Biology subjects

Kim, C.

Publications and source records attributed to Kim, C..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

Stable nebulization and muco-trapping properties of Regdanvimab/IN-006 support its development as a potent, dose-saving inhaled therapy for COVID-19

The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries around the world, can effectively trap SARS-CoV-2 virus-like-particles in fresh human airway mucus. IN-006, a reformulation of Regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.

bioengineering↗

bioRxiv2022

Characterization of altered molecular mechanisms in Parkinson disease through cell type-resolved multi-omics analyses

Parkinsons disease (PD) is a progressive neurodegenerative disorder. However, cell type-dependent transcriptional regulatory programs responsible for PD pathogenesis remain elusive. Here, we establish transcriptomic and epigenomic landscapes of the substantia nigra (SN) by profiling 87,733 nuclei obtained from healthy controls and PD patients. Our multi-omic data integration provides functional annotation of 128,724 cis-regulatory elements (cREs) and uncovers cell-type specific dysregulated cREs with a strong transcriptional influence on genes implicated in PD. The establishment of high-resolution three-dimensional chromatin contact maps identifies 656 target genes of dysregulated cREs and genetic risk loci, including both novel candidates and known PD risk genes. Notably, these new PD candidate genes exhibit modular gene expression patterns with unique molecular signatures in distinct cell types. Thus, our single-cell transcriptome and epigenome uncover cell type-specific disrupted transcriptional regulations in PD. TeaserSingle-cell transcriptome and epigenome uncover cell type-specific disrupted transcriptional regulations in Parkinsons disease.

genomics↗

bioRxiv2022

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

immunology↗

bioRxiv2022

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Tpp49Aa1 from Lysinibacillus sphaericus is a Toxin_10 family protein that - in combination with Cry48Aa1, a 3-domain crystal protein - has potent mosquitocidal activity, specifically against Culex quinquefasciatus mosquitoes. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser, allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 1.62 [A] resolution from native nanocrystals. This revealed the packing of Tpp49Aa1 within these nanocrystals, isolated from sporulated bacteria, as a homodimer with a large intermolecular interface, shedding light on natural crystallization. Complementary experiments conducted at varied pH also enabled investigations of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals. Using modelling, we propose a potential interaction between Tpp49Aa1 and Cry48Aa1 that may play a role in their codependency and broaden our understanding of this two-component system. We expand the known target range, demonstrating Tpp49Aa1/Cry48Aa1 susceptibility of larvae from Anopheles stephensi, Aedes albopictus and Culex tarsalis - substantially increasing the potential use of this toxin pair in mosquito control. Further functional insights are gained using Culex cell lines to characterise cellular models for future investigations into Cry48Aa1/Tpp49Aa1 mechanism of action and to demonstrate transient detrimental effects of individual toxin components. Significance StatementThe Tpp49Aa1/Cry48Aa1 protein pair kills mosquito larvae. Innovative use of nano-focused X-ray free electron laser to match the size of natural Tpp49Aa1 nanocrystals and the highest beam intensity available in any XFEL for high-throughput data collection, allowed structural resolution to 1.62 [A]. Tpp proteins show a range of interactions with different partners to elicit toxicity. To gain insight into Tpp49Aa1, its interaction with Cry48Aa1 was modelled. We also establish cell-based assays of Tpp49Aa1/Cry48Aa1 activity. We expand the known target range to include three more mosquito species: Anopheles stephensi, Aedes albopictus and Culex tarsalis. This study will underpin future Tpp mode of action investigations and aid insecticide optimization against mosquito vectors of emerging diseases such as West Nile Virus and malaria.

biochemistry↗

bioRxiv2022

Structural basis of Omicron neutralization by affinity-matured public antibodies

The SARS-CoV-2 Omicron1 Variant of Concern (B.1.1.529) has spread rapidly in many countries. With a spike that is highly diverged from that of the pandemic founder, it escapes most available monoclonal antibody therapeutics2,3 and erodes vaccine protection4. A public class of IGHV3-53-using SARS-CoV-2 neutralizing antibodies5,6 typically fails to neutralize variants carrying mutations in the receptor-binding motif7-11, including Omicron. As antibodies from this class are likely elicited in most people following SARS-CoV-2 infection or vaccination, their subsequent affinity maturation is of particular interest. Here, we isolated IGHV3-53-using antibodies from an individual seven months after infection and identified several antibodies capable of broad and potent SARS-CoV-2 neutralization, extending to Omicron without loss of potency. By introducing select somatic hypermutations into a germline-reverted form of one such antibody, CAB-A17, we demonstrate the potential for commonly elicited antibodies to develop broad cross-neutralization through affinity maturation. Further, we resolved the structure of CAB-A17 Fab in complex with Omicron spike at an overall resolution of 2.6 [A] by cryo-electron microscopy and defined the structural basis for this breadth. Thus, public SARS-CoV-2 neutralizing antibodies can, without modified spike vaccines, mature to cross-neutralize exceptionally antigenically diverged SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Variable loss of antibody potency against SARS-CoV-2 B.1.1.529 (Omicron)

The recently-emerged SARS-CoV-2 B.1.1.529 variant (Omicron) is spreading rapidly in many countries, with a spike that is highly diverged from the pandemic founder, raising fears that it may evade neutralizing antibody responses. We cloned the Omicron spike from a diagnostic sample which allowed us to rapidly establish an Omicron pseudotyped virus neutralization assay, sharing initial neutralization results only 13 days after the variant was first reported to the WHO, 8 days after receiving the sample. Here we show that Omicron is substantially resistant to neutralization by several monoclonal antibodies that form part of clinical cocktails. Further, we find neutralizing antibody responses in pooled reference sera sampled shortly after infection or vaccination are substantially less potent against Omicron, with neutralizing antibody titers reduced by up to 45 fold compared to those for the pandemic founder. Similarly, in a cohort of convalescent sera prior to vaccination, neutralization of Omicron was low to undetectable. However, in recent samples from two cohorts from Stockholm, Sweden, antibody responses capable of cross-neutralizing Omicron were prevalent. Sera from infected-then-vaccinated healthcare workers exhibited robust cross-neutralization of Omicron, with an average potency reduction of only 5-fold relative to the pandemic founder variant, and some donors showing no loss at all. A similar pattern was observed in randomly sampled recent blood donors, with an average 7-fold loss of potency. Both cohorts showed substantial between-donor heterogeneity in their ability to neutralize Omicron. Together, these data highlight the extensive but incomplete evasion of neutralizing antibody responses by the Omicron variant, and suggest that increasing the magnitude of neutralizing antibody responses by boosting with unmodified vaccines may suffice to raise titers to levels that are protective.

immunology↗

bioRxiv2021

CV-containing vesicle budding from chloroplast inner envelope membrane is mediated by clathrin but inhibited by GAPC

Clathrin-mediated vesicular formation and trafficking are highly conserved in eukaryotic cells and are responsible for molecular cargo transport and signal transduction among organelles. It remains largely unknown whether clathrin-coated vesicles can be generated from chloroplasts. CHLOROPLAST VESICULATION (CV)-containing vesicles (CVVs) generate from chloroplasts and mediate chloroplast degradation under abiotic stress. In this study, we showed that CV interacted with the clathrin heavy chain (CHC) and induced vesicle budding from the chloroplast inner envelope membrane. Defects on CHC2 and the dynamin-encoding DRP1A gene affected CVV budding and releasing from chloroplast. CHC2 is also required for CV-induced chloroplast degradation and hypersensitivity to water stress. Moreover, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) interacts with CV and impairs the CV-CHC2 interaction. GAPC1 overexpression inhibited CV-mediated chloroplast degradation and hypersensitivity to water stress. CV silencing alleviated the hypersensitivity of gapc1gapc2 plant to water stress. Together, our work revealed a pathway of clathrin-assisted CVV budding from the chloroplast inner envelope membrane, which mediated the stress-induced chloroplast degradation and stress response.

plant biology↗

bioRxiv2021

Rare alternative second line injectable drug resistance markers identified by gene-wise genome wide association in M. tuberculosis with unexplained resistance

Point mutations in the rrs gene and eis promoter are known to confer resistance to second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer a majority of SLID-resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1184 clinical M. tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, 270 AMK-R, CAP-R, and KAN-R isolates, 264 (81.2%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 111 of the phenotypically resistant isolates. A gene-wise method identified three genes and promoters with mutations that, on aggregate, associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 promoter mutations with KAN resistance, supporting clinical relevance for the previously demonstrated role of whiB7 overexpression in KAN resistance. We also provide evidence for the novel association of ppe51 (a gene previously associated with various antimicrobial compounds) with AMK resistance, and for the novel association of thrB with AMK and CAP resistance. The use of gene-wise association can provide additional insight, and therefore is recommended for identification of rare mechanisms of resistance when individual mutations carry insufficient statistical power.

genomics↗

bioRxiv2021

Fluorescence Activation Mechanism and Imaging of Drug Permeation with New Sensors for Smoking-Cessation Ligands

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ("iDrugSnFRs") for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

pharmacology and toxicology↗

bioRxiv2021

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alphas small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic. IMPORTANCEThe global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have resulted in the emergence of different variants. Many of these variants have become more virulent and infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological understanding of these variants remains poor. Here, we directly compared the plaque size, stability, and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at physiological temperatures. Our observations may help to characterize each variant in terms of their interactions with host factors and responses to environmental conditions. We also believe that our evaluations will improve understanding of the emergence of new variants and contribute to controlling their spread.

microbiology↗

bioRxiv2021

Integrated gene analyses of de novo mutations from 46,612 trios with autism and developmental disorders

Most genetic studies consider autism spectrum disorder (ASD) and developmental disorder (DD) separately despite overwhelming comorbidity and shared genetic etiology. Here we analyzed de novo mutations (DNMs) from 15,560 ASD (6,557 are new) and 31,052 DD trios independently and combined as broader neurodevelopmental disorders (NDD) using three models. We identify 615 candidate genes (FDR 5%, 189 potentially novel) by one or more models, including 138 reaching exome-wide significance (p < 3.64e-07) in all models. We find no evidence for ASD-specific genes in contrast to 18 genes significantly enriched for DD. There are 53 genes show particular mutational-bias including enrichments for missense (n=41) or truncating DNM (n=12). We find 22 genes with evidence of sex-bias including five X chromosome genes also with significant female burden (DDX3X, MECP2, SMC1A, WDR45, and HDAC8). NDD risk genes group into five functional networks associating with different brain developmental lineages based on single-cell nuclei transcriptomic data, which provides important insights into disease subtypes and future functional studies.

genetics↗

bioRxiv2021

PGP1 personal genome assembly - a hybrid assembly dataset using ONT's PromethION and PacBio's HiFi sequencing

PGP1 is the first participant of Personal Genome Project. We present the PGP1s chromosome-scale genome assembly. It was constructed using 255 Gb ultra-long PromethION reads and 97 Gb short paired-end reads. For reducing base calling errors, we corrected PromethION reads using 72 Gb PacBio HiFi reads. 327 Gb Hi-C chromosomal mapping data were utilized to maximize the assemblys contiguity. PGP1s contig assembly was 3.01 Gb in length comprising of 4,234 contigs with an N50 value of 33.8 Mb. After scaffolding with Hi-C data and extensive manual curation, we obtained a chromosome-scale assembly that represents 3,880 scaffolds with an N50 value of 142 Mb. From the Merqury assessment, PGP1 assembly achieved a high QV score of Q45.45. For a gene annotation, we predicted 106,789 genes with a liftover from the Gencode 38 and an assembly of transcriptome data.

genomics↗

bioRxiv2021

Pro70 Hydroxylation of Actin Impairs Cell Motility Through Binding with VHL and Blocking His73 Methylation

Actin filaments generate intrinsic forces that are essential for cell motility. This process is tightly regulated by various posttranslational modifications (PTMs), including acetylation, arginylation, and oxidation. However, the role of actin hydroxylation in regulating its dynamics remains poorly understood. Here, we demonstrate that the inhibition of Prolyl Hydroxylase Domain-containing proteins (PHDs) activity significantly promotes actin polymerization and enhances cell motility. Using hydroxylation proteomics, we identified actin as a substrate for PHDs, with hydroxylation occurring at multiple proline residues, especially at proline 70 (Pro70). This modification recruits the von Hippel-Lindau (VHL) tumor suppressor protein, which then leads to the disruption of the interaction between actin and the histidine methyltransferase SET Domain Containing 3 (SETD3). Consequently, SETD3-mediated methylation of histidine 73 (His73) on actin is suppressed, impairing actin polymerization and compromising cell motility. Notably, genetic loss of VHL or pharmacological inhibition of PHDs restores His73 methylation, enhances actin filament formation, and promotes cell motility. Together, our findings uncover a novel regulatory crosstalk between hydroxylation and methylation on actin, establishing a critical mechanism by which PTMs fine-tune actin dynamics to govern cell motility. HighlightsO_LIMK-8617 augments cell motility and actin polymerization in VHL dependent manner; C_LIO_LIHydroxylation proteomics reveals that actin is hydroxylated by PHDs; C_LIO_LIHydroxylation at Pro70 recruits VHL, disrupting actins interaction with SETD3 and reducing His73 methylation; C_LIO_LIGenetic loss of VHL in clear cell renal cell carcinoma (ccRCC) potentially promotes cell motility by increasing His73 methylation on actin. C_LI

biochemistry↗

bioRxiv2021

Quantitative Phosphoproteomic Analyses Identify STK11IP as a Lysosome-Specific Substrate of mTORC1 that Regulates Lysosomal Acidification

The evolutionarily conserved serine/threonine kinase mTORC1 is a central regulator of cell growth and proliferation. mTORC1 is activated on the lysosome surface. However, once mTORC1 is activated, it is unclear whether mTORC1 phosphorylates local lysosomal proteins to regulate specific aspects of lysosomal biology. Through cross-reference analyses of lysosome proteomic with mTORC1-regulated phosphoproteomic, we identified STK11IP as a novel lysosome-specific substrate of mTORC1. mTORC1 directly phosphorylates STK11IP at S404. Knockout of STK11IP led to a robust increase of autophagosome-lysosome fusion and autophagy flux. Dephosphorylation of STK11IP at S404 represses the role of STK11IP as an autophagy inhibitor. Mechanistically, STK11IP binds to V-ATPase, and regulates the activity of V-ATPase. Knockout of STK11IP protects mice from fasting and Methionine and Choline-Deficient Diet (MCD) diet induced fatty liver. Thus, our study demonstrates that STK11IP phosphorylation represents a novel mechanism for mTORC1 to regulate lysosomal acidification, and points to STK11IP as a promising therapeutic target for the amelioration of diseases with aberrant autophagy signaling.

cell biology↗

bioRxiv2021

Core Protein-Directed Antivirals and Importinβ Can Synergistically Disrupt HBV Capsids

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Imp{beta}) via Cps C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Imp{beta} and trypsin in vitro. We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Imp{beta} synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Imp{beta}. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCEThe HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.

microbiology↗

bioRxiv2021

A Gene Replacement Humanization Platform for Rapid Functional Testing of Clinical Variants in Epilepsy-associated STXBP1.

PurposeFunctional evidence is a pillar of variant interpretation according to ACMG guidelines. Functional evidence can be obtained in a variety of models and assay systems, including patient-derived tissues and iPSCs, in vitro cellular assays, and in vivo assays. Here we evaluate the reliability and practicality of variant interpretation in the small animal model, C. elegans, through a series of experiments evaluating the function of syntaxin binding protein, STXBP1, a well-known causative gene for Early infantile epileptic encephalopathy 1 (EIEE1). MethodsUsing CRISPR, we replaced the coding sequence for unc-18 with the coding sequence for the human ortholog STXBP1. Next, we used CRISPR to introduce precise point mutations in the human STXBP1 coding sequence, reflecting three clinical categories (benign, pathogenic, and variants of uncertain significance (VUS)). We quantified 26 features of the resulting worms movement to train Random Forest (RF) and Support Vector Machines (SVM) machine learning classifiers on known pathogenic and benign variants. We characterized the classifiers, and then used the behavioral data from the VUS-expressing animals to predict the categorization of the VUS. ResultsWhereas knock-out worms without unc-18 are severely impaired in motor function, worms expressing STXBP1 in its place have restored motor function. We produced worms with STXBP1 variants previously classified by ACMG criteria, including 25 benign variants, 32 pathogenic, and 24 variants of uncertain significance (VUS). Using either SVM or RF classifiers, we were able to obtain a sensitivity of 0.84-0.97 on known benign and pathogenic strains. By comparing multiple ML classification methods, we were able to classify 9 of the VUS as functionally abnormal, suggesting that these VUS are likely to be pathogenic. ConclusionsWe demonstrate that automated analysis of a small animal system is an effective, scalable, and fast way to understand functional consequences of variants in STXBP1, one of the most common causes of genetic epilepsies and neurodevelopmental disorders.

genetics↗

bioRxiv2021

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 g/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

immunology↗

bioRxiv2021

The in vitro and in vivo potency of CT-P59 against Delta and its associated variants of SARS-CoV-2

The Delta variant originally from India is rapidly spreading across the world and causes to resurge infections of SARS-CoV-2. We previously reported that CT-P59 presented its in vivo potency against Beta and Gamma variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on the Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal study. CT-P59 showed reduced antiviral activity but enabled neutralization against Delta, Epsilon, and Kappa variants in cells. In line with in vitro results, the mouse challenge experiment with the Delta variant substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against the Delta variant infection, hinting that CT-P59 has therapeutic potency for patients infected with Delta and its associated variants. HighlightsO_LICT-P59 exerts the antiviral effect on authentic Delta, Epsilon and Kappa variants in cell-based experiments. C_LIO_LICT-P59 showed neutralizing potency against variants including Delta, Epsilon, Kappa, L452R, T478K and P681H pseudovirus variants. C_LIO_LIThe administration of clinically relevant dose of CT-P59 showed in vivo C_LIO_LIprotection against Delta variants in animal challenge experiment. C_LI

microbiology↗