Holly Ramage

Bio: Holly Ramage is an academic researcher from University of Pennsylvania. The author has contributed to research in topics: Coronavirus & Innate immune system. The author has an hindex of 14, co-authored 21 publications receiving 2264 citations. Previous affiliations of Holly Ramage include Leibniz Association & Gladstone Institutes.

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications

Abstract: Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.

Comprehensive Functional Analysis of Mycobacterium tuberculosis Toxin-Antitoxin Systems: Implications for Pathogenesis, Stress Responses, and Evolution

Abstract: Toxin-antitoxin (TA) systems, stress-responsive genetic elements ubiquitous in microbial genomes, are unusually abundant in the major human pathogen Mycobacterium tuberculosis. Why M. tuberculosis has so many TA systems and what role they play in the unique biology of the pathogen is unknown. To address these questions, we have taken a comprehensive approach to identify and functionally characterize all the TA systems encoded in the M. tuberculosis genome. Here we show that 88 putative TA system candidates are present in M. tuberculosis, considerably more than previously thought. Comparative genomic analysis revealed that the vast majority of these systems are conserved in the M. tuberculosis complex (MTBC), but largely absent from other mycobacteria, including close relatives of M. tuberculosis. We found that many of the M. tuberculosis TA systems are located within discernable genomic islands and were thus likely acquired recently via horizontal gene transfer. We discovered a novel TA system located in the core genome that is conserved across the genus, suggesting that it may fulfill a role common to all mycobacteria. By expressing each of the putative TA systems in M. smegmatis, we demonstrate that 30 encode a functional toxin and its cognate antitoxin. We show that the toxins of the largest family of TA systems, VapBC, act by inhibiting translation via mRNA cleavage. Expression profiling demonstrated that four systems are specifically activated during stresses likely encountered in vivo, including hypoxia and phagocytosis by macrophages. The expansion and maintenance of TA genes in the MTBC, coupled with the finding that a subset is transcriptionally activated by stress, suggests that TA systems are important for M. tuberculosis pathogenesis.

A CRISPR screen defines a signal peptide processing pathway required by flaviviruses

Abstract: Flaviviruses infect hundreds of millions of people annually, and no antiviral therapy is available. We performed a genome-wide CRISPR/Cas9-based screen to identify host genes that, when edited, resulted in reduced flavivirus infection. Here, we validated nine human genes required for flavivirus infectivity, and these were associated with endoplasmic reticulum functions including translocation, protein degradation, and N-linked glycosylation. In particular, a subset of endoplasmic reticulum-associated signal peptidase complex (SPCS) proteins was necessary for proper cleavage of the flavivirus structural proteins (prM and E) and secretion of viral particles. Loss of SPCS1 expression resulted in markedly reduced yield of all Flaviviridae family members tested (West Nile, Dengue, Zika, yellow fever, Japanese encephalitis, and hepatitis C viruses), but had little impact on alphavirus, bunyavirus, or rhabdovirus infection or the surface expression or secretion of diverse host proteins. We found that SPCS1 dependence could be bypassed by replacing the native prM protein leader sequences with a class I major histocompatibility complex (MHC) antigen leader sequence. Thus, SPCS1, either directly or indirectly via its interactions with unknown host proteins, preferentially promotes the processing of specific protein cargo, and Flaviviridae have a unique dependence on this signal peptide processing pathway. SPCS1 and other signal processing pathway members could represent pharmacological targets for inhibiting infection by the expanding number of flaviviruses of medical concern.

Controllable protein phase separation and modular recruitment to form responsive membraneless organelles.

Abstract: Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

Abstract: A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.

Longitudinal analyses reveal immunological misfiring in severe COVID-19.

Abstract: Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

Cytokine Storm.

Abstract: From the Department of Medicine, Division of Translational Medicine and Human Genetics, Center for Cytokine Storm Treatment and Laboratory (D.C.F.), and the Center for Cellular Immunotherapies and the Parker Institute for Cancer Immunotherapy (C.H.J.), Perelman School of Medicine, University of Pennsylvania, Philadelphia. Address reprint requests to Dr. Fajgenbaum at davidfa@ pennmedicine . upenn . edu or to Dr. June at cjune@ upenn . edu.