Publications by authors named "Edward P Browne"

31 Publications

Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors.

PLoS Pathog 2021 Feb 26;17(2):e1009346. Epub 2021 Feb 26.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.
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http://dx.doi.org/10.1371/journal.ppat.1009346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946360PMC
February 2021

Epitope spreading toward wild-type melanocyte-lineage antigens rescues suboptimal immune checkpoint blockade responses.

Sci Transl Med 2021 Feb;13(581)

Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8 T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.
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http://dx.doi.org/10.1126/scitranslmed.abd8636DOI Listing
February 2021

SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.

Nature 2021 03 9;591(7850):451-457. Epub 2021 Feb 9.

International Center for the Advancement of Translational Science, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

All coronaviruses known to have recently emerged as human pathogens probably originated in bats. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.
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http://dx.doi.org/10.1038/s41586-021-03312-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979515PMC
March 2021

The Role of Toll-Like Receptors in Retroviral Infection.

Authors:
Edward P Browne

Microorganisms 2020 Nov 14;8(11). Epub 2020 Nov 14.

Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.

Toll-like receptors (TLRs) are key pathogen sensing receptors that respond to diverse microbial ligands, and trigger both innate and adaptive immune responses to infection. Since their discovery, a growing body of evidence has pointed to an important role for TLRs in retroviral infection and pathogenesis. These data suggest that multiple TLRs contribute to the anti-retroviral response, and that TLR engagement by retroviruses can have complex and divergent outcomes for infection. Despite this progress, numerous questions remain about the role of TLRs in retroviral infection. In this review, I summarize existing evidence for TLR-retrovirus interactions and the functional roles these receptors play in immunity and pathogenesis, with particular focus on human immunodeficiency virus (HIV).
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http://dx.doi.org/10.3390/microorganisms8111787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697840PMC
November 2020

HSF1 Activation Can Restrict HIV Replication.

ACS Infect Dis 2020 07 10;6(7):1659-1666. Epub 2020 Jun 10.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

Host protein folding stress responses can play important roles in RNA virus replication and evolution. Prior work suggested a complicated interplay between the cytosolic proteostasis stress response, controlled by the transcriptional master regulator heat shock factor 1 (HSF1), and human immunodeficiency virus-1 (HIV-1). We sought to uncouple HSF1 transcription factor activity from cytotoxic proteostasis stress and thereby better elucidate the proposed role(s) of HSF1 in the HIV-1 lifecycle. To achieve this objective, we used chemical genetic, stress-independent control of HSF1 activity to establish whether and how HSF1 influences HIV-1 replication. Stress-independent HSF1 induction decreased both the total quantity and infectivity of HIV-1 virions. Moreover, HIV-1 was unable to escape HSF1-mediated restriction over the course of several serial passages. These results clarify the interplay between the host's heat shock response and HIV-1 infection and motivate continued investigation of chaperones as potential antiviral therapeutic targets.
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http://dx.doi.org/10.1021/acsinfecdis.0c00166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434488PMC
July 2020

Evaluation of EED Inhibitors as a Class of PRC2-Targeted Small Molecules for HIV Latency Reversal.

ACS Infect Dis 2020 07 30;6(7):1719-1733. Epub 2020 May 30.

UNC HIV Cure Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599, United States.

A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.
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http://dx.doi.org/10.1021/acsinfecdis.9b00514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359025PMC
July 2020

Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo.

Nature 2020 02 22;578(7793):160-165. Epub 2020 Jan 22.

International Center for the Advancement of Translational Science, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Long-lasting, latently infected resting CD4 T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4 T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.
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http://dx.doi.org/10.1038/s41586-020-1951-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111210PMC
February 2020

The immune cell landscape in kidneys of patients with lupus nephritis.

Nat Immunol 2019 07 17;20(7):902-914. Epub 2019 Jun 17.

Center for Autoimmune and Musculoskeletal Diseases, The Feinstein Institute for Medical Research, Northwell Health, Manhasset, NY, USA.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.
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http://dx.doi.org/10.1038/s41590-019-0398-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726437PMC
July 2019

Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures.

Sci Adv 2019 01 23;5(1):eaau9223. Epub 2019 Jan 23.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.
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http://dx.doi.org/10.1126/sciadv.aau9223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357748PMC
January 2019

A secreted PD-L1 splice variant that covalently dimerizes and mediates immunosuppression.

Cancer Immunol Immunother 2019 Mar 18;68(3):421-432. Epub 2018 Dec 18.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Ave., Boston, MA, 02215, USA.

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.
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http://dx.doi.org/10.1007/s00262-018-2282-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426808PMC
March 2019

Single-Cell Analysis of Quiescent HIV Infection Reveals Host Transcriptional Profiles that Regulate Proviral Latency.

Cell Rep 2018 10;25(1):107-117.e3

UNC HIV Cure Center and Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

A detailed understanding of the mechanisms that establish or maintain the latent reservoir of HIV will guide approaches to eliminate persistent infection. We used a cell line and primary cell models of HIV latency to investigate viral RNA (vRNA) expression and the role of the host transcriptome using single-cell approaches. Single-cell vRNA quantitation identified distinct populations of cells expressing various levels of vRNA, including completely silent populations. Strikingly, single-cell RNA-seq of latently infected primary cells demonstrated that HIV downregulation occurred in diverse transcriptomic environments but was significantly associated with expression of a specific set of cellular genes. In particular, latency was more frequent in cells expressing a transcriptional signature that included markers of naive and central memory T cells. These data reveal that expression of HIV proviruses within the latent reservoir are influenced by the host cell transcriptional program. Therapeutic modulation of these programs may reverse or enforce HIV latency.
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http://dx.doi.org/10.1016/j.celrep.2018.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258175PMC
October 2018

Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

Arthritis Res Ther 2018 07 11;20(1):139. Epub 2018 Jul 11.

University of Rochester Medical Center, Rochester, NY, 14642, USA.

Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples.

Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq.

Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4 and CD8 T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified.

Conclusions: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.
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http://dx.doi.org/10.1186/s13075-018-1631-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042350PMC
July 2018

Erratum: "Prediction uncertainty and optimal experimental design for learning dynamical systems" [Chaos 26, 063110 (2016)].

Chaos 2017 06;27(6):069901

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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http://dx.doi.org/10.1063/1.4986799DOI Listing
June 2017

Prediction uncertainty and optimal experimental design for learning dynamical systems.

Chaos 2016 06;26(6):063110

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Dynamical systems are frequently used to model biological systems. When these models are fit to data, it is necessary to ascertain the uncertainty in the model fit. Here, we present prediction deviation, a metric of uncertainty that determines the extent to which observed data have constrained the model's predictions. This is accomplished by solving an optimization problem that searches for a pair of models that each provides a good fit for the observed data, yet has maximally different predictions. We develop a method for estimating a priori the impact that additional experiments would have on the prediction deviation, allowing the experimenter to design a set of experiments that would most reduce uncertainty. We use prediction deviation to assess uncertainty in a model of interferon-alpha inhibition of viral infection, and to select a sequence of experiments that reduces this uncertainty. Finally, we prove a theoretical result which shows that prediction deviation provides bounds on the trajectories of the underlying true model. These results show that prediction deviation is a meaningful metric of uncertainty that can be used for optimal experimental design.
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http://dx.doi.org/10.1063/1.4953795DOI Listing
June 2016

The effect of HIV-1 Vif polymorphisms on A3G anti-viral activity in an in vivo mouse model.

Retrovirology 2016 06 30;13(1):45. Epub 2016 Jun 30.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.
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http://dx.doi.org/10.1186/s12977-016-0280-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929759PMC
June 2016

A Computational Model of Inhibition of HIV-1 by Interferon-Alpha.

PLoS One 2016 24;11(3):e0152316. Epub 2016 Mar 24.

Operations Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152316PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807028PMC
August 2016

Visualizing antibody affinity maturation in germinal centers.

Science 2016 03 18;351(6277):1048-54. Epub 2016 Feb 18.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.

Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza.
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http://dx.doi.org/10.1126/science.aad3439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938154PMC
March 2016

An interleukin-1 beta-encoding retrovirus exhibits enhanced replication in vivo.

Authors:
Edward P Browne

J Virol 2015 Jan 15;89(1):155-64. Epub 2014 Oct 15.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

Unlabelled: Interleukin-1 beta (IL-1β) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1β secretion through the DNA-sensing molecule IFI16, the effect of IL-1β on the course of infection is unknown. To test whether IL-1β secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL-1β) that encodes the mature form of IL-1β. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1β exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1β-infected mice correlated with a large expansion of Gr-1(+) CD11b(+) myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1β infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1β was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1β secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1β represents a new model of proinflammatory retroviral infection.

Importance: Interleukin-1 beta (IL-1β) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1β on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL-1β) that encodes IL-1β and promotes abundant IL-1β secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1β as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.
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http://dx.doi.org/10.1128/JVI.02314-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301099PMC
January 2015

Different modes of retrovirus restriction by human APOBEC3A and APOBEC3G in vivo.

PLoS Pathog 2014 May 22;10(5):e1004145. Epub 2014 May 22.

Department of Microbiology, Institute for Immunology and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.
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http://dx.doi.org/10.1371/journal.ppat.1004145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031197PMC
May 2014

Toll-like receptor 7 inhibits early acute retroviral infection through rapid lymphocyte responses.

Authors:
Edward P Browne

J Virol 2013 Jul 24;87(13):7357-66. Epub 2013 Apr 24.

Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Early events during retroviral infection play a critical role in determining the course of infection and pathogenesis, but the mechanisms that regulate this phase of infection are poorly understood. Toll-like receptor 7 (TLR7) is required for promoting germinal center reactions and virus-specific neutralizing antibodies at later time points postinfection, but TLR7's role in early acute infection has not been determined. By infecting TLR7-deficient mice with a retroviral pathogen, Friend virus (FV), I determined that TLR7 potently inhibits retroviral replication during the first 5 days of infection and is required for rapid secretion of virus-specific IgM and interleukin-10 (IL-10) in response to infection. Although the IgM response was nonneutralizing, plasmas from wild-type mice but not TLR7-deficient mice inhibited FV replication when passively transferred to infected mice, suggesting an indirect mechanism of antibody function. Interestingly, IL-10 was secreted primarily by CD4 T cells, and IL-10-deficient mice also exhibited accelerated early virus spread, demonstrating that this response inhibits acute infection. Surprisingly, TLR7-deficient mice exhibited normal or elevated secretion of proinflammatory cytokines during acute infection, revealing the existence of a TLR7-independent retrovirus-sensing pathway that drives inflammatory cytokine secretion. Together, these results establish a previously unappreciated role for lymphocytes in mediating rapid TLR7-dependent inhibition of early retroviral infection through nonneutralizing IgM and IL-10.
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http://dx.doi.org/10.1128/JVI.00788-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700304PMC
July 2013

Regulation of B-cell responses by Toll-like receptors.

Authors:
Edward P Browne

Immunology 2012 Aug;136(4):370-9

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The discovery of host-encoded gene products that sense molecular patterns in infectious microbes, and the demonstration of their role in triggering innate and adaptive immune responses, has been a key milestone in our understanding of immunology. Twenty-three years after Janeway first outlined the fundamental concepts of the 'pattern recognition' model, and 15 years since the identification of Toll-like receptors (TLRs) as pattern recognition receptors (PRRs), new insights continue to be revealed, and questions remain. For example, innate immune responses to microbes that are mediated by PRRs have historically been viewed as the domain of innate immune cell populations such as dendritic cells and macrophages. New evidence, however, has pointed to the role of B-cell-intrinsic TLR activation in shaping antibody responses. These studies have revealed that TLRs regulate a complex transcriptional network that controls multiple steps in the development of antigen-specific antibodies. This review covers these recent developments regarding the role of TLRs in B-cell gene expression and function in vitro and in vivo, and highlights the remaining challenges in the field, with particular emphasis on the role of TLRs in antibody responses to viral infection. A more complete understanding of how TLRs regulate antibody responses will lead to improved vaccine design.
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http://dx.doi.org/10.1111/j.1365-2567.2012.03587.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401975PMC
August 2012

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin.

Biotechnol Lett 2012 May 30;34(5):853-6. Epub 2011 Dec 30.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin-sepharose beads and successively mixing an HIV-1 solution with fresh beads.
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http://dx.doi.org/10.1007/s10529-011-0840-0DOI Listing
May 2012

Toll-like receptor 7 controls the anti-retroviral germinal center response.

Authors:
Edward P Browne

PLoS Pathog 2011 Oct 6;7(10):e1002293. Epub 2011 Oct 6.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR) signaling in control of a model retroviral pathogen, Friend virus (FV), I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC) or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.
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http://dx.doi.org/10.1371/journal.ppat.1002293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3188541PMC
October 2011

Restriction of HIV-1 by APOBEC3G is cytidine deaminase-dependent.

Virology 2009 May 21;387(2):313-21. Epub 2009 Mar 21.

Department of Microbiology, New York University School of Medicine, New York, 10016, USA.

Cytidine deamination is the primary mechanism by which APOBEC3G restricts HIV-1; however, several studies have reported that APOBEC3G also inhibits virus replication via a mechanism that is independent of deamination. Using active site APOBEC3G mutants, we have re-evaluated the biological relevance of deaminase-independent APOBEC3G-mediated restriction of HIV-1. APOBEC3G proteins with Glu-->Ala mutations in AS1, AS2 or AS1 and AS2 were stably expressed at physiological levels in CEM-SS T cells and 293T cells and the ability of the cells to support Deltavif HIV-1 replication was then tested. The AS2 and AS1/AS2 mutants were packaged efficiently into virions but in single-cycle or multi-cycle HIV-1 replication assays, were found to lack antiviral activity. The AS1 mutant, which retained deaminase activity, maintained near wild-type antiviral function. To determine the potency of APOBEC3G antiviral activity, cell lines were established that that expressed low levels of wild-type APOBEC3G and generated virions that contained as few as 1-2 APOBEC3G molecules. Even at very low copy number, APOBEC3G caused a significant reduction in infectivity, suggesting that a single molecule of packaged APOBEC3G inactivates the virus. The high potency of APOBEC3G is consistent with a catalytic mechanism of restriction in which a single molecule can induce a string of mutations but difficult to reconcile with a deaminase-independent, non-catalytic mechanism. Analysis of the reverse transcript sequences showed that the G-->A mutations were clustered, likely reflecting the action of single APOBEC3G molecules acting processively. We conclude that cytidine deamination is the mechanism by which APOBEC3G restricts HIV-1.
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http://dx.doi.org/10.1016/j.virol.2009.02.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3708462PMC
May 2009

Myd88 is required for an antibody response to retroviral infection.

PLoS Pathog 2009 Feb 13;5(2):e1000298. Epub 2009 Feb 13.

The Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, New York, United States of America.

Although retroviruses have been extensively studied for many years, basic questions about how retroviral infections are detected by the immune system and which innate pathways are required for the generation of immune responses remain unanswered. Defining these pathways and how they contribute to the anti-retroviral immune responses would assist in the development of more effective vaccines for retroviral pathogens such as HIV. We have investigated the roles played by CD11c(+) dendritic cells (DCs) and by Toll-like receptor (TLR) signaling pathways in the generation of an anti-retroviral immune response against a mouse retroviral pathogen, Friend murine leukemia virus (F-MLV). Specific deletion of DCs during F-MLV infection caused a significant increase in viral titers at 14 days post-infection, indicating the importance of DCs in immune control of the infection. Similarly, Myd88 knockout mice failed to control F-MLV, and sustained high viral titers (10(7) foci/spleen) for several months after infection. Strikingly, both DC-depleted mice and Myd88 knockout mice exhibited only a partial reduction of CD8(+) T cell responses, while the IgG antibody response to F-MLV was completely lost. Furthermore, passive transfer of immune serum from wild-type mice to Myd88 knockout mice rescued control of F-MLV. These results identify TLR signaling and CD11c(+) DCs as playing critical roles in the humoral response to retroviruses.
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http://dx.doi.org/10.1371/journal.ppat.1000298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633609PMC
February 2009

Species-specific restriction of apobec3-mediated hypermutation.

J Virol 2008 Feb 21;82(3):1305-13. Epub 2007 Nov 21.

The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, 540 First Ave., Second Floor, New York, NY 10016, USA.

Apobec proteins are a family of cellular cytidine deaminases, among which several members have been shown to have potent antiviral properties. This antiviral activity is associated with the ability to cause hypermutation of retroviral cDNA. However, recent research has indicated that Apobec proteins are also able to inhibit retroviruses by other mechanisms that are independent of their deaminase activity. We have compared the antiviral activities of human and murine Apobec3 (A3) proteins, and we have found that, consistent with previous reports, human immunodeficiency virus (HIV) is able to resist human A3G but is sensitive to murine A3, whereas murine leukemia virus (MLV) is relatively resistant to murine A3 (mA3) but sensitive to human A3G. In contrast to previous studies, we observed that mA3 is packaged efficiently into MLV particles. The C-terminal cytidine deaminase domain (CDD2) is required for packaging of mA3 into MLV particles, and packaging did not depend on the MLV viral RNA. However, mA3 packed into MLV particles failed to cause hypermutation of viral DNA, indicating that its deaminase activity is blocked or inhibited. hA3G also caused significantly less hypermutation of MLV than of HIV DNA. Both mA3 and the splice variant mA3Delta5 exhibited some residual antiviral activity against MLV and caused a reduction in the ability of MLV particles to generate reverse transcription products. These results suggest that MLV has evolved specific mechanisms to block the ability of Apobec proteins to mediate deaminase-dependent hypermutation.
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http://dx.doi.org/10.1128/JVI.01371-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224457PMC
February 2008

Virus-host interactions: new insights from the small RNA world.

Genome Biol 2005 31;6(11):238. Epub 2005 Oct 31.

Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

RNA silencing has a known role in the antiviral responses of plants and insects. Recent evidence, including the finding that the Tat protein of human immunodeficiency virus (HIV) can suppress the host's RNA-silencing pathway and may thus counteract host antiviral RNAs, suggests that RNA-silencing pathways could also have key roles in mammalian virus-host interactions.
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http://dx.doi.org/10.1186/gb-2005-6-11-238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1297642PMC
June 2006

Evaluation of the host transcriptional response to human cytomegalovirus infection.

Physiol Genomics 2004 Jun 17;18(1):51-62. Epub 2004 Jun 17.

Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.

Gene expression data from human cytomegalovirus (HCMV)-infected cells were analyzed using DNA-Chip Analyzer (dChip) followed by singular value decomposition (SVD) and compared with a previous analysis of the same data that employed GeneChip software and a fold change filtering approach. dChip and SVD analysis revealed two clusters of coexpressed human genes responding differently to HCMV infection: one containing some genes identified previously, and another that was largely unique to this analysis. Annotating these genes, we identified several functional categories important to host cell responses to HCMV infection. These categories included genes involved in transcriptional regulation, oncogenesis, and cell cycle regulation, which were more prevalent in cluster 1, and genes involved in immune system regulation, signal transduction, and cell adhesion, which were more prevalent in cluster 2. Within these categories, we found genes involved in the host response to HCMV infection (mainly in cluster 1), as well as genes targeted by HCMV's immune evasion strategies (mainly in cluster 2). As the second group of genes identified by the dChip and SVD approach was statistically and biologically significant, our results point out the advantages of using different methods to analyze gene expression data.
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http://dx.doi.org/10.1152/physiolgenomics.00155.2003DOI Listing
June 2004

Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells.

Proc Natl Acad Sci U S A 2003 Sep 12;100(20):11439-44. Epub 2003 Sep 12.

Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA.

The initial interaction of human cytomegalovirus with fibroblasts triggers, and then partially blocks, an innate immune response pathway that leads to the induction of IFN-responsive genes and proinflammatory chemokines. Infection of fibroblasts with human cytomegalovirus inhibited their ability to respond to exogenous IFN. Consistent with the observation that the block did not depend on de novo viral protein synthesis, ectopic expression of the viral UL83-coded pp65, an abundant virion protein, inhibited IFN signaling. Furthermore, DNA array analysis showed that infection with a pp65-deficient mutant virus caused a much stronger induction of many IFN-response and proinflammatory chemokine RNAs than infection with wild-type virus. The nuclear DNA-binding activities of transcription factors NF-kappaB and IRF1 were induced to a much greater extent after infection with the pp65-deficient mutant than with wild-type virus. IFN-stimulated gene factor 3 DNA-binding was modestly enhanced, whereas IRF3 activity was not affected by mutation of pp65. Together, these results imply that pp65, which is delivered to newly infected cells in the virion, antagonizes a pathway that affects NF-kappaB and IRF1 and prevents the accumulation of mRNAs encoded by numerous cellular antiviral genes.
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http://dx.doi.org/10.1073/pnas.1534570100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208776PMC
September 2003