Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

Anti-phospholipase A2 receptor in Non-lupus Patients with Membranous Nephropathy and Crescents

Introduction: Anti-phospholipase A2 receptor (PLA2R) is detected in approximately 70% of biopsies of “primary” membranous nephropathy (MN). Crescents in MN in non-lupus patients suggest additional injury, such as antineutrophil cytoplasmic antibody (ANCA) or anti-glomerular basement membrane (anti-GBM)-associated glomerulonephritis and is postulated to reflect injury by a mechanism that unmasks cryptic epitopes leading to the second autoantibody. Methods: We studied PLA2R staining in non-lupus patients with MN and crescents. Native renal biopsies in 16 non-lupus patients with MN and crescents were stained for PLA2R. Results: The patients included 5 women and 11 men, with mean age 61 yrs and elevated serum creatinine (mean 4.68 mg/dL). Hematuria and proteinuria (mean 4.97 g/d) were documented in 13 patients. Two patients had positive serum anti-GBM antibody. Nine of eleven patients tested for ANCA were positive, with p-ANCA (n=4), c-ANCA (n=2), or both (n=1), with two not specified. On average, 27% of glomeruli had crescents. One patient had an initial biopsy with MN, 4 years later had MN with crescent, and 7 years later had rebiopsy with persistent MN with crescents. One patient had ANCA-associated vasculitis, and 5 years later had MN and crescent. The remaining 14 patients had concurrent diagnoses of MN and crescents. PLA2R was positive in 5 cases, 3 with ANCA positivity, 2 with unknown ANCA status, and none with anti-GBM disease. The patient with initial MN preceding crescent was PLA2R positive; the patient with initial ANCA-associated vasculitis preceding MN was PLA2R negative. Conclusions: Most patients (64%) presented with concomitant MN and crescents, with rare occurrence of an initial disease process followed later by the second injury. PLA2R was positive in 31% of patients, suggesting most are secondary MN. Further study to determine the cryptic epitopes may shed light on the triggering mechanisms for these rare but unlikely coincidental glomerular injuries.

Antibody Therapy: Past, Present and Future

The emergence of pandemics like SARS-CoV-2 and a gradual increase in Multidrug Resistant (MDR) infections highlights the need of innovation in therapeutics. Antibodies are one of the potential solutions for long. Antibody therapy has come very long way from the fight against infectious diseases, bacterial toxins to hybridoma technology and monoclonal antibodies. Hybridoma cells receive a deserving attention due to their antigen-specificity. But, as they were murine in origin, Human Anti Murine Antibody (HAMA) emerged. To achieve this, phage display was introduced. The emergence of molecular cloning lead to the generation of genetically engineered recombinant antibodies such as Fab, Fc, Variable Fragment (Fv), Single Chain Variable Fragments (scFv), single domain antibodies, diabodies; like scFv fragments to different moieties, such as drugs toxins, radionuclides, liposomes or quantum dots etc. Minimized antibodies have several advantages like rapid blood clearance, reduced immunogenicity, low retention time in non-target tissues, access to cryptic epitopes facilitating tumor penetration, rapid growth facilitating higher yield and lower production cost. This paper gives an overview of the history of development of antibodies and its fragments as potential therapeutic agents for the treatment of infectious diseases, one of the biggest challenges of humanity.

Rapid Identification of Neutralizing Antibodies against SARS-CoV-2 Variants by mRNA Display

The increasing prevalence of SARS-CoV-2 variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly-reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identified a set of antibodies against SARS-CoV-2 spike (S) proteins and characterized the structures of nAbs that recognized epitopes in the S1 subunit of the S glycoprotein. These structural studies revealed distinct binding modes for several antibodies, including targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interacts with angiotensin- converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. A potent ACE2-blocking nAb was further engineered to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is a promising approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

Laminin-521 is a Novel Target of Autoantibodies Associated with Lung Hemorrhage in Anti-GBM Disease

BackgroundAntiglomerular basement membrane (anti-GBM) disease is characterized by GN and often pulmonary hemorrhage, mediated by autoantibodies that typically recognize cryptic epitopes within α345(IV) collagen—a major component of the glomerular and alveolar basement membranes. Laminin-521 is another major GBM component and a proven target of pathogenic antibodies mediating GN in animal models. Whether laminin-521 is a target of autoimmunity in human anti-GBM disease is not yet known.MethodsA retrospective study of circulating autoantibodies from 101 patients with anti-GBM/Goodpasture’s disease and 85 controls used a solid-phase immunoassay to measure IgG binding to human recombinant laminin-521 with native-like structure and activity.ResultsCirculating IgG autoantibodies binding to laminin-521 were found in about one third of patients with anti-GBM antibody GN, but were not detected in healthy controls or in patients with other glomerular diseases. Autoreactivity toward laminin-521 was significantly more common in patients with anti-GBM GN and lung hemorrhage, compared with those with kidney-limited disease (51.5% versus 23.5%, P=0.005). Antilaminin-521 autoantibodies were predominantly of IgG1 and IgG4 subclasses and significantly associated with lung hemorrhage (P=0.005), hemoptysis (P=0.008), and smoking (P=0.01), although not with proteinuria or serum creatinine at diagnosis.ConclusionsBesides α345(IV) collagen, laminin-521 is another major autoantigen targeted in anti-GBM disease. Autoantibodies to laminin-521 may have the potential to promote lung injury in anti-GBM disease by increasing the total amount of IgG bound to the alveolar basement membranes.

Molecular mechanisms of induction and acceleration of autoimmunity by microorganisms

Infectious agents are well-known ecological factors inducing/accelerating human autoimmune diseases. Host infection by a pathogen can lead to autoimmunity via multiple mechanisms: molecular mimicry; epitope spreading and presentation of cryptic epitopes of self-antigen owing to lysis of self-tissue by persisting pathogen or immune cells; bystander activation, adjuvant effect of pathogens as a result of non-specific activation of immune system; polyclonal activation of B-cells by chronic infection; activation of T-cells by bacterial superantigens. Infectious agents and nonpathogenic microorganisms can also protect from autoimmune diseases via activation of regulatory T-cells and displacement of balance between two classes of T helper cells in favor of Th2. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Institute of Bioorganic Сhemistry, National Academy of Sciences of Belarus.

SARS-CoV-2 Simulations Go Exascale to Capture Spike Opening and Reveal Cryptic Pockets Across the Proteome

SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression, and replication, which depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate an unprecedented 0.1 seconds of the viral proteome. Our simulations capture dramatic opening of the apo Spike complex, far beyond that seen experimentally, which explains and successfully predicts the existence of ‘cryptic’ epitopes. Different Spike homologues modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also observe dramatic conformational changes across the proteome, which reveal over 50 ‘cryptic’ pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.