and in 10/14 of those subjects post treatment values were reduced (P=0.068), allowing a calculated intact proviral half life of 0.6 years (95% CI 0.3, 2.5), compared to 7.1 years (95% CI 3.9, 18, p=0.004) in historical controls. Differentiation Quantitative Viral Outgrowth Assays (dQVOA) identified measurable proviruses in 15 subjects pre-treatment; post-treatment values were numerically reduced in 9, but overall differences were not significantly different. INTERPRETATION: Our study successfully met its primary endpoint of demonstrating the safety of ixazomib for 24 weeks in HIV infected persons. Exploratory analyses suggest that the effects observed ex vivo of latency reversal and reductions in HIV reservoir size, also occur in vivo. Future controlled studies of ixazomib are warranted. FUNDING: This study was funded by Millennium Pharmaceuticals Inc..; the Mayo Clinic Foundation; the National Institutes of Health, including the National Institute of Allergy and Infectious Diseases, Division of AIDS, the National Heart, Lung and Blood Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Neurological Disorders and Stroke, and the National Institute on Drug Abuse. Mayo Clinic also acknowledges generous funding support from Mr. Joseph T. and Mrs. Michele P. Betten.

Functional impairment of HIV-specific CD8+ T cells precedes aborted spontaneous control of viremia.

Spontaneous control of HIV infection has been repeatedly linked to antiviral CD8+ T cells but is not always permanent. To address mechanisms of durable and aborted control of viremia, we evaluated immunologic and virologic parameters longitudinally among 34 HIV-infected subjects with differential outcomes. Despite sustained recognition of autologous virus, HIV-specific proliferative and cytolytic T cell effector functions became selectively and intrinsically impaired prior to aborted control. Longitudinal transcriptomic profiling of functionally impaired HIV-specific CD8+ T cells revealed altered expression of genes related to activation, cytokine-mediated signaling, and cell cycle regulation, including increased expression of the antiproliferative transcription factor KLF2 but not of genes associated with canonical exhaustion. Lymphoid HIV-specific CD8+ T cells also exhibited poor functionality during aborted control relative to durable control. Our results identify selective functional impairment of HIV-specific CD8+ T cells as prognostic of impending aborted HIV control, with implications for clinical monitoring and immunotherapeutic strategies.

Long noncoding RNA MIR4435-2HG enhances metabolic function of myeloid dendritic cells from HIV-1 elite controllers.

Restriction of HIV-1 replication in elite controllers (ECs) is frequently attributed to T cell-mediated immune responses, while the specific contribution of innate immune cells is less clear. Here, we demonstrate an upregulation of the host long noncoding RNA (lncRNA) MIR4435-2HG in primary myeloid dendritic cells (mDCs) from ECs. Elevated expression of this lncRNA in mDCs was associated with a distinct immunometabolic profile, characterized by increased oxidative phosphorylation and glycolysis activities in response to TLR3 stimulation. Using functional assays, we show that MIR4435-2HG directly influenced the metabolic state of mDCs, likely through epigenetic mechanisms involving H3K27ac enrichment at an intronic enhancer in the RPTOR gene locus, the main component of the mammalian target of rapamycin complex 1 (mTORC1). Together, these results suggest a role of MIR4435-2HG for enhancing immunometabolic activities of mDCs in ECs through targeted epigenetic modifications of a member of the mTOR signaling pathway.

Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.

The Loss of Bcl-6 Expressing T Follicular Helper Cells and the Absence of Germinal Centers in COVID-19.

Humoral responses in COVID-19 disease are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined postmortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers, a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+TFH cell differentiation together with an increase in T-bet+TH1 cells and aberrant extra-follicular TNF-a accumulation.  Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections and suggest that achieving herd immunity through natural infection may be difficult. Funding: This work was supported by NIH U19 AI110495 to SP, NIH R01 AI146779 to AGS, NIH R01AI137057 and DP2DA042422 to DL, BMH was supported by NIGMS T32 GM007753, TMC was supported by T32 AI007245. Funding for these studies from the Massachusetts Consortium of Pathogen Readiness, the Mark and Lisa Schwartz Foundation and Enid Schwartz is also acknowledged. Conflict of Interest: None. Ethical Approval: This study was performed with the approval of the Institutional Review Boards at the Massachusetts General Hospital and the Brigham and Women's Hospital.