Biology subjects

Mechaly, A.

Publications and source records attributed to Mechaly, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Assessing Corynebacterium glutamicum as a surrogate of Mycobacterium tuberculosis for DNA gyrase inhibitor design.

DNA gyrase is an essential bacterial enzyme and a clinically validated target for the treatment of tuberculosis. However, the discovery of new inhibitors remains limited by the many challenges regarding the manipulation on pathogenic mycobacteria. This study validates Corynebacterium glutamicum (Cglu) as a safe, non-pathogenic surrogate for Mycobacterium tuberculosis (Mtb) to investigate DNA gyrase and facilitate the identification of new inhibitors. Using Cglu as a target allows for fast whole-cell screening under safe conditions while ensuring efficient drug uptake. Cglu shares key physiological features with Mtb, including genome size, complex cell wall structure, and a single type I and type II topoisomerase. Structural and functional comparisons emphasize the similarity of Cglu and Mtb gyrases, which share 70% sequence identity and show comparable catalytic properties and responsiveness to known inhibitors. Thus, the cryo-EM structure of the Cglu gyrase-DNA complex at 3.2 [A] resolution reveals highly conserved drug-binding pockets for known anti-gyrase inhibitors and the genetic depletion of gyrA or gyrB in Cglu causes severe growth and morphological defects, mirroring the effects of chemical inhibition and allowing to link gyrase function to cellular phenotypes. Comparative imaging of different inhibitor classes (fluoroquinolones, aminocoumarins, NBTIs) uncovers distinct morphological signatures that reflect each compounds mode of action. Finally, cross-species complementation confirms functional conservation but also highlights subtle structural differences affecting efficiency. Together, these findings establish Cglu as a robust and biosafe model for dissecting gyrase function, visualizing DNA topology dynamics, and accelerating the discovery of gyrase-targeting antimicrobials. More generally, our studies demonstrate the feasibility of using Cglu as a cell-based screening platform to discover new anti-tuberculous compounds targeting conserved mechanisms, not only for validated TB drug targets such as DNA gyrase but also for new, yet to be identified, targets.

microbiology↗

bioRxiv2026

A Seven-Protein Assembly Promotes Stability, Neutralisation and Secretion of the T7SSb LXG-effector TelE

Streptococcus gallolyticus subsp. gallolyticus (SGG) is a gut pathobiont associated with colorectal cancer. Like many Firmicutes, SGG utilizes a specialized Type VII Secretion System (T7SSb) to export WXG100 and LXG proteins, the latter involved in bacterial competition. We previously identified TelE, an LXG protein whose C-terminus mediates membrane pore formation in Escherichia coli. In SGG UCN34, TelE (Gallo_0562) is co-expressed with six proteins (Gallo_0559 to Gallo_0565), including its immunity protein TipE (Gallo_0565). Here we show that the absence of those co-expressed proteins affects TelE stability and secretion. These proteins associate with TelE to form a soluble and stable seven-protein complex. Gallo_0559 and Gallo_0560 interact with the N-terminal LXG domain of TelE, Gallo_0561 binds to its central region, while the six-transmembrane protein Gallo_0563, together with Gallo_0564 and TipE associates with its C-terminal domain. These findings describe a new modular complex that stabilizes TelE while reducing its toxicity and optimizing its T7SSb-mediated delivery.

microbiology↗

bioRxiv2026

Structural insights into the inactive state of the adhesion GPCR ADGRV1

Adhesion G protein-coupled receptors (aGPCRs) are involved in numerous physiological processes, including cell-cell and cell-matrix interactions, and are associated with several human diseases. ADGRV1 is a member of the aGPCR family and plays a significant role in the sensorineural systems. Mutations of ADGRV1 are linked to the Usher syndrome, a genetic disorder causing deafness and blindness in human. However, the molecular mechanisms that control the activity of ADGRV1 remain unclear. In this study, we present the high-resolution cryo-electron microscopy structure of the inactive ADGRV1 receptor in complex with the nanobody RE02, providing detailed insights into its transmembrane domain, intracellular loop conformations and the inactive orthosteric site. Functional cellular assays revealed that ADGRV1 exhibits a weak constitutive activation independent of the tethered agonist peptide, primarily coupling with Gi proteins. These findings suggest that ADGRV1 may employ an alternative activation mechanism, distinct that from other aGPCRs reported so far. Our structural analysis highlights that ADGRV1 does not follow the conventional tethered agonist activation mechanism. This is due to the divergent sequence of its tethered agonist peptide, as well as the lack of residues critical for conformational transitions toward the active state in other aGPCRs. Moreover, the large intracellular loop 3 (ICL3) adopts a closed conformation, tightly packed against the transmembrane region of ADGRV1. This suggests that the ICL3 loop can compete with G-protein binding, ultimately acting as an additional barrier for ADGRV1 activation. Significance StatementAdhesion G protein-coupled receptors (aGPCRs) regulate essential processes such as cell communication and sensory function, yet the mechanisms controlling many family members remain poorly understood. We report the first high-resolution cryo-electron microscopy structure of the human receptor ADGRV1, a protein linked to Usher syndrome, a major genetic cause of deafness and blindness. Structural and functional analyses reveal that ADGRV1 displays weak constitutive signaling and likely operates through an activation mechanism distinct from the canonical tethered agonist model that defines most aGPCRs. Unique structural features, including a closed intracellular loop (ICL3) that may limit G-protein engagement, suggest an alternative regulatory strategy. These findings expand the conceptual framework of aGPCR activation and provide a foundation for understanding ADGRV1 function in sensory physiology and disease.

biochemistry↗

bioRxiv2026

Structure-guided generative design of peptides targeting the FtsQBL divisome complex inhibit Escherichia coli cell division.

The discovery of antibiotics targeting Gram-negative bacteria remains limited in part by the difficulty of pharmacologically modulating protein-protein interactions essential for bacterial physiology. The divisome complex formed by FtsQ, FtsB and FtsL represents an attractive but challenging target, as its assembly relies on {beta}-strand-mediated interface interactions within the bacterial periplasm. Here, we combined interpretable interface mapping using InDeep with hotspot-constrained RFdiffusion design to generate peptides targeting the FtsB-binding site of Escherichia coli FtsQ. The designed peptides mimic the native {beta}-augmentation interaction and selectively engage the FtsQ interface both in bacterial cells and in vitro. X-ray crystallography of one of these peptides in complex with FtsQ reveals that it accurately adopts the native binding geometry while introducing additional stabilizing interactions within a hydrophobic pocket. Complementary NMR analyses further show that optimized peptides adopt pre-organized {beta}-hairpin conformations in solution consistent with the bound state. Several of these peptides disrupt bacterial cell division and inhibit growth in an E. coli strain exhibiting increased outer membrane permeability. Together, these results establish a structurally validated framework in which predictive interface analysis and generative design can be combined to target cooperative protein-protein interfaces in bacteria and provide a foundation for the development of divisome-targeting antibacterial strategies.

microbiology↗

bioRxiv2025

Energy-guided combinatorial co-optimization of antibody affinity and stability

Affinity maturation is an essential process in antibody engineering. Although powerful, it is iterative, time-consuming, and can result in trade-offs, where affinity is gained at the cost of important properties, such as specificity and stability. We present a scalable strategy, called LAffAb, that starts from a crystallographic structure of the antibody-antigen complex and introduces combinations of mutations to optimize its energy. A combinatorial library comprising 7,000 variants with up to nine mutations results in gains of up to 30-fold in affinity while co-optimizing stability. Surprisingly, the library does not converge on a single solution, instead favoring diverse variants with a high mutational load. Small-scale screening of 10 designs against a potential drug target results in an order-of-magnitude improvement in affinity while maintaining high developability. We also apply LAffAb to improve the developability of a therapeutic antibody without degrading affinity. We envision that LAffAb can be used to design stable, specific, and high-affinity binders and to improve our understanding of sequence, structure, and function relationships in antibodies.

synthetic biology↗

bioRxiv2025

Architecture of a peptidoglycan peptidase complex involved in morphological transition in Helicobacter pylori.

Peptidoglycan is a meshwork macromolecule, made of polysaccharide strands cross-linked by short peptides, which encases the cytoplasmic membrane of bacteria and protects them against turgor pressure. Peptidoglycan peptidases are membrane or periplasmic enzymes that cleave these peptides, either lowering the cross-linking level of peptidoglycan to sculpt bacterial shape or allowing cell elongation by making space for the insertion of neosynthesized glycan strands. In the pathogen Helicobacter pylori, shape is important for virulence, and transition to a coccoid form after prolonged growth enables immune evasion. One particular endopeptidase, HdpA, is known to be involved in the maintenance of cell shape and in the transition to coccoids. Here we show that along growth, HdpA is constantly associated with LhiA, an inner membrane chaperone lipoprotein that keeps it in check while protecting it from fast proteolysis. The crystal structure of the HdpA-LhiA complex suggests that this interaction freezes the autoinhibitory interaction between the first domain of HdpA and the third, catalytic domain. Analysis of the evolution of the HdpA and LhiA protein levels over growth suggests that transition to coccoids is not triggered by a burst in HdpA activity but rather by a gradual weakening of the sacculus caused by the small fraction of free HdpA in equilibrium with LhiA-sequestered HdpA. SIGNIFICANCE STATEMENTThe cell wall, the exoskeleton of bacteria, is the target of numerous antibiotics. Its principal component, peptidoglycan, is remodeled by an interplay between peptidoglycan synthases and hydrolases to accomodate bacterial growth. Because their activity can be harmful, hydrolases have to be tightly regulated. We discovered the dedicated inhibitory chaperone of a H. pylori peptidoglycan hydrolase and showed how the chaperone keeps the hydrolase in check in the cytoplasmic membrane and releases it slowly to allow it to perform its task, ultimately triggering a shape transition to a spherical form important for immune evasion. The crystal structure of the complex gives clues to the mechanism of peptidase activation, suggesting strategies to design agonists that could be used as antibacterials.

microbiology↗

bioRxiv2025

Unraveling the prevalence and multifaceted roles of accessory peptide deformylases in bacterial adaptation and resistance

Peptide deformylases (PDFs) are enzymes that are essential for bacterial viability and attractive targets for antibiotic development. Yet, despite their conserved function, many bacteria encode multiple PDFs, a genomic feature whose prevalence and implications remain largely unexplored. Here, we reveal that nearly half of all bacterial genomes carry more than one PDF gene, frequently embedded within mobile genetic elements such as plasmids and integrons. In Vibrio cholerae, the accessory PDF (Def2VCH) confers reduced susceptibility to actinonin (ACT), the most studied PDF inhibitor, while still supporting bacterial growth in absence of the canonical PDF copies (Def1VCH). Crystallographic analysis shows that this reduced susceptibility stems from an arginine-to-tyrosine substitution that probably reduces ACT binding. Strikingly, this resistance signature is shared by integron-encoded PDFs, and transfer of an integron-encoded PDF cassette from Pseudoxanthomonas into a susceptible V. cholerae is sufficient to abolish ACT susceptibility. These findings expose a cryptic reservoir of resistance within the bacterial mobilome and highlight a challenge to the therapeutic potential of PDF-targeting antibiotics: resistance may not only emerge, but is already encoded, mobile, and ready to spread.

microbiology↗

bioRxiv2023

CREBBP lysine acetyltransferase domain mutations create zombie enzymes that alter chromatin loading dynamics and prevent EP300 redundancy

Epigenetic regulation of gene expression is fundamental to cell state transitions. A prominent example of this is the epigenetically-regulated generation of transitory cells states within the germinal center (GC) reaction that are required for humoral immunity. The deregulation of these processes through somatic mutations can give rise to B-cell lymphoma, thus studying these mutations can provide insight into fundamental mechanisms underlying epigenetic regulation of gene expression as well as GC B-cell and lymphoma biology. Here we show that classes of mutations in the CREBBP lysine acetyltransferase gene result in different structure, catalytic activity and function. We discover that CREBBP and its paralog EP300 are dynamically and reciprocally loaded onto chromatin during a signal-responsive cell state transitions within the GC. Mutation of CREBBP in GC-derived lymphoma interrupt signal-responsive CREBBP loading onto chromatin, while also inhibiting EP300 redundancy. These observations lead to a model in which CREBBP or EP300 loss of function through acetyltransferase domain mutations, which occur frequently in both hematopoietic and solid tumors, create a zombie enzyme that retards the function of the active paralog by binding limiting concentrations of transcription factor substrate and preventing signal-responsive enhancer activation.

cancer biology↗

bioRxiv2023

Ton Motor Conformational Switch and Peptidoglycan Role in Bacterial Nutrient Uptake

Active nutrient uptake is fundamental for survival and pathogenicity of Gram-negative bacteria, which operate a multi-protein Ton system to transport essential nutrients like metals and vitamins. This system harnesses the proton motive force at the inner membrane to energize the import through the outer membrane, but the mechanism of energy transfer remains enigmatic. Here, we study the periplasmic domain of ExbD, a crucial component of the proton channel of the Ton system. We show that this domain is a dynamic dimer switching between two conformations representing the proton channels open and closed states. By in vivo phenotypic assays we demonstrate that this conformational switch is essential for the nutrient uptake by bacteria. The open state of ExbD triggers a disorder to order transition of TonB, enabling TonB to supply energy to the nutrient transporter. We also reveal the anchoring role of the peptidoglycan layer in this mechanism. Herein, we propose a mechanistic model for the Ton system, emphasizing ExbD duality and the pivotal catalytic role of peptidoglycan. Sequence analysis suggests that this mechanism is conserved in other systems energizing gliding motility and membrane integrity. Our study fills important gaps in understanding bacterial motor mechanism and proposes novel antibacterial strategies.

microbiology↗

bioRxiv2023

Interactions of the Protein Tyrosine Phosphatase PTPN3 with Viral and Cellular Partners through its PDZ Domain: Insights into Structural Determinants and Phosphatase Activity

The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a phosphatase containing a PDZ (PSD-95/Dlg/ZO-1) domain that has been found to play both tumor-suppressive and tumor-promoting roles in various cancers, despite limited knowledge of its cellular partners and signaling functions. Notably, the high-risk genital human papillomavirus (HPV) types 16 and 18 and the hepatitis B virus (HBV) target the PDZ domain of PTPN3 through PDZ-binding motifs (PBMs) in their E6 and HBc proteins respectively. This study focuses on the interactions between the PTPN3 PDZ domain (PTPN3-PDZ) and PBMs of viral and cellular protein partners. The solved X-ray structures of complexes between PTPN3-PDZ and PBMs of E6 of HPV18 and the tumor necrosis factor-alpha converting enzyme (TACE) reveal two novel interactions. We provide new insights into key structural determinants of PBM recognition by PTPN3 by screening the selectivity of PTPN3-PDZ recognition of PBMs, and by comparing the PDZome binding profiles of PTPN3-recognized PBMs and the interactome of PTPN3-PDZ. The PDZ domain of PTPN3 was known to auto-inhibit the proteins phosphatase activity. We discovered that the linker connecting the PDZ and phosphatase domains is involved in this inhibition, and that the binding of PBMs does not impact this catalytic regulation. Overall, the study sheds light on the interactions and structural determinants of PTPN3 with its cellular and viral partners, as well as on the inhibitory role of its PDZ domain on its phosphatase activity.

biophysics↗

bioRxiv2023

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Actinobacteria possess unique ways to regulate the oxoglutarate node located in the central position of the tricarboxylic acid cycle, a crossroad between energy conservation and nitrogen metabolism. Here, we studied the decarboxylative oxidation route that leads, through the 2-oxoglutarate dehydrogenase (ODH) complex, to the generation of succinyl-CoA and reduced equivalents to feed the respiratory chain. Compared to most organisms in which the oxidative decarboxylation and reductive acylation steps are carried out by different enzymes within the ODH complex, actinobacteria rely on an all-in-one protein (OdhA) in which both activities are carried out by the same polypeptide. We describe high-resolution cryo-EM and X-ray crystallography snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the FHA (Fork-Head Associated) signalling protein OdhI, delivering new molecular details on how this actinobacterial-specific switching mechanism operates. Overall, the quaternary organization of OdhA represents a new piece of the fascinating puzzle of the synergistic, mixed pyruvate dehydrogenase/2-oxoglutarate dehydrogenase actinobacterial supercomplex.

biochemistry↗

bioRxiv2022

Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system

Type II secretion systems (T2SS) allow diderm bacteria to secrete hydrolytic enzymes, adhesins or toxins important for growth and virulence. In T2SS, secretion of folded proteins from the periplasm to the cell surface requires assembly of periplasmic filaments called pseudopili. Like the related type IV pili, pseudopili are polymerized in the inner membrane through addition of subunits at the filament base, mediated by the essential assembly platform (AP). To understand the structure and molecular role of the AP, we focused on its components PulL and PulM from the Klebsiella oxytoca T2SS. By combining biophysical methods, NMR and X-ray crystallography we studied the structure and associations of their periplasmic domains. We describe the first structure of the heterodimer complex formed by the PulL and PulM ferredoxin-like domains and show how their structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and cross-linking of transmembrane segments provided additional constraints to build a structural model of the PulL-PulM complex and assembly in the cellular context. Together with the relative abundance of PulL, PulM and their partners our findings suggest a model of the AP as a dynamic hub that orchestrates pseudopilus polymerization.

microbiology↗

bioRxiv2022

Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

Calmodulin (CaM) is a eukaryotic multifunctional, calcium-modulated protein that regulates the activity of numerous effector proteins involved in a variety of physiological processes. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Here, we report the structural characterization of CaM:CDZ complexes using combined SAXS, X-ray crystallography, HDX-MS and NMR approaches. Our results provide molecular insights into the CDZ-induced dynamics and structural changes of CaM leading to its inhibition. CDZ-binding induces an open-to-closed conformational change of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from protein targets, despite their distant chemical nature. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. These results open the way to rationally design new CaM-selective drugs. Figure and text for the Table of Contents (ToC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/474921v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@b0c76org.highwire.dtl.DTLVardef@15f462forg.highwire.dtl.DTLVardef@1f8e57forg.highwire.dtl.DTLVardef@1a33575_HPS_FORMAT_FIGEXP M_FIG C_FIG Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results open the way to rationally design new and more selective inhibitors of calmodulin.

biophysics↗

bioRxiv2021

Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins

The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.

biochemistry↗

bioRxiv2021

Isolation and characterization of a highly specific monoclonal antibody targeting the botulinum neurotoxin type E exposed SNAP-25 neoepitope

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. Specific detection and quantification of BoNT/E-cleaved SNAP-25 neoepitope is essential for diagnosis of BoNT/E intoxication as well as for characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. Immunized rabbits developed a specific and robust polyclonal antibody response, whereas immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. Sequence alignment of the isolated clones revealed high similarity between both heavy and light chains, with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled sensitive detection of the cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. This novel antibody can be further used to develop an in vitro cell-based assay to diagnose BoNT/E intoxication and to characterize antitoxin preparations, thus eliminating the use of animals in the standard mouse bioassay.

biochemistry↗

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

Post-exposure protection of SARS-CoV-2 lethal infected K18-hACE2 transgenic mice by neutralizing human monoclonal antibody

Coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterized and further evaluated the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection while administration of the MD65 antibody as late as 3 days after exposure, rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data unprecedentedly demonstrate, the therapeutic value of human monoclonal antibodies as a life-saving treatment of severe COVID-19 infection.

immunology↗