Publications by authors named "Frederic D Bushman"

244 Publications

Signatures of COVID-19 severity and immune response in the respiratory tract microbiome.

medRxiv 2021 Apr 5. Epub 2021 Apr 5.

Rationale: Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease.

Objective: To define the respiratory tract microbiome in COVID-19 and relationship disease severity, systemic immunologic features, and outcomes.

Methods And Measurements: We examined 507 oropharyngeal, nasopharyngeal and endotracheal samples from 83 hospitalized COVID-19 patients, along with non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, commensal DNA viruses and were quantified by qPCR, and immune features were characterized by lymphocyte/neutrophil (L/N) ratios and deep immune profiling of peripheral blood mononuclear cells (PBMC).

Main Results: COVID-19 patients had upper respiratory microbiome dysbiosis, and greater change over time than critically ill patients without COVID-19. Diversity at the first time point correlated inversely with disease severity during hospitalization, and microbiome composition was associated with L/N ratios and PBMC profiles in blood. Intubated patients showed patient-specific and dynamic lung microbiome communities, with prominence of . and showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity.

Conclusions: The respiratory tract microbiome and commensal virome are disturbed in COVID-19, correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, possible use as biomarkers, and role of bacterial and viral taxa identified here in COVID-19 pathogenesis.
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http://dx.doi.org/10.1101/2021.04.02.21254514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043476PMC
April 2021

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Dynamics Tracking.

medRxiv 2021 Mar 26. Epub 2021 Mar 26.

Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples.

Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients.

Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections.

Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.
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http://dx.doi.org/10.1101/2021.03.17.21253847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010739PMC
March 2021

Decreased Intestinal Microbiome Diversity in Pediatric Sepsis: A Conceptual Framework for Intestinal Dysbiosis to Influence Immunometabolic Function.

Crit Care Explor 2021 Mar 17;3(3):e0360. Epub 2021 Mar 17.

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

Objectives: The intestinal microbiome can modulate immune function through production of microbial-derived short-chain fatty acids. We explored whether intestinal dysbiosis in children with sepsis leads to changes in microbial-derived short-chain fatty acids in plasma and stool that are associated with immunometabolic dysfunction in peripheral blood mononuclear cells.

Design: Prospective observational pilot study.

Setting: Single academic PICU.

Patients: Forty-three children with sepsis/septic shock and 44 healthy controls.

Measurements And Main Results: Stool and plasma samples were serially collected for sepsis patients; stool was collected once for controls. The intestinal microbiome was assessed using 16S ribosomal RNA sequencing and alpha- and beta-diversity were determined. We measured short-chain fatty acids using liquid chromatography, peripheral blood mononuclear cell mitochondrial respiration using high-resolution respirometry, and immune function using ex vivo lipopolysaccharide-stimulated whole blood tumor necrosis factor-α. Sepsis patients exhibited reduced microbial diversity compared with healthy controls, with lower alpha- and beta-diversity. Reduced microbial diversity among sepsis patients (mainly from lower abundance of commensal obligate anaerobes) was associated with increased acetic and propionic acid and decreased butyric, isobutyric, and caproic acid. Decreased levels of plasma butyric acid were further associated with lower peripheral blood mononuclear cell mitochondrial respiration, which in turn, was associated with lower lipopolysaccharide-stimulated tumor necrosis factor-α. However, neither intestinal dysbiosis nor specific patterns of short-chain fatty acids were associated with lipopolysaccharide-stimulated tumor necrosis factor-α.

Conclusions: Intestinal dysbiosis was associated with altered short-chain fatty acid metabolites in children with sepsis, but these findings were not linked directly to mitochondrial or immunologic changes. More detailed mechanistic studies are needed to test the role of microbial-derived short-chain fatty acids in the progression of sepsis.
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http://dx.doi.org/10.1097/CCE.0000000000000360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994045PMC
March 2021

The human virome: assembly, composition and host interactions.

Nat Rev Microbiol 2021 Mar 30. Epub 2021 Mar 30.

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

The human body hosts vast microbial communities, termed the microbiome. Less well known is the fact that the human body also hosts vast numbers of different viruses, collectively termed the 'virome'. Viruses are believed to be the most abundant and diverse biological entities on our planet, with an estimated 10 particles on Earth. The human virome is similarly vast and complex, consisting of approximately 10 particles per human individual, with great heterogeneity. In recent years, studies of the human virome using metagenomic sequencing and other methods have clarified aspects of human virome diversity at different body sites, the relationships to disease states and mechanisms of establishment of the human virome during early life. Despite increasing focus, it remains the case that the majority of sequence data in a typical virome study remain unidentified, highlighting the extent of unexplored viral 'dark matter'. Nevertheless, it is now clear that viral community states can be associated with adverse outcomes for the human host, whereas other states are characteristic of health. In this Review, we provide an overview of research on the human virome and highlight outstanding recent studies that explore the assembly, composition and dynamics of the human virome as well as host-virome interactions in health and disease.
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http://dx.doi.org/10.1038/s41579-021-00536-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008777PMC
March 2021

NPM-ALK induces reprogramming of mature TCR-stimulated T cells resulting in de-differentiation and malignant transformation.

Cancer Res 2021 Feb 22. Epub 2021 Feb 22.

Microbiology and Center for Cellular Immunotherapies, University of Pennsylvania

Fusion genes including NPM-ALK can promote T cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM-ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T cell effector programs, re-emergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM-ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Lastly, TCR-generated signals were required to achieve T cell transformation, explaining how healthy individuals can harbor T cells with NPM-ALK translocations. These findings describe the fundamental mechanisms of NPM-ALK-mediated oncogenesis, and may serve as a model to better understand factors that regulate tumor formation.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2297DOI Listing
February 2021

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Mol Ther 2021 04 29;29(4):1625-1638. Epub 2021 Jan 29.

Division of Hematology, Children's Hospital of Philadelphia (CHOP), Philadelphia, PA, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Cell and Molecular Biology Affinity Group (CAMB), University of Pennsylvania, Philadelphia, PA, USA; Raymond G. Perelman Center for Cellular and Molecular Therapeutics, CHOP, Philadelphia, PA, USA; Penn Center for Musculoskeletal Disorders, CHOP, Philadelphia, PA, USA. Electronic address:

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34 cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.
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http://dx.doi.org/10.1016/j.ymthe.2020.12.036DOI Listing
April 2021

Role of dietary fiber in the recovery of the human gut microbiome and its metabolome.

Cell Host Microbe 2021 03 12;29(3):394-407.e5. Epub 2021 Jan 12.

Division of Gastroenterology and Hepatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Gut microbiota metabolites may be important for host health, yet few studies investigate the correlation between human gut microbiome and production of fecal metabolites and their impact on the plasma metabolome. Since gut microbiota metabolites are influenced by diet, we performed a longitudinal analysis of the impact of three divergent diets, vegan, omnivore, and a synthetic enteral nutrition (EEN) diet lacking fiber, on the human gut microbiome and its metabolome, including after a microbiota depletion intervention. Omnivore and vegan, but not EEN, diets altered fecal amino acid levels by supporting the growth of Firmicutes capable of amino acid metabolism. This correlated with relative abundance of a sizable number of fecal amino acid metabolites, some not previously associated with the gut microbiota. The effect on the plasma metabolome, in contrast, were modest. The impact of diet, particularly fiber, on the human microbiome influences broad classes of metabolites that may modify health.
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http://dx.doi.org/10.1016/j.chom.2020.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022197PMC
March 2021

Antigen-driven clonal selection shapes the persistence of HIV-1-infected CD4+ T cells in vivo.

J Clin Invest 2021 Feb;131(3)

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor β-chain (TCRβ) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRβ repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRβ and integration site analysis showed that infection could occur early or late in the course of a clone's response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.
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http://dx.doi.org/10.1172/JCI145254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843227PMC
February 2021

ICTV Virus Taxonomy Profile: .

J Gen Virol 2021 Jan 27;102(1). Epub 2020 Nov 27.

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

Viruses in the family have a circular genome of 3.0 kb with three open reading frames. The packaged genome is inferred to be single-stranded DNA by analogy to related viruses. Redondoviruses were discovered through metagenomic sequencing methods in samples from human subjects and are inferred to replicate in humans. Evidence of redondovirus infection is associated with periodontitis and critical illness, but redondoviruses have not been shown to be the causative agent of any diseases. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family , which is available at ictv.global/report/redondoviridae.
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http://dx.doi.org/10.1099/jgv.0.001526DOI Listing
January 2021

A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.

Nat Biotechnol 2021 01 16;39(1):47-55. Epub 2020 Nov 16.

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.
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http://dx.doi.org/10.1038/s41587-020-0741-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855056PMC
January 2021

Investigating hospital Mycobacterium chelonae infection using whole genome sequencing and hybrid assembly.

PLoS One 2020 9;15(11):e0236533. Epub 2020 Nov 9.

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

Mycobacterium chelonae is a rapidly growing nontuberculous mycobacterium that is a common cause of nosocomial infections. Here we describe investigation of a possible nosocomial transmission of M. chelonae at the Hospital of the University of Pennsylvania (HUP). M. chelonae strains with similar high-level antibiotic resistance patterns were isolated from two patients who developed post-operative infections at HUP in 2017, suggesting a possible point source infection. The isolates, along with other clinical isolates from other patients, were sequenced using the Illumina and Oxford Nanopore technologies. The resulting short and long reads were hybrid assembled into draft genomes. The genomes were compared by quantifying single nucleotide variants in the core genome and assessed using a control dataset to quantify error rates in comparisons of identical genomes. We show that all M. chelonae isolates tested were highly dissimilar, as indicated by high pairwise SNV values, consistent with environmental acquisition and not a nosocomial point source. Our control dataset determined a threshold for evaluating identity between strains while controlling for sequencing error. Finally, antibiotic resistance genes were predicted for our isolates, and several single nucleotide variants were identified that have the potential to modulated drug resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236533PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652343PMC
December 2020

Mitochondrial dysfunction in inflammatory bowel disease alters intestinal epithelial metabolism of hepatic acylcarnitines.

J Clin Invest 2021 Jan;131(1)

Department of Medicine, Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

As the interface between the gut microbiota and the mucosal immune system, there has been great interest in the maintenance of colonic epithelial integrity through mitochondrial oxidation of butyrate, a short-chain fatty acid produced by the gut microbiota. Herein, we showed that the intestinal epithelium could also oxidize long-chain fatty acids, and that luminally delivered acylcarnitines in bile could be consumed via apical absorption by the intestinal epithelium, resulting in mitochondrial oxidation. Finally, intestinal inflammation led to mitochondrial dysfunction in the apical domain of the surface epithelium that may reduce the consumption of fatty acids, contributing to higher concentrations of fecal acylcarnitines in murine Citrobacter rodentium-induced colitis and human inflammatory bowel disease. These results emphasized the importance of both the gut microbiota and the liver in the delivery of energy substrates for mitochondrial metabolism by the intestinal epithelium.
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http://dx.doi.org/10.1172/JCI133371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773399PMC
January 2021

Assessment of HIV-1 integration in tissues and subsets across infection stages.

JCI Insight 2020 10 15;5(20). Epub 2020 Oct 15.

Department of Microbiology and.

The integration of HIV DNA into the host genome contributes to lifelong infection in most individuals. Few studies have examined integration in lymphoid tissue, where HIV predominantly persists before and after antiretroviral treatment (ART). Of particular interest is whether integration site distributions differ between infection stages with paired blood and tissue comparisons. Here, we profiled HIV integration site distributions in sorted memory, tissue-resident, and/or follicular helper CD4+ T cell subsets from paired blood and lymphoid tissue samples from acute, chronic, and ART-treated individuals. We observed minor differences in the frequency of nonintronic and nondistal intergenic sites, varying with tissue and residency phenotypes during ART. Genomic and epigenetic annotations were generally similar. Clonal expansion of cells marked by identical integration sites was detected, with increased detection in chronic and ART-treated individuals. However, overlap between or within CD4+ T cell subsets or tissue compartments was only observed in 8 unique sites of the 3540 sites studied. Together, these findings suggest that shared integration sites between blood and tissue may, depending on the tissue site, be the exception rather than the rule and indicate that additional studies are necessary to fully understand the heterogeneity of tissue-sequestered HIV reservoirs.
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http://dx.doi.org/10.1172/jci.insight.139783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605534PMC
October 2020

Influence of the amino-terminal sequence on the structure and function of HIV integrase.

Retrovirology 2020 08 31;17(1):28. Epub 2020 Aug 31.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Background: Antiretroviral therapy (ART) can mitigate the morbidity and mortality caused by the human immunodeficiency virus (HIV). Successful development of ART can be accelerated by accurate structural and biochemical data on targets and their responses to inhibitors. One important ART target, HIV integrase (IN), has historically been studied in vitro in a modified form adapted to bacterial overexpression, with a methionine or a longer fusion protein sequence at the N-terminus. In contrast, IN present in viral particles is produced by proteolytic cleavage of the Pol polyprotein, which leaves a phenylalanine at the N-terminus (IN 1F). Inspection of available structures suggested that added residues on the N-terminus might disrupt proper protein folding and formation of multimeric complexes.

Results: We purified HIV-1 IN 1F and solved its structure at 2.4 Å resolution, which showed extension of an N-terminal helix compared to the published structure of IN. Full-length IN 1F showed increased in vitro catalytic activity in assays of coupled joining of the two viral DNA ends compared to two IN variants containing additional N-terminal residues. IN 1F was also altered in its sensitivity to inhibitors, showing decreased sensitivity to the strand-transfer inhibitor raltegravir and increased sensitivity to allosteric integrase inhibitors. In solution, IN 1F exists as monomers and dimers, in contrast to other IN preparations which exist as higher-order oligomers.

Conclusions: The structural, biochemical, and biophysical characterization of IN 1F reveals the conformation of the native HIV-1 IN N-terminus and accompanying unique biochemical and biophysical properties. IN 1F thus represents an improved reagent for use in integration reactions in vitro and the development of antiretroviral agents.
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http://dx.doi.org/10.1186/s12977-020-00537-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457537PMC
August 2020

Multi-omic Analysis of the Interaction between Clostridioides difficile Infection and Pediatric Inflammatory Bowel Disease.

Cell Host Microbe 2020 09 20;28(3):422-433.e7. Epub 2020 Aug 20.

Division of Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Children with inflammatory bowel diseases (IBD) are particularly vulnerable to infection with Clostridioides difficile (CDI). IBD and IBD + CDI have overlapping symptoms but respond to distinctive treatments, highlighting the need for diagnostic biomarkers. Here, we studied pediatric patients with IBD and IBD + CDI, comparing longitudinal data on the gut microbiome, metabolome, and other measures. The microbiome is dysbiotic and heterogeneous in both disease states, but the metabolome reveals disease-specific patterns. The IBD group shows increased concentrations of markers of inflammation and tissue damage compared with healthy controls, and metabolic changes associate with susceptibility to CDI. In IBD + CDI, we detect both metabolites associated with inflammation/tissue damage and fermentation products produced by C. difficile. The most discriminating metabolite found is isocaproyltaurine, a covalent conjugate of a distinctive C. difficile fermentation product (isocaproate) and an amino acid associated with tissue damage (taurine), which may be useful as a joint marker of the two disease processes.
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http://dx.doi.org/10.1016/j.chom.2020.07.020DOI Listing
September 2020

A Summary of the Fifth Annual Virology Education HIV Microbiome Workshop.

AIDS Res Hum Retroviruses 2020 11 7;36(11):886-895. Epub 2020 Sep 7.

Department of Epidemiology, The George Washington University, Washington, District of Columbia, USA.

In October of 2019, researchers and community members from around the world met at the NIH for the fifth annual International Workshop on Microbiome in HIV. New research was presented on the role of the microbiome on chronic inflammation and vaccine design, interactions of genetics, environment, sexual practice and HIV infection with the microbiome and the development and clinical trials of microbiome-based therapeutic approaches intended to decrease the probability of HIV acquisition/transmission or ameliorate sequelae of HIV. The keynote address by Dr. Jacques Ravel focused on his work on the vaginal microbiome and efforts to improve the analysis and resolution of microbiome data.
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http://dx.doi.org/10.1089/AID.2020.0121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869876PMC
November 2020

The stepwise assembly of the neonatal virome is modulated by breastfeeding.

Nature 2020 05 15;581(7809):470-474. Epub 2020 Apr 15.

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

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which-in some cases-leads to gastrointestinal disorders. Here we show that the assembly of the viral community in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium or early stool samples shows few or no particles, but by one month of life particle numbers increase to 10 per gram, and these numbers seem to persist throughout life. We investigated the origin of these viral populations using shotgun metagenomic sequencing of virus-enriched preparations and whole microbial communities, followed by targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut and by one month prophages induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies who were exclusively fed on formula milk compared with those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections. Bacteriophage populations also differed depending on whether or not the infant was breastfed. We show that the colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells; this second phase is modulated by breastfeeding.
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http://dx.doi.org/10.1038/s41586-020-2192-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263352PMC
May 2020

Lifestyle and the presence of helminths is associated with gut microbiome composition in Cameroonians.

Genome Biol 2020 05 25;21(1):122. Epub 2020 May 25.

Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Background: African populations provide a unique opportunity to interrogate host-microbe co-evolution and its impact on adaptive phenotypes due to their genomic, phenotypic, and cultural diversity. We integrate gut microbiome 16S rRNA amplicon and shotgun metagenomic sequence data with quantification of pathogen burden and measures of immune parameters for 575 ethnically diverse Africans from Cameroon. Subjects followed pastoralist, agropastoralist, and hunter-gatherer lifestyles and were compared to an urban US population from Philadelphia.

Results: We observe significant differences in gut microbiome composition across populations that correlate with subsistence strategy and country. After these, the variable most strongly associated with gut microbiome structure in Cameroonians is the presence of gut parasites. Hunter-gatherers have high frequencies of parasites relative to agropastoralists and pastoralists. Ascaris lumbricoides, Necator americanus, Trichuris trichiura, and Strongyloides stercoralis soil-transmitted helminths ("ANTS" parasites) significantly co-occur, and increased frequency of gut parasites correlates with increased gut microbial diversity. Gut microbiome composition predicts ANTS positivity with 80% accuracy. Colonization with ANTS, in turn, is associated with elevated levels of TH1, TH2, and proinflammatory cytokines, indicating an association with multiple immune mechanisms. The unprecedented size of this dataset allowed interrogation of additional questions-for example, we find that Fulani pastoralists, who consume high levels of milk, possess an enrichment of gut bacteria that catabolize galactose, an end product of lactose metabolism, and of bacteria that metabolize lipids.

Conclusions: These data document associations of bacterial microbiota and eukaryotic parasites with each other and with host immune responses; each of these is further correlated with subsistence practices.
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http://dx.doi.org/10.1186/s13059-020-02020-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249393PMC
May 2020

Dynamics of the Stool Virome in Very Early-Onset Inflammatory Bowel Disease.

J Crohns Colitis 2020 Nov;14(11):1600-1610

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

Background And Aims: Dysbiosis of the gut microbiota is a well-known correlate of the pathogenesis of inflammatory bowel disease [IBD]. However, few studies have examined the microbiome in very early-onset [VEO] IBD, which is defined as onset of IBD before 6 years of age. Here we focus on the viral portion of the microbiome-the virome-to assess possible viral associations with disease processes, reasoning that any viruses potentially associated with IBD might grow more robustly in younger subjects, and so be more detectable.

Methods: Virus-like particles [VLPs] were purified from stool samples collected from patients with VEO-IBD [n = 54] and healthy controls [n = 23], and characterized by DNA and RNA sequencing and VLP particle counts.

Results: The total number of VLPs was not significantly different between VEO-IBD and healthy controls. For bacterial viruses, the VEO-IBD subjects were found to have a higher ratio of Caudovirales vs to Microviridae compared to healthy controls. An increase in Caudovirales was also associated with immunosuppressive therapy. For viruses infecting human cells, Anelloviridae showed higher prevalence in VEO-IBD compared to healthy controls. Within the VEO-IBD group, higher levels of Anelloviridae DNA were also positively associated with immunosuppressive treatment. To search for new viruses, short sequences enriched in VEO-IBD samples were identified, and some could be validated in an independent cohort, although none was clearly viral; this provides sequence tags to interrogate in future studies.

Conclusions: These data thus document perturbations to normal viral populations associated with VEO-IBD, and provide a biomarker-Anelloviridae DNA levels-potentially useful for reporting the effectiveness of immunosuppression.
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http://dx.doi.org/10.1093/ecco-jcc/jjaa094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648169PMC
November 2020

Single-cell transcriptional landscapes reveal HIV-1-driven aberrant host gene transcription as a potential therapeutic target.

Sci Transl Med 2020 05;12(543)

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06519, USA.

Understanding HIV-1-host interactions can identify the cellular environment supporting HIV-1 reactivation and mechanisms of clonal expansion. We developed HIV-1 SortSeq to isolate rare HIV-1-infected cells from virally suppressed, HIV-1-infected individuals upon early latency reversal. Single-cell transcriptome analysis of HIV-1 SortSeq cells revealed enrichment of nonsense-mediated RNA decay and viral transcription pathways. HIV-1 SortSeq cells up-regulated cellular factors that can support HIV-1 transcription ( and ) or promote cellular survival ( and ). HIV-1-host RNA landscape analysis at the integration site revealed that HIV-1 drives high aberrant host gene transcription downstream, but not upstream, of the integration site through HIV-1-to-host aberrant splicing, in which HIV-1 RNA splices into the host RNA and aberrantly drives host RNA transcription. HIV-1-induced aberrant transcription was driven by the HIV-1 promoter as shown by CRISPR-dCas9-mediated HIV-1-specific activation and could be suppressed by CRISPR-dCas9-mediated inhibition of HIV-1 5' long terminal repeat. Overall, we identified cellular factors supporting HIV-1 reactivation and HIV-1-driven aberrant host gene transcription as potential therapeutic targets to disrupt HIV-1 persistence.
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http://dx.doi.org/10.1126/scitranslmed.aaz0802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453882PMC
May 2020

Bacterial colonization reprograms the neonatal gut metabolome.

Nat Microbiol 2020 06 13;5(6):838-847. Epub 2020 Apr 13.

Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Initial microbial colonization and later succession in the gut of human infants are linked to health and disease later in life. The timing of the appearance of the first gut microbiome, and the consequences for the early life metabolome, are just starting to be defined. Here, we evaluated the gut microbiome, proteome and metabolome in 88 African-American newborns using faecal samples collected in the first few days of life. Gut bacteria became detectable using molecular methods by 16 h after birth. Detailed analysis of the three most common species, Escherichia coli, Enterococcus faecalis and Bacteroides vulgatus, did not suggest a genomic signature for neonatal gut colonization. The appearance of bacteria was associated with reduced abundance of approximately 50 human proteins, decreased levels of free amino acids and an increase in products of bacterial fermentation, including acetate and succinate. Using flux balance modelling and in vitro experiments, we provide evidence that fermentation of amino acids provides a mechanism for the initial growth of E. coli, the most common early colonizer, under anaerobic conditions. These results provide a deep characterization of the first microbes in the human gut and show how the biochemical environment is altered by their appearance.
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http://dx.doi.org/10.1038/s41564-020-0694-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052915PMC
June 2020

grabseqs: simple downloading of reads and metadata from multiple next-generation sequencing data repositories.

Bioinformatics 2020 06;36(11):3607-3609

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania.

Summary: High-throughput sequencing is a powerful technique for addressing biological questions. Grabseqs streamlines access to publicly available metagenomic data by providing a single, easy-to-use interface to download data and metadata from multiple repositories, including the Sequence Read Archive, the Metagenomics Rapid Annotation through Subsystems Technology server and iMicrobe. Users can download data and metadata in a standardized format from any number of samples or projects from a given repository with a single grabseqs command.

Availability And Implementation: Grabseqs is an open-source tool implemented in Python and licensed under the MIT license. The source code is freely available at https://github.com/louiejtaylor/grabseqs, the Python Package Index and Anaconda Cloud repository.

Contact: bushman@pennmedicine.upenn.edu.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267817PMC
June 2020

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs.

Blood 2020 04;135(15):1219-1231

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

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.
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http://dx.doi.org/10.1182/blood.2019002350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146019PMC
April 2020

CRISPR-engineered T cells in patients with refractory cancer.

Science 2020 02 6;367(6481). Epub 2020 Feb 6.

Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα () and TCRβ (), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; ), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.
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http://dx.doi.org/10.1126/science.aba7365DOI Listing
February 2020

Lentiviral gene therapy for X-linked chronic granulomatous disease.

Nat Med 2020 02 27;26(2):200-206. Epub 2020 Jan 27.

Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust, London, UK.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34 hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.
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http://dx.doi.org/10.1038/s41591-019-0735-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115833PMC
February 2020

Retroviral Insertional Mutagenesis in Humans: Evidence for Four Genetic Mechanisms Promoting Expansion of Cell Clones.

Mol Ther 2020 02 7;28(2):352-356. Epub 2020 Jan 7.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA. Electronic address:

Integration of new DNA into a cellular chromosome can alter the activity of nearby genes, sometimes affecting subsequent cell growth. A potent form of insertional mutagenesis involves integration of retroviral DNA produced by reverse transcription, a required step in the replication of retroviruses. In recent years retroviral replication has been adapted to allow new gene addition by retroviral vectors. Early in the history of retrovirus research, analysis of insertional mutagenesis in laboratory animals was found at times to result in transformation, leading to the discovery of cellular proto-oncogenes. In-depth analysis of the genetic consequences showed that integration of retroviral DNA could alter the gene activity in a variety of ways. Mechanisms of retroviral insertional mutagenesis in humans are much less well documented. However, recent work from the gene therapy and HIV fields now specify four genetic mechanisms of retroviral insertional mutagenesis in humans: (1) gene activation by integration of an enhancer sequence encoded in a retroviral vector (enhancer insertion), (2) gene activation by promoter insertion, (3) gene inactivation by insertional disruption, and (4) gene activation by mRNA 3' end substitution. In each example, integration in patients was associated with clonal expansion or frank transformation.
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http://dx.doi.org/10.1016/j.ymthe.2019.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001082PMC
February 2020

Improving natural product research translation: From source to clinical trial.

FASEB J 2020 01 10;34(1):41-65. Epub 2019 Dec 10.

CENAPT and PCRPS, University of Illinois at Chicago College of Pharmacy, Chicago, IL, USA.

While great interest in health effects of natural product (NP) including dietary supplements and foods persists, promising preclinical NP research is not consistently translating into actionable clinical trial (CT) outcomes. Generally considered the gold standard for assessing safety and efficacy, CTs, especially phase III CTs, are costly and require rigorous planning to optimize the value of the information obtained. More effective bridging from NP research to CT was the goal of a September, 2018 transdisciplinary workshop. Participants emphasized that replicability and likelihood of successful translation depend on rigor in experimental design, interpretation, and reporting across the continuum of NP research. Discussions spanned good practices for NP characterization and quality control; use and interpretation of models (computational through in vivo) with strong clinical predictive validity; controls for experimental artefacts, especially for in vitro interrogation of bioactivity and mechanisms of action; rigorous assessment and interpretation of prior research; transparency in all reporting; and prioritization of research questions. Natural product clinical trials prioritized based on rigorous, convergent supporting data and current public health needs are most likely to be informative and ultimately affect public health. Thoughtful, coordinated implementation of these practices should enhance the knowledge gained from future NP research.
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http://dx.doi.org/10.1096/fj.201902143RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470648PMC
January 2020

CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration.

J Clin Invest 2020 02;130(2):673-685

Department of Microbiology.

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.
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http://dx.doi.org/10.1172/JCI130144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994131PMC
February 2020

Detecting contamination in viromes using ViromeQC.

Nat Biotechnol 2019 12;37(12):1408-1412

Department CIBIO, University of Trento, Trento, Italy.

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http://dx.doi.org/10.1038/s41587-019-0334-5DOI Listing
December 2019