Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity

IntroductionThe success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance.MethodsWe report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls.ResultsIn contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients.DiscussionThese features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges

Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named “zymoxins”. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected

Tracking Down the Epigenetic Footprint of HCV-Induced Hepatocarcinogenesis

Hepatitis C virus (HCV) is a major cause of death and morbidity globally and is a leading cause of hepatocellular carcinoma (HCC). Incidence of HCV infections, as well as HCV-related liver diseases, are increasing. Although now, with new direct acting antivirals (DAAs) therapy available, HCV is a curable cancer-associated infectious agent, HCC prevalence is expected to continue to rise because HCC risk still persists after HCV cure. Understanding the factors that lead from HCV infection to HCC pre- and post-cure may open-up opportunities to novel strategies for HCC prevention. Herein, we provide an overview of the reported evidence for the induction of alterations in the transcriptome of host cells via epigenetic dysregulation by HCV infection and describe recent reports linking the residual risk for HCC post-cure with a persistent HCV-induced epigenetic signature. Specifically, we discuss the contribution of the epigenetic changes identified following HCV infection to HCC risk pre- and post-cure, the molecular pathways that are epigenetically altered, the downstream effects on expression of cancer-related genes, the identification of targets to prevent or revert this cancer-inducing epigenetic signature, and the potential contribution of these studies to early prognosis and prevention of HCC as an approach for reducing HCC-related mortality

Colony formation assay for the assessment of “mCherry-NS3 activated MazF” cytotoxicity toward naïve cells.

<p>A day before transfection, 7.5×10⁵ HEK293 T-REx cells where seeded per well in 6 wells plate and subsequently transfected with 2 µg of plasmids encoding either mCherry-NS3 activated MazF, mCherry (only the fluorescent protein) or EGFP- MazF (where MazF is not fused to its inhibitory peptide). 48 hours later, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. Transfected cells were than trypsinized, counted and seeded in 3 fold dilutions (starting from 150,000 cells/well) in 6 well plates and were incubated for 10 days in the presence of 1 mg/ml of G418 (to which all three plasmids confer resistance). Surviving colonies were fixed and stained with Giemsa (upper panel). Number of surviving Colonies from wells that were seeded with 5556 cells was determined by manual counting. Each bar represents the mean ± standard deviation (SD) of a set of data from two wells (lower panel).</p

Eradication of NS3 expressing cells by mCherry-NS3 activated MazF.

<p>Upper panel: Tet-inducible full NS3-4A (No MazF), Tet-NS3/activated MazF (NS3-activated MazF) or Tet-NS3/uncleavable MazF (uncleavable MazF) cells were seeded in 96 well plates (2×10⁴ cells per well). After 24 h, cells were supplemented with 3 fold dilutions of tetracycline starting with concentration of 1000 ng/ml, or left untreated. 72 hours later, the fraction of viable cells (relatively to the untreated controls) was determined using an enzymatic MTT assay. Each bar represents the mean ±SD of a set of data from six wells. Lower panel: 30 ng of total protein from lysates of Tet-NS3/uncleavable MazF cells that were supplemented with 3 fold dilutions of tetracycline for 48 h were analyzed by immunoblotting with mouse anti-GFP (for the detection of EGFP-NS3) and mouse anti-actin antibodies (loading control) followed by HRP-conjugated secondary antibodies and ECL development.</p

Schematic representation of the construct “mCherry-NS3 activated MazF” and hypothetical mechanism of activation by NS3 protease.

<p>The NS3-activated MazF zymoxin was constructed by fusing 5 elements in the following order (from the N terminus): monomeric red fluorescence protein mCherry, <i>E. coli</i> MazF ribonuclease, HCV P10-P10' NS3 cleavage sequence derived from 2a genotype (strain JFH1) NS5A/B junction, a short inhibitory peptide corresponding to MazE C-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (MazEp) that has been described by Li <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032320#pone.0032320-Li1" target="_blank">[25]</a>) and the C-terminal ER membrane anchor of the tyrosine phosphatase PTP1B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032320#pone.0032320-Anderie1" target="_blank">[28]</a>. After being anchored to the ER membrane, the NS3 cleavage site that is located between the ribonuclease and the inhibitory peptide in the “mCherry-NS3 activated MazF” construct (which is active as a dimer but for convenience is illustrated here in its monomeric form) is cleaved by the HCV- NS3 protease which is also localized to the cytoplasmic side of the ER membrane. The toxic ribonuclease, no longer covalently tethered to its ER-anchored inhibitor, is now free to diffuse to the cytoplasm (which lacks the antidote) and exert its destructive activity.</p

Eradication of NS3-expressing Huh7.5 cells by recombinant adenovirus-mediated delivery of mCherry-NS3 activated MazF encoding cassette.

<p>1×10⁴ wild-type (W.T) or EGFP-full NS3-4A expressing Huh7.5 cells were seeded per well in 96 well plates. After 24 h, recombinant adenoviruses encoding for mCherry-fused NS3 activated MazF or uncleavable-MazF zymoxins were added at the indicated MOI's. Control cells remained untreated. (A) MTT viability assay: 4 days post infection, the fraction of viable cells (relatively to uninfected controls) was determined using an enzymatic MTT assay. A representative graph of three independent experiments is shown. Each bar represents the mean ±SD of a set of data determined in triplicates. (B) Microscopic examination: 4 days post infection, wild-type (lower panel) or EGFP-full NS3-4A expressing Huh7.5 cells (upper panel), uninfected or infected with recombinant adenoviruses encoding for mCherry fused NS3-activated MazF zymoxin (at MOI of ∼3), were fixed and subjected to microscopic examination. The bar represents 200 µm.</p

Expression of mCherry-NS3 activated MazF results in growth inhibition and morphological changes in NS3-expressing cells.

<p>1×10⁵ Tet-NS3/activated MazF or Tet-NS3/uncleavable MazF cells were seeded on poly-L-lysine coated cover-slips in a 24 well-plate. 12 h later, cells were supplemented with 10 ng/ml or 1000 ng/ml of tetracycline, or left untreated. 36 h later, cells were fixed. Following nuclear staining by Hoechst 33258 (Blue), slides were examined by fluorescence microscopy. The bar represents 50 µm.</p

Treatment of HCV infected/uninfected mixed cell culture with recombinant adenovirus-delivered MazF based zymoxin.

<p>Uninfected (HCV-negative) Huh7.5 cells and a mixed culture of HCV infected and uninfected cells at 1∶1 ratio (50% infected culture) were seeded in 96-well plates (1×10⁴ cells/well). After 24 h, cells were treated with recombinant adenoviruses (MOI of ∼3) encoding for the mCherry fused NS3-activated MazF or uncleavable-MazF zymoxins. Control cells remained untreated. Upper panel: MTT viability assay: 72 h post treatment, the fraction of viable cells (relatively to untreated controls) was determined using an enzymatic MTT assay. A representative graph of three independent experiments is shown. Each bar represents the mean ±SD of a set of data determined in triplicates. Lower panel: Microscopic examination: 72 h post treatment, the uninfected (HCV-negative) Huh7.5 cells, the mixed culture of HCV infected and uninfected cells and the control untreated cells were fixed and subjected to microscopic examination. Hollow arrows point to cells that are characterized by a “typical” Huh7.5 cell morphology. Filled arrows point to partially detached cells with round, condensed or distorted shape. The bar represents 200 µm.</p