Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

Llamanade: an open-source computational pipeline for robust nanobody humanization

Nanobodies (Nbs) have recently emerged as a promising class of antibody fragments for biomedical and therapeutic applications. Despite having marked physicochemical properties, Nbs are derived from camelids and may require "humanization" to improve translational potentials for clinical trials. Here we have systematically analyzed the sequence and structural properties of Nbs based on NGS (next-generation sequencing) databases and high-resolution structures. Our analysis reveals substantial framework diversities and underscores the key differences between Nbs and human Immunoglobulin G (IgG) antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen-binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade, a user-friendly, open-source to facilitate rational humanization of Nbs. Using Nb sequence as input, Llamanade provides information on the sequence features, model structures, and optimizes solutions to humanize Nbs. The full analysis for a given Nb takes less than a minute on a local computer. To demonstrate the robustness of this tool, we applied it to successfully humanize a cohort of structurally diverse and highly potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a web server to support the development of a rapidly expanding repertoire of therapeutic Nbs into safe and effective trials. Author SummaryCamelid Nbs are characterized by small size, excellent pharmacological properties and high flexibility in bioengineering for therapeutic development. However, Nbs are "xeno" antibodies, which require "humanization" to improve their translational potential. Currently, there is a lack of systematic investigation of Nbs to rationally guide humanization. No dedicated software has been developed for this purpose. Here, we report the development of Llamanade, an open-source computational pipeline and the first dedicated software to facilitate rational humanization of Nbs. To subjectively evaluate Llamanade, we used it to humanize a cohort of structurally diverse and ultrapotent antiviral Nbs against SARS-CoV-2. Robust humanization by Llamanade significantly improved the humanness level of Nbs to closely resemble fully human IgGs. Importantly, these highly humanized antiviral Nbs remained excellent solubility and comparably high bioactivities to the non-humanized Nb precursors. We envision that Llamanade will help advance Nb research into therapeutic development.

Impact of South African 501.V2 Variant on SARS-CoV-2 Spike Infectivity and Neutralization: A Structure-based Computational Assessment

MotivationThe SARS-CoV-2 variants emerging from South Africa (501.V2) and the UK (B.1.1.7) necessitate rapid assessment of the effects of the corresponding amino acid substitutions in the spike (S) receptor-binding domain (RBD) of the variants on the interactions with the human ACE2 receptor and monoclonal antibodies (mAbs) reported earlier to neutralize the spike. ResultsMolecular modeling and simulations reveal that N501Y, shared by both variants, increases ACE2 binding affinity, and may impact the collective dynamics of the ACE2-RBD complex, occupying a central hinge site that modulates the overall dynamics of the complex. In contrast, the substitutions K417N and E484K in the South African variant 501.V2 would reduce the ACE2-binding affinity by abolishing two interfacial salt bridges that facilitate RBD binding to ACE2, K417(S)-D30(ACE2) and E484 (S)-K31(ACE2). These two mutations may thus be more than compensating the attractive effect induced by N501Y, overall resulting in an ACE2-binding affinity comparable to that of the wildtype RBD. Further analysis of the impact of these mutations on the interactions with mAbs targeting the spike indicate that the substitutions K417N and E484K may also abolish the salt bridges between the spike and selected mAbs, such as REGN10933, BD23, H11_H4, and C105, thus reducing the binding affinity and effectiveness of these mAbs. Contactbahar@pitt.edu Supplementary informationSupplementary data are available at Bioinformatics online.

A monoclonal antibody against staphylococcal enterotoxin B superantigen inhibits SARS-CoV-2 entry in vitro

We recently discovered a superantigen-like motif, similar to Staphylococcal enterotoxin B (SEB), near the S1/S2 cleavage site of SARS-CoV-2 Spike protein, which might explain the multisystem-inflammatory syndrome (MIS-C) observed in children and cytokine storm in severe COVID-19 patients. We show here that an anti-SEB monoclonal antibody (mAb), 6D3, can bind this viral motif, and in particular its PRRA insert, to inhibit infection by blocking the access of host cell proteases, TMPRSS2 or furin, to the cleavage site. The high affinity of 6D3 for the furin-cleavage site originates from a poly-acidic segment at its heavy chain CDR2, a feature shared with SARS-CoV-2-neutralizing mAb 4A8. The affinity of 6D3 and 4A8 for this site points to their potential utility as therapeutics for treating COVID-19, MIS-C, or common cold caused by human coronaviruses (HCoVs) that possess a furin-like cleavage site.

Identification of a unique TCR repertoire, consistent with a superantigen selection process in Children with Multi-system Inflammatory Syndrome

Multisystem Inflammatory Syndrome in Children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares many clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. The superantigen specificity for binding different V{beta}-chains results in V{beta}-skewing, whereby T cells with specific V{beta}-chains and diverse antigen specificity are overrepresented in the TCR repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCR Beta Variable gene (TRBV)11-2. Furthermore, TRBV11-2 skewing was remarkably correlated with MIS-C severity and serum cytokine levels. Further analysis of TRBJ gene usage and CDR3 length distribution of MIS-C expanding TRBV11-2 clones revealed extensive junctional diversity, indicating a superantigen-mediated selection process for TRBV expansion. In silico modelling indicates that polyacidic residues in TCR V{beta}11-2 engage in strong interactions with the superantigen-like motif of SARS-CoV-2 spike glycoprotein. Overall, our data indicate that the immune response in MIS-C is consistent with superantigenic activation. HighlightsO_LIMultisystem Inflammatory Disease in Children (MIS-C) patients exhibit T cell receptor (TCR) repertoire skewing, with expansion of T cell Receptor Beta Variable gene (TRBV)11-2 C_LIO_LITRBV11-2 skewing correlates with MIS-C severity and cytokine storm C_LIO_LIJ gene/CDR3 diversity in MIS-C patients is compatible with a superantigen selection process C_LIO_LIIn silico modelling indicates TCR V{beta}11-2 engages in CDR3-independent interactions with the polybasic insert P681RRAR in the SAg-like motif of SARS-CoV-2 spike C_LI

Decoding of persistent multiscale structures in complex biological networks

In any omics study, the scale of analysis can dramatically affect the outcome. For instance, when clustering single-cell transcriptomes, is the analysis tuned to discover broad or specific cell types? Likewise, protein communities revealed from protein networks can vary widely in sizes depending on the method. Here we use the concept of "persistent homology", drawn from mathematical topology, to identify robust structures in data at all scales simultaneously. Application to mouse single-cell transcriptomes significantly expands the catalog of identified cell types, while analysis of SARS-COV-2 protein interactions suggests hijacking of WNT. The method, HiDeF, is available via Python and Cytoscape.

An insertion unique to SARS-CoV-2 exhibits superantigenic character strengthened by recent mutations

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with Coronavirus Disease 2019 (COVID-19) is a newly recognized condition in which children with recent SARS-CoV-2 infection present with a constellation of symptoms including hypotension, multiorgan involvement, and elevated inflammatory markers. These symptoms and the associated laboratory values strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to MHCII molecules and T cell receptors (TCRs). Here, we used structure-based computational models to demonstrate that the SARS-CoV-2 spike (S) exhibits a high-affinity motif for binding TCR, interacting closely with both the - and {beta}-chains variable domains complementarity-determining regions. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in any other SARS coronavirus), which is highly similar in both sequence and structure to bacterial superantigens. Further examination revealed that this interaction between the virus and human T cells is strengthened in the context of a recently reported rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from a motif shared between the SARS viruses from the 2003 and 2019 pandemics, which has intracellular adhesion molecule (ICAM)-like character. These data suggest that the SARS-CoV-2 S may act as a superantigen to drive the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches. SignificanceAlthough children have been largely spared from severe COVID-19 disease, a rare hyperinflammatory syndrome has been described in Europe and the East Coast of the United States, termed Multisystem Inflammatory Syndrome in Children (MISC). The symptoms and diagnostic lab values of MIS-C resemble those of toxic shock, typically caused by pathogenic superantigens stimulating excessive activation of the adaptive immune system. We show that SARS-CoV-2 spike has a sequence and structure motif highly similar to those of bacterial superantigens, and may directly bind to the T cell receptors. This sequence motif, not present in other coronaviruses, may explain the unique potential for SARS-CoV-2 to cause both MIS-C and the cytokine storm observed in adult COVID-19 patients.