Biology subjects

Dvorkin, S.

Publications and source records attributed to Dvorkin, S..

8 recordsLinked to original sources

bioRxiv2023

Cellular RNA interacts with MAVS to promote antiviral signaling

Immune signaling needs to be well-regulated to promote clearance of pathogens, while preventing aberrant inflammation. Interferons (IFNs) and antiviral genes are activated by the detection of viral RNA by RIG-I-like receptors (RLRs). Signal transduction downstream of RLRs proceeds through a multi-protein complex organized around the central adaptor protein MAVS. Recent work has shown that protein complex function can be modulated by RNA molecules providing allosteric regulation or acting as molecular guides or scaffolds. Thus, we hypothesized that RNA plays a role in organizing MAVS signaling platforms. Here, we show that MAVS, through its central intrinsically disordered domain, directly interacts with the 3' untranslated regions of cellular mRNAs. Importantly, elimination of RNA by RNase treatment disrupts the MAVS signalosome, including newly identified regulators of RLR signaling, and inhibits phosphorylation of the transcription factor IRF3. This supports the hypothesis that RNA molecules scaffold proteins in the MAVS signalosome to induce IFNs. Together, this work uncovers a function for cellular RNA in promoting signaling through MAVS and highlights a generalizable principle of RNA regulatory control of cytoplasmic immune signaling complexes.

immunology↗

bioRxiv2021

Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum, amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

bioengineering↗

bioRxiv2021

Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

bioengineering↗

bioRxiv2020

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors

The number of new cases world-wide for the COVID-19 disease is increasing dramatically, while efforts to contain Severe Acute Respiratory Syndrome Coronavirus 2 is producing varied results in different countries. There are three key SARS-CoV-2 enzymes potentially targetable with antivirals: papain-like protease (PLpro), main protease (Mpro), and RNA-dependent RNA polymerase. Of these, PLpro is an especially attractive target because it plays an essential role in several viral replication processes, including cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex (RTC), and disruption of host viral response machinery to facilitate viral proliferation and replication. Moreover, this enzyme is conserved across different coronaviruses and promising inhibitors have already been discovered for its SARS-CoV variant. Here we report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the enzyme from SARS-CoV-2 in both wild-type and mutant forms. These efforts include the first structures of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors, determined at 1.60-2.70 Angstroms. This collection of structures provides fundamental molecular and mechanistic insight to PLpro, and critically, illustrates details for inhibitors recognition and interactions. All presented compounds inhibit the peptidase activity of PLpro in vitro, and some molecules block SARS-CoV-2 replication in cell culture assays. These collated findings will accelerate further structure-based drug design efforts targeting PLpro, with the ultimate goal of identifying high-affinity inhibitors of clinical value for SARS-CoV-2.

molecular biology↗

bioRxiv2020

Autoencoder based local T cell repertoire density can be used to classify samples and T cell receptors

Recent advances in T cell repertoire (TCR) sequencing allow for characterization of repertoire properties, as well as the frequency and sharing of specific TCR. However, there is no efficient measure for the local density of a given TCR. TCRs are often described either through their Complementary Determining region 3 (CDR3) sequences, or theirV/J usage or their clone size. We here show that the local repertoire density can be estimated using a combined representation of these components through distance conserving autoencoders and Kernel Density Estimates (KDE). We present ELATE - an Encoder based LocAl Tcr dEnsity and show that the resulting density of a sample can be used as a novel measure to study repertoire properties. The cross-density between two samples can be used as a similarity matrix to fully characterize samples from the same host. Finally, the same projection in combination with machine learning algorithms can be used to predict TCR-peptide binding through the local density of known TCRs binding a specific target. Code availability- https://github.com/louzounlab/Autoencoder

immunology↗

bioRxiv2020

CDR3 and V- genes show distinct reconstitution patterns in T-cell repertoire post allogenic bone marrow transplantation

BackgroundRestoration of T-cell repertoire diversity after allogeneic bone marrow transplantation (allo-BMT) is crucial for immune recovery. T-cell diversity is produced by rearrangements of germline gene segments (V (D) and J) of the T-cell receptor (TCR) and {beta} chains, and selection induced by binding of TCRs to MHC-peptide complexes. This diversity can be measured by many measures. We here focus on the V gene usage and the CDR3 sequences of the beta chain. We compared multiple T-cell repertoires to follow T cell repertoire changes post allo-BMT in HLA-matched related donor and recipient pairs. ResultsOur analyses of the differences between donor and recipient complementarity determining region 3 (CDR3) beta composition and V-gene profile show that the CDR sequence composition does not change during restoration, implying its dependence on the HLA typing. In contrast, V gene usage followed a time-dependent pattern, got following the donor profile post transplant and then shifting back to the recipients profile. The final long-term repertoire was more similar to that of the recipients original one than the donors.; some recipients converged within months while others took multiple years. ConclusionBased on the results of our analyses, we propose that donor-recipient V-gene distribution differences may serve as clinical biomarkers for monitoring immune recovery.

immunology↗

bioRxiv2019

Prediction of specific TCR-peptide binding from large dictionaries of TCR-peptide pairs

Current sequencing methods allow for detailed samples of T cell receptors (TCR) repertoires. To determine from a repertoire whether its host had been exposed to a target, computational tools that predict TCR-epitope binding are required. Currents tools are based on conserved motifs and are applied to peptides with many known binding TCRs. Given any TCR and peptide, we employ new NLP-based methods to predict whether they bind. We combined large-scale TCR-peptide dictionaries with deep learning methods to produce ERGO (pEptide tcR matchinG predictiOn), a highly specific and generic TCR-peptide binding predictor. A set of standard tests are defined for the performance of peptide-TCR binding, including the detection of TCRs binding to a given peptide/antigen, choosing among a set of candidate peptides for a given TCR and determining whether any pair of TCR-peptide bind. ERGO significantly outperforms current methods in these tests even when not trained specifically for each test. The software implementation and data sets are available at https://github.com/louzounlab/ERGO

immunology↗