Biology subjects

Hanke, L.

Publications and source records attributed to Hanke, L..

7 recordsLinked to original sources

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

Driving potent neutralization of a SARS-CoV-2 Variant of Concern with a heterotypic boost

The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet known whether heterotypic vaccine boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein. Passive immunization with plasma sampled following this boost protected K18-hACE2 mice from lethal challenge with a 501Y.V2 clinical isolate, whereas only partial protection was afforded by plasma sampled after two Wu-Hu-1 spike immunizations.

immunology↗

bioRxiv2021

A bispecific monomeric nanobody induces SARS-COV-2 spike trimer dimers

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we isolated a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the B.1.351 variant, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the B.1.351 variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes a novel strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Caprin-1 binding to the critical stress granule protein G3BP1 is regulated by pH

G3BP is the central hub within the protein-RNA interaction network of stress-induced bio-molecular condensates known as stress granules (SG). The SG-associated proteins Caprin-1 and USP10 exhibit mutually exclusive binding to the structured NTF2-domain of G3BP1, thereby regulating G3BP1-mediated condensation, but with opposite effects: Caprin-1 promotes but USP10 inhibits SG formation. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1 derived short linear motif (SLiM), which provides a molecular understanding for the mutually exclusive binding of USP10 and Caprin-1 to G3BP1. Caprin-1 but not USP10 contacts two G3BP1-NTF2 histidine residues, which was confirmed using biochemical, biophysical and cellular biological binding assays. G3BP1/Caprin-1 interactions disrupted via point mutations resulted in fewer and smaller SG condensates. In addition, biochemical binding assays demonstrated reduced binding of Caprin-1 to G3BP1 at lower pH values. Finally, ratiometric pH sensitive measurements of SGs revealed a substantial drop in pH compared to the adjacent cytosol, suggesting that reduced pH can fine-tune and regulate the G3BP1-mediated interaction network via a NTF2-mediated pH-sensitive SLiM-selection mechanism.

biochemistry↗

bioRxiv2020

SARS-CoV-2 protein subunit vaccination elicits potent neutralizing antibody responses

The outbreak and spread of SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2), the cause of coronavirus disease 2019 (COVID-19), is a current global health emergency and a prophylactic vaccine is needed urgently. The spike glycoprotein of SARS-CoV-2 mediates entry into host cells, and thus is a target for neutralizing antibodies and vaccine design. Here we show that adjuvanted protein immunization with SARS-CoV-2 spike trimers, stabilized in prefusion conformation 1, results in potent antibody responses in mice and rhesus macaques with neutralizing antibody titers orders of magnitude greater than those typically measured in serum from SARS-CoV-2 seropositive humans. Neutralizing antibody responses were observed after a single dose, with exceptionally high titers achieved after boosting. Furthermore, neutralizing antibody titers elicited by a dose-sparing regimen in mice were similar to those obtained from a high dose regimen. Taken together, these data strongly support the development of adjuvanted SARS-CoV-2 prefusion-stabilized spike protein subunit vaccines.

immunology↗

bioRxiv2020

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 {micro}g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the up ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

biophysics↗

bioRxiv2020

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

SARS-CoV-2 is the etiologic agent of COVID-19, currently causing a devastating pandemic for which pharmacological interventions are urgently needed. The virus enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. The nanobody binds with high affinity in the low nM range to the RBD, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations and that Ty1 sterically hinders RBD-ACE2 binding. This 12.8 kDa nanobody does not need an Fc domain to neutralize SARS-CoV-2, and can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗