Biology subjects

Weiss, S.

Publications and source records attributed to Weiss, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Nonclinical Safety and Immunogenicity of an rVSV-ΔG-SARS-CoV-2-S vaccine in mice, hamsters, rabbits and pigs

rVSV-{Delta}G-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. Nonclinical safety, immunogenicity and efficacy studies were conducted in 4 animal species, using multiple dose levels (up to 108 PFU/animal) and various dosing regimens. There were no treatment related mortalities in any study, or any noticeable clinical signs. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings gave cause for safety concern in any of the studies. There was no viral shedding in urine, nor viral RNA detected in whole blood or serum samples 7 days post vaccination. The rVSV-{Delta}G-SARS-CoV-2-S vaccine immune response gave rise to neutralizing antibodies, cellular immune response, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph node. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive IFNAR KO mice. Vaccine virus replication and distribution in K18 hACE2 transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The rVSV-{Delta}G-SARS-CoV-2-S vaccine was well tolerated locally and systemically and elicited an effective immunogenic response up to the highest dose tested, supporting further clinical development.

pharmacology and toxicology↗

bioRxiv2021

Rapid Ensemble Measurement of Protein Diffusion, Probe Blinking and Photobleaching Dynamics in the Complex Cellular Space

We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.

biophysics↗

bioRxiv2021

Image quantification technique reveals novel lung cancer cytoskeletal phenotype with partial EMT signature

Epithelial-mesenchymal Transition (EMT) is a multi-step process that involves cytoskeletal rearrangement. Here, using novel image quantification tools, we have identified an intermediate EMT state with a specific cytoskeletal signature. We have been able to partition EMT into two steps: (1) initial formation of transverse arcs and dorsal stress fibers and (2) their subsequent conversion to ventral stress fibers with a concurrent alignment of fibers. Using the Orientational Order Parameter (OOP) as a figure of merit, we have been able to track EMT progression in live cells as well as characterize and quantify drug responses. Our technique has improved throughput and is non-destructive, making it a viable candidate for studying a broad range of biological processes. Further, owing to the increased stiffness (and hence invasiveness) of the intermediate phenotype compared to mesenchymal cells, our work can be instrumental in aiding the search for new treatment strategies that combat metastasis by specifically targeting the fiber alignment process.

biophysics↗

bioRxiv2021

Monitoring group activity of hamsters and mice as a novel tool to evaluate COVID-19 progression, convalescence and rVSV-ΔG-spike vaccination efficacy

COVID-19 pandemic initiated a worldwide race toward the development of treatments and vaccines. Small animal models were the Syrian golden hamster and the K18-hACE2 mice infected with SARS-CoV-2 to display a disease state with some aspects of the human COVID-19. Group activity of animals in their home cage continuously monitored by the HCMS100 was used as a sensitive marker of disease, successfully detecting morbidity symptoms of SARS-CoV-2 infection in hamsters and in K18-hACE2 mice. COVID-19 convalescent hamsters re-challenged with SARS-CoV-2, exhibited minor reduction in group activity compared to naive hamsters. To evaluate rVSV-{Delta}G-spike vaccination efficacy against SARS-CoV-2, we used the HCMS100 to monitor group activity of hamsters in their home cage. Single-dose rVSV-{Delta}G-spike vaccination of immunized group showed a faster recovery compared to the non-immunized infected hamsters, substantiating the efficacy of rVSV-{Delta}G-spike vaccine. HCMS100 offers non-intrusive, hands-free monitoring of a number of home cages of hamsters or mice modeling COVID-19.

immunology↗

bioRxiv2021

Optical Probing of Local Membrane Potential with Fluorescent Polystyrene Beads

Studying the electrical activity in single cells and in local circuits of excitable cells, like neurons, requires an easy to use and high throughput methodology that enables the measurement of membrane potential. Studying the electrical properties in particular sub-compartments of neurons, or in a specific type of neurons produces additional complexity. An optical voltage-imaging technique that allows high spatial and temporal resolution could be an ideal solution. However, most of the valid voltage imaging techniques are nonspecific; The ones that are more site-directed require much pre-work and specific adaptations in addition to other disadvantages. Here, a new technique for membrane voltage imaging, based on FRET between fluorescent polystyrene (FPS) beads and Dipicrylamine (DPA) is explored. Not only fluorescent intensity is demonstrated to be correlated with membrane potential, but more importantly, single particle voltage detection is demonstrated. Among other advantages, FPS beads can be synthesized with functional surface groups, and be further targeted to specific proteins via conjugation of recognition molecules. Therefore, FPS beads, in the presence of DPA, constitute single-particle detectors for membrane voltage, with a potential to be localized to specific membrane compartments. This new and accessible platform for targeted optical voltage imaging may further elucidate the mechanisms of neuronal electrical activity.

biophysics↗

bioRxiv2021

Point-localized and site-specific membrane potential optical recording by engineered single fluorescent nanodiscs

Nanodisc technology was implemented as a platform for voltage nanosensors. A FRET-based voltage sensing scheme employing fluorescent nanodiscs and the hydrophobic ion dipicrylamine (DPA) was developed and utilized to optically record membrane potentials on the single nanodisc level. Ensemble- and single- nanosensor recordings were demonstrated for HEK293 cells and primary cortical neuron cells. Conjugation of nanodiscs to anti-GABA-A antibodies allowed for site specific membrane potential measurements from post synaptic sites.

biophysics↗

bioRxiv2021

Multi-parameter photon-by-photon hidden Markov modeling

Single-molecule Forster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacro-molecules. Photon-by-photon hidden Markov modelling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may introduce artefacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin.

biophysics↗

bioRxiv2021

The neutralization potency of anti-SARS-CoV-2 therapeutic human monoclonal antibodies is retained against novel viral variants

A wide range of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) were reported to date, most of which target the spike glycoprotein and in particular its receptor binding domain (RBD) and N-terminal domain (NTD) of the S1 subunit. The therapeutic implementation of these antibodies has been recently challenged by emerging SARS-CoV-2 variants that harbor extensively mutated spike versions. Consequently, the re-assessment of mAbs, previously reported to neutralize the original early-version of the virus, is of high priority. Four previously selected mAbs targeting non-overlapping epitopes, were evaluated for their binding potency to RBD versions harboring individual mutations at spike positions 417, 439, 453, 477, 484 and 501. Mutations at these positions represent the prevailing worldwide distributed modifications of the RBD, previously reported to mediate escape from antibody neutralization. Additionally, the in vitro neutralization potencies of the four RBD-specific mAbs, as well as two NTD-specific mAbs, were evaluated against two frequent SARS-CoV-2 variants of concern (VOCs): (i) the B.1.1.7 variant, emerged in the UK and (ii) the B.1.351 variant, emerged in South Africa. Variant B.1.351 was previously suggested to escape many therapeutic mAbs, including those authorized for clinical use. The possible impact of RBD mutations on recognition by mAbs is addressed by comparative structural modelling. Finally, we demonstrate the therapeutic potential of three selected mAbs by treatment of K18-hACE2 transgenic mice two days post infection with each of the virus strains. Our results clearly indicate that despite the accumulation of spike mutations, some neutralizing mAbs preserve their potency against SARS-CoV-2. In particular, the highly potent MD65 and BL6 mAbs are shown to retain their ability to bind the prevalent novel viral mutations and to effectively protect against B.1.1.7 and B.1.351 variants of high clinical concern.

immunology↗

bioRxiv2021

Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

Since the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

immunology↗

bioRxiv2020

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms.

IntroductionCardiac L-type voltage-gated CaV1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of CaV1.2 starts with activation of {beta}-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through CaV1.2 channels. To date, this cascade has never been fully reconstituted in heterologous systems; even partial reconstitution proved challenging and controversial. A recent study identified Rad, a calcium channel inhibitory protein, as an essential component of the adrenergic signaling cascade. We corroborated this finding, further characterized, and fully reconstituted, the complete {beta}-AR CaV1.2 modulation cascade in a heterologous expression system. ObjectiveOur primary goal was to heterologously reconstitute the complete {beta}-adrenergic cascade, and to investigate the role of Rad and additional molecular determinants in adrenergic regulation of cardiac CaV1.2. Methods and ResultsWe utilized the Xenopus oocyte heterologous expression system. We expressed CaV1.2 channel subunits, without or with Rad and {beta}1-AR or {beta}2-AR. To activate PKA, we injected cyclic AMP (cAMP) into the oocytes, or extracellularly applied isoproterenol (Iso) to stimulate {beta}-AR. Whole-cell Ba2+ currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of CaV1.2: Rad-dependent (~80% of total) and Rad-independent. We separate the two mechanisms by showing distinct requirements for the cytosolic N- and distal C- termini of 1C and for the CaV{beta} subunit. Finally, for the first time, we reconstitute the complete pathway using agonist activation of either {beta}1-AR or {beta}2-AR. The reconstituted system reproduces the known features of {beta}-AR regulation in cardiomyocytes, such as a >2-fold increase in CaV1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of {beta}2-AR. ConclusionsThe adrenergic modulation of CaV1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the {beta}-AR-induced enhancement of CaV1.2 activity, crucially depends on CaV{beta} subunit, and is differently regulated by {beta}1-AR and {beta}2-AR. The reconstitution of the full {beta}-AR cascade provides the means to address central unresolved issues related to roles of auxiliary proteins in the cascade, CaV1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.

physiology↗

bioRxiv2020

Disruption of Glial Ca2+ Oscillations at the Drosophila Blood-Brain Barrier Predisposes to Seizure-Like Behavior

Glia play key roles in regulating multiple aspects of neuronal development and function from invertebrates to humans. We recently found microdomain Ca2+ signaling in Drosophila cortex glia and astrocytes regulate extracellular K+ buffering and neurotransmitter uptake, respectively. Here we identify a role for ER store-operated Ca2+ entry (SOCE) in perineurial glia (PG), a distinct population that contributes to the blood-brain barrier (BBB). PG show a diverse range of Ca2+ oscillatory activity that varies based on their locale within the brain. Unlike cortex glia and astrocytes, PG Ca2+ oscillations do not require extracellular Ca2+ and are blocked by inhibition of SOCE or gap junctions. Disruption of these components triggers heat shock and mechanical-induced seizure-like episodes without effecting PG morphology or large molecule BBB permeability. These findings indicate SOCE-mediated Ca2+ oscillations in PG increase the susceptibility of seizure-like episodes in Drosophila, providing an additional link between glial Ca2+ signaling and neuronal activity.

neuroscience↗

bioRxiv2020

A single dose of recombinant VSV-{triangleup}G-spike vaccine provides protection against SARS-CoV-2 challenge

The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-{Delta}G-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-{Delta}G-spike indicated expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in vivo model for COVID-19 was implemented. We show that vaccination of hamsters with recombinant VSV-{Delta}G-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-{Delta}G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction

SARS-CoV-2 genetic identification is based on viral RNA extraction prior to RT-qPCR assay, however recent studies support the elimination of the extraction step. Herein, we assessed the RNA extraction necessity, by comparing RT-qPCR efficacy in several direct approaches vs. the gold standard RNA extraction, in detection of SARS-CoV-2 from laboratory samples as well as clinical Oro-nasopharyngeal SARS-CoV-2 swabs. Our findings show advantage for the extraction procedure, however a direct no-buffer approach might be an alternative, since it identified up to 70% of positive clinical specimens.

microbiology↗

bioRxiv2020

sPLINK: A Federated, Privacy-Preserving Tool as a Robust Alternative to Meta-Analysis in Genome-Wide Association Studies

Genome-wide association studies (GWAS) have been widely used to unravel connections between genetic variants and diseases. Larger sample sizes in GWAS can lead to discovering more associations and more accurate genetic predictors. However, sharing and combining distributed genomic data to increase the sample size is often challenging or even impossible due to privacy concerns and privacy protection laws such as the GDPR. While meta-analysis has been established as an effective approach to combine summary statistics of several GWAS, its accuracy can be attenuated in the presence of cross-study heterogeneity. Here, we present sPLINK (safe PLINK), a user-friendly tool, which performs federated GWAS on distributed datasets while preserving the privacy of data and the accuracy of the results. sPLINK neither exchanges raw data nor does it rely on summary statistics. Instead, it performs model training in a federated manner, communicating only model parameters between cohorts and a central server. We verify that the federated results from sPLINK are the same as those from aggregated analyses conducted with PLINK. We demonstrate that sPLINK is robust against heterogeneous data (phenotype and confounding factors) distributions across cohorts while existing meta-analysis tools considerably lose accuracy in such scenarios. We also show that sPLINK achieves practical runtime, in order of minutes or hours, and acceptable network bandwidth consumption for chi-square and linear/logistic regression tests. Federated analysis with sPLINK, thus, has the potential to replace meta-analysis as the gold standard for collaborative GWAS. The user-friendly, readily usable sPLINK tool is available at https://exbio.wzw.tum.de/splink.

bioinformatics↗

bioRxiv2020

Multi-ancestry genome-wide gene-sleep interactions identify novel loci for blood pressure

Long and short sleep duration are associated with elevated blood pressure (BP), possibly through effects on molecular pathways that influence neuroendocrine and vascular systems. To gain new insights into the genetic basis of sleep-related BP variation, we performed genome-wide gene by short or long sleep duration interaction analyses on four BP traits (systolic BP, diastolic BP, mean arterial pressure, and pulse pressure) across five ancestry groups using 1 degree of freedom (1df) interaction and 2df joint tests. Primary multi-ancestry analyses in 62,969 individuals in stage 1 identified 3 novel loci that were replicated in an additional 59,296 individuals in stage 2, including rs7955964 (FIGNL2/ANKRD33) showing significant 1df interactions with long sleep duration and rs73493041 (SNORA26/C9orf170) and rs10406644 (KCTD15/LSM14A) showing significant 1df interactions with short sleep duration (Pint < 5x10-8). Secondary ancestry-specific two-stage analyses and combined stage 1 and 2 analyses additionally identified 23 novel loci that need external replication, including 3 and 5 loci showing significant 1df interactions with long and short sleep duration, respectively (Pint < 5x10-8). Multiple genes mapped to our 26 novel loci have known functions in sleep-wake regulation, nervous and cardiometabolic systems. We also identified new gene by long sleep interactions near five known BP loci ([≤]1Mb) including NME7, FAM208A, MKLN1, CEP164, and RGL3/ELAVL3 (Pint < 5x10-8). This study indicates that sleep and primary mechanisms regulating BP may interact to elevate BP level, suggesting novel insights into sleep-related BP regulation.

genomics↗

bioRxiv2020

Tiger team: a panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug, specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD, therefore they represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

immunology↗

bioRxiv2020

The coding capacity of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic 1,2. In order to understand SARS-CoV-2 pathogenicity and antigenic potential, and to develop diagnostic and therapeutic tools, it is essential to portray the full repertoire of its expressed proteins. The SARS-CoV-2 coding capacity map is currently based on computational predictions and relies on homology to other coronaviruses. Since coronaviruses differ in their protein array, especially in the variety of accessory proteins, it is crucial to characterize the specific collection of SARS-CoV-2 proteins in an unbiased and open-ended manner. Utilizing a suite of ribosome profiling techniques 3-8, we present a high-resolution map of the SARS-CoV-2 coding regions, allowing us to accurately quantify the expression of canonical viral open reading frames (ORF)s and to identify 23 novel unannotated viral translated ORFs. These ORFs include upstream ORFs (uORFs) that are likely playing a regulatory role, several in-frame internal ORFs lying within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; rather, virus translation dominates host translation due to high levels of viral transcripts. Overall, our work reveals the full coding capacity of SARS-CoV-2 genome, providing a rich resource, which will form the basis of future functional studies and diagnostic efforts.

microbiology↗

bioRxiv2020

Unsupervised cluster analysis of SARS-CoV-2 genomes reflects its geographic progression and identifies distinct genetic subgroups of SARS-CoV-2 virus

Over 10,000 viral genome sequences of the SARS-CoV-2 virus have been made readily available during the ongoing coronavirus pandemic since the initial genome sequence of the virus was released on the open access Virological website (http://virological.org/) early on January 11. We utilize the published data on the single stranded RNAs of 11, 132 SARS-CoV-2 patients in the GISAID (Elbe and Buckland-Merrett, 2017; Shu and McCauley, 2017) database, which contains fully or partially sequenced SARS-CoV-2 samples from laboratories around the world. Among many important research questions which are currently being investigated, one aspect pertains to the genetic characterization/classification of the virus. We analyze data on the nucleotide sequencing of the virus and geographic information of a subset of 7, 640 SARS-CoV-2 patients without missing entries that are available in the GISAID database. Instead of modelling the mutation rate, applying phylogenetic tree approaches, etc., we here utilize a model-free clustering approach that compares the viruses at a genome-wide level. We apply principal component analysis to a similarity matrix that compares all pairs of these SARS-CoV-2 nucleotide sequences at all loci simultaneously, using the Jaccard index (Jaccard, 1901; Tan et al., 2005; Prokopenko et al., 2016; Schlauch et al., 2017). Our analysis results of the SARS-CoV-2 genome data illustrates the geographic and chronological progression of the virus, starting from the first cases that were observed in China to the current wave of cases in Europe and North America. This is in line with a phylogenetic analysis which we use to contrast our results. We also observe that, based on their sequence data, the SARS-CoV-2 viruses cluster in distinct genetic subgroups. It is the subject of ongoing research to examine whether the genetic subgroup could be related to diseases outcome and its potential implications for vaccine development.

genomics↗