Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with ACE2 interface, which gives 2G1 ability to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar IC50 in vitro. In SARS-CoV-2 and Beta- and Delta- variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 could be potentially capable of dealing with emerging SARS-CoV-2 variants in future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

Competitive SARS-CoV-2 Serology Reveals Most Antibodies Targeting the Spike Receptor-Binding Domain Compete for ACE2 Binding

ABSTRACT As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Here, we present an efficient, competitive serological assay that can simultaneously determine an individual’s seroreactivity against the SARS-CoV-2 Spike protein and determine the proportion of anti-Spike antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. In this approach based on the use of enzyme-linked immunosorbent assays (ELISA), we present natively folded viral Spike protein receptor-binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then competed with soluble ACE2-Fc, or with a higher-affinity variant thereof, to determine the proportion of ACE2 blocking anti-RBD antibodies. Assessment of sera from 144 SARS-CoV-2 patients ultimately revealed that a remarkably consistent and high proportion of antibodies in the anti-RBD pool targeted the epitope responsible for ACE2 engagement (83% ± 11%; 50% to 107% signal inhibition in our largest cohort), further underscoring the importance of tailoring vaccines to promote the development of such antibodies. IMPORTANCE With the emergence and continued spread of the SARS-CoV-2 virus, and of the associated disease, coronavirus disease 2019 (COVID-19), there is an urgent need for improved understanding of how the body mounts an immune response to the virus. Here, we developed a competitive SARS-CoV-2 serological assay that can simultaneously determine whether an individual has developed antibodies against the SARS-CoV-2 Spike protein receptor-binding domain (RBD) and measure the proportion of these antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. Using this assay and 144 SARS-CoV-2 patient serum samples, we found that a majority of anti-RBD antibodies compete for ACE2 binding. These results not only highlight the need to design vaccines to generate such blocking antibodies but also demonstrate the utility of this assay to rapidly screen patient sera for potentially neutralizing antibodies.

A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential

The outbreak of COVID-19 has emerged as a global pandemic. The unprecedented scale and severity call for rapid development of effective prophylactics or therapeutics. We here reported Nanobody (Nb) phage display libraries derived from four camels immunized with the SARS-CoV-2 spike receptor-binding domain (RBD), from which 381 Nbs were identified to recognize SARS-CoV-2-RBD. Furthermore, seven Nbs were shown to block interaction of human angiotensin converting enzyme 2 (ACE2) with SARS-CoV-2-RBD-variants, bat-SL-CoV-WIV1-RBD and SARS-CoV-1-RBD. Among the seven candidates, Nb11-59 exhibited the highest activity against authentic SARS-CoV-2 with ND50 of 0.55 μg/mL. Nb11-59 can be produced on a large-scale in Pichia pastoris, with 20 g/L titer and 99.36% purity. It also showed good stability profile, and nebulization did not impact its stability. Overall, Nb11-59 might be a promising prophylactic and therapeutic molecule against COVID-19, especially through inhalation delivery.Graphical Abstract

A SARS-CoV-2 serological assay to determine the presence of blocking antibodies that compete for human ACE2 binding

ABSTRACTAs SARS-CoV-2 continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Convalescent serum is being used for treatment and for isolation of patient-derived antibodies. However, currently there is not a simple means to estimate serum bulk neutralizing capability. Here we present an efficient competitive serological assay that can simultaneously determine an individual’s seropositivity against the SARS-CoV-2 Spike protein and estimate the neutralizing capacity of anti-Spike antibodies to block interaction with the human angiotensin converting enzyme 2 (ACE2) required for viral entry. In this ELISA-based assay, we present natively-folded viral Spike protein receptor binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then supplemented with soluble ACE2-Fc to compete for RBD-binding serum antibodies, and antibody binding quantified. Comparison of signal from untreated serum and ACE2-Fc-treated serum reveals the presence of antibodies that compete with ACE2 for RBD binding, as evidenced by loss of signal with ACE2-Fc treatment. In our test cohort of nine convalescent SARS-CoV-2 patients, we found all patients had developed anti-RBD antibodies targeting the epitope responsible for ACE2 engagement. This assay provides a simple and high-throughput method to screen patient sera for potentially neutralizing anti-Spike antibodies to enable identification of candidate sera for therapeutic use.

Use of a Common Garden Experiment in Selecting Adapted Beech Provenances for Artificial Stand Restoration

Increased frequency of extreme weather events has seriously affected forestry operations in south-eastern Europe. A precondition for effective artificial restoration of disturbed forest stands is site-adapted forest reproductive material (FRM). Common garden experiments (provenance trials) may assist in selecting such FRM. The main objective of this study was to establish among-provenance variation pattern using data from a beech provenance trial. Usefulness of the results in selecting seed sources for restoration of European beech stands is discussed. The trial was set up in 2007, at a slope of Medvednica mount facing north-west at 730-750 m above sea level. Plant heights were measured and survival scored in 2008 and 2015. Height increments were calculated and processed to determine variance components due to various effects. Highly significant provenance-by-block interaction was revealed, indicating strong microsite effects on provenance performances. Therefore, corrections were made and provenance mean height increments recalculated. Provenance mean height increment multiplied with survival was used as a measure of a provenance’s adaptedness. Regression tree (RT) analysis was used to determine the pattern of among-provenance variations. A set of provenance clus­ters was grown using climatic variables related to the provenance stands of origin as criteria. All analyzed effects were significant (provenance: F=2.07, p<0.05; block: F=5.07, p<0.05; provenance by block interaction: F=7.32, p<0.001). Data corrections reduced the interaction effect, thereby increasing reliability of calculated provenance adaptedness indices (AI). Provenances were grouped into 4 clusters due to elevation, mean July temperature and summer heat-to-moisture index (SHM). Cluster 4, containing provenances from the highest altitudes (>750m), had the highest mean AI (143.9±8.4 cm). The lowest mean AI (106.7±14.8 cm) had cluster 1, containing provenances from lower altitudes with lower mean July temperatures (≤18.4°C). Provenances originating from lower elevations with higher mean July temperatures (>18.4°C) were further divided into two clusters due to the SHM variable. Cluster 2 had the second highest mean AI (141.2±1.5 cm) and contained provenances from relatively wetter habitats (SHM≤48.2). Cluster 3, containing provenances from relatively arid habitats (SHM>48.2), had significantly lower mean AI (116.8±8.6 cm). Established among-provenance variation pattern might be used as a tool in selecting seed sources for artificial restoration of beech stands at mount Medvednica. It is advisable to use FRM from higher altitudes and/or from slightly lower altitudes (up to 150 m lower than a restoring site) but featured with warmer and drier conditions compared to the trial. Generally, provenance trials should be utilized as a valuable decision tool in restoring disturbed forest stands but may also be misleading if not well designed and analyzed.