Functional studies on the interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia

Background: With increasing number of vaccinations against SARS-CoV-2, rare but life threatening thrombotic events at unusual sites have been reported, and collectively this phenomenon is termed as vaccine-induced immune thrombotic thrombocytopenia (VITT). Pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT), and associated with platelet-activating antibodies against platelet factor 4 (PF4). Aim(s): Current guidelines for anticoagulation in VITT patients are issued accordingly, with a focus on non-heparin anticoagulants. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux and argatroban with VITT-Ab/ PF4 complexes. Method(s): We utilized an in-house enzyme immunoassays (EIA) to estimate antibody binding, inhibition and dissociation of preformed PF4-VITT complexes. Using biolayer interferometry (BLI), we analyzed binding kinetics and dissociation of complexes in real time. In a flow-based ex vivo model, we assessed the impact of anticoagulants on VITT-mediated thrombus formation. Result(s): We found that heparin and danaparoid not only inhibited VITT IgG binding to PF4 but were also able to effectively dissociate preformed PF4/IgG complexes in EIA. In BLI, binding of PF4 specific antibodies was observed for all VITT samples tested, and we found remarkable changes in their dissociation after addition of various anticoagulants. Furthermore, IgGs from VITT patients induce increased thrombus formation in comparison to the healthy controls (mean % SAC +/- SEM: 11.59 +/- 0.57 vs. 1.99 +/- 0.34 respectively, p < 0.001), which can further be effectively inhibited with danaparoid and heparin (mean % SAC +/- SEM 2.82 +/- 0.50 and 1.85 +/- 0.56. p < 0.001). Fondaparinux and argatroban inhibited thrombus formation;however, they did not affect antibody binding. Conclusion(s): Taken together, our data shed a light on suitability of anticoagulants in VITT, and indicate that negatively charged anticoagulants can disrupt VITT-Ab/ PF4 interactions, which might serve as an approach to reduce antibody-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulation in VITT patients could be made.

An Experimental Framework for Developing Point-of-Need Biosensors: Connecting Bio-Layer Interferometry and Electrochemical Impedance Spectroscopy.

Biolayer interferometry (BLI) is a well-established laboratory technique for studying biomolecular interactions important for applications such as drug development. Currently, there are interesting opportunities for expanding the use of BLI in other fields, including the development of rapid diagnostic tools. To date, there are no detailed frameworks for implementing BLI in target-recognition studies that are pivotal for developing point-of-need biosensors. Here, we attempt to bridge these domains by providing a framework that connects output(s) of molecular interaction studies with key performance indicators used in the development of point-of-need biosensors. First, we briefly review the governing theory for protein-ligand interactions, and we then summarize the approach for real-time kinetic quantification using various techniques. The 2020 PRISMA guideline was used for all governing theory reviews and meta-analyses. Using the information from the meta-analysis, we introduce an experimental framework for connecting outcomes from BLI experiments (KD, kon, koff) with electrochemical (capacitive) biosensor design. As a first step in the development of a larger framework, we specifically focus on mapping BLI outcomes to five biosensor key performance indicators (sensitivity, selectivity, response time, hysteresis, operating range). The applicability of our framework was demonstrated in a study of case based on published literature related to SARS-CoV-2 spike protein to show the development of a capacitive biosensor based on truncated angiotensin-converting enzyme 2 (ACE2) as the receptor. The case study focuses on non-specific binding and selectivity as research goals. The proposed framework proved to be an important first step toward modeling/simulation efforts that map molecular interactions to sensor design.

Integration of Power-Free and Self-Contained Microfluidic Chip with Fiber Optic Particle Plasmon Resonance Aptasensor for Rapid Detection of SARS-CoV-2 Nucleocapsid Protein.

The global pandemic of COVID-19 has created an unrivalled need for sensitive and rapid point-of-care testing (POCT) methods for the detection of infectious viruses. For the novel coronavirus SARS-CoV-2, the nucleocapsid protein (N-protein) is one of the most abundant structural proteins of the virus and it serves as a useful diagnostic marker for detection. Herein, we report a fiber optic particle plasmon resonance (FOPPR) biosensor which employed a single-stranded DNA (ssDNA) aptamer as the recognition element to detect the SARS-CoV-2 N-protein in 15 min with a limit of detection (LOD) of 2.8 nM, meeting the acceptable LOD of 106 copies/mL set by the WHO target product profile. The sensor chip is a microfluidic chip based on the balance between the gravitational potential and the capillary force to control fluid loading, thus enabling the power-free auto-flowing function. It also has a risk-free self-contained design to avoid the risk of the virus leaking into the environment. These findings demonstrate the potential for designing a low-cost and robust POCT device towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.

Simulation of Microfluidic Biosensor for Binding Kinetics of SARS-CoV-2 S Protein Using the Electrothermal Effect

To contribute to the fight versus the coronavirus disease 2019, great efforts have been made by scientists around the world to improve the performance of detection devices so that they can efficiently and quickly detect the virus responsible for this disease. In this context we performed a two-dimensional finite element simulation on the binding kinetics of SARS-CoV-2 S protein of a biosensor using the alternating current electrothermal (ACET) effect. The ACET flow can produce vortex patterns, thereby improving the transportation of the target analyte to the binding surface and thus enhancing the performance of the biosensor. The results showed that the detection time can be improved under the electrothermal effect. The effect of certain design parameters concerning the reaction surface, such as its length as well as its position on the top wall of the microchannel, on the biosensor efficiency were also presented. Results showed that the decrease in the length of the binding surface can lead to an increase in the rate of the binding reaction and therefore decrease the biosensor response time. Also, moving the sensitive surface from an optimal position, which is opposite the electrodes, decreases the performance of the biosensor. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.

Biophysical and structural characterizations of the effects of mutations on the structure-activity relationships of SARS-CoV-2 spike protein.

Mutations on the spike (S) protein of SARS-CoV-2 could induce structural changes that help increase viral transmissibility and enhance resistance to antibody neutralization. Here, we report a robust workflow to prepare recombinant S protein variants and its host receptor angiotensin-convert enzyme 2 (ACE2) by using a mammalian cell expression system. The functional states of the S protein variants are investigated by cryo-electron microscopy (cryo-EM) and negative staining electron microscopy (NSEM) to visualize their molecular structures in response to mutations, receptor binding, antibody binding, and environmental changes. The folding stabilities of the S protein variants can be deduced from morphological changes based on NSEM imaging analysis. Differential scanning calorimetry provides thermodynamic information to complement NSEM. Impacts of the mutations on host receptor binding and antibody neutralization are in vitro by kinetic binding analyses in addition to atomic insights gleaned from cryo-electron microscopy (cryo-EM). This experimental strategy is generally applicable to studying the molecular basis of host-pathogen interactions.

Humoral immune response characterization of heterologous prime-boost vaccination with CoronaVac and BNT162b2.

BACKGROUND: Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be a successful strategy for prevent severe infections. CoronaVac and BNT162b2 are the most used vaccines worldwide, but their use in heterologous vaccination schedules is still subjected to evaluation. METHODS: Fifty healthy individuals who received heterologous prime-boost vaccination with CoronaVac and BNT162b2 were enrolled in a post-vaccination serological follow-up longitudinal prospective study. We evaluated specific serum anti-receptor binding domain (RBD) IgG antibody levels, and their capacity to block RBD-ACE2 interaction with a surrogate neutralization assay. In 20 participants, we assessed antibody binding kinetics by surface plasmon resonance, and Fc-mediated functions by ADCC and ADCP reporter assays. RESULTS: Our baseline seronegative cohort, displayed seroconversion after two doses of CoronaVac and an important decrease in serum anti-RBD IgG antibodies levels 80 days post-second dose. These levels increased significantly early after the third dose with BNT162b2, but 73 days after the booster we found a new fall. Immunoglobulin functionalities showed a similar behavior. CONCLUSIONS: The heterologous prime-boost vaccination with CoronaVac and BNT162b2 generated an impressive increase in serum anti-RBD specific antibody levels followed by a drop. Nevertheless, these titers remained well above those found in individuals only vaccinated with CoronaVac in the same elapsed time. Serum IgG levels showed high correlation with antibody binding analysis, their capacity to block RBD-ACE2 interaction, and Fc-effectors mechanisms. Our work sheds light on the humoral immune response to heterologous vaccination with CoronaVac and BNT162b2, to define a post-vaccination correlate of protection against SARS-CoV-2 infection and to discuss the scheduling of future vaccine boosters in general population.

ANTIGENICITY of the MU (B.1.621) and A.2.5 SARS-CoV-2 SPIKES

Background: The rapid emergence of SARS-CoV-2 variants is fueling the recent waves of the COVID-19 pandemic. Recently identified Mu (B.1.621) and A.2.5 variants carry some mutations shared by other variants of concerns (VOCs). For example, N501Y and E484K mutations in the receptor-binding domain (RBD) domain detected in B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) are now present within the Mu variant. Similarly, the L452R mutation of B.1.617.2 (Delta) variant is now present in A.2.5. Here, we evaluated the capacity of Mu and A.2.5 Spikes to interact with angiotensin-converting enzyme 2 (ACE2) and performed binding and neutralization assays with plasma from vaccinated individuals. In addition, to better understand their antigenic properties, we compared both Mu and A.2.5 with Alpha, Beta, Gamma and Delta VOCs Spikes. Methods: Cells expressing the different Spikes were interrogated for their capacity to interact with the ACE2 receptor using a recombinant ACE2-Fc recombinant protein. We also evaluated their recognition by plasma from BNT162b2 vaccinated individuals. Biolayer interferometry (BLI) was used to measure the binding kinetics of selected RBD mutants to soluble ACE2 (sACE2). Finally, we evaluated the susceptibility of pseudoviral particles bearing the different Spikes to neutralization by plasma from vaccinated individuals. Results: All SARS-CoV-2 S-glycoprotein variants were recognized less efficiently by plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals compared to D614G Spike with the exception of B.1.1.7 S-glycoprotein. Enhanced ACE2 interaction by the Spikes tested was associated with a decrease in the off-rate of the ACE2-RBD interaction. Pseudoviral particles bearing the Spike of Mu variant were similarly neutralized by plasma from vaccinated individuals than those carrying the Beta and Delta Spikes. Conclusion: Plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals efficiently recognized all the Spikes tested. The decreased neutralization susceptibility of pseudoviral particles expressing the Mu Spike was similar to Beta and Delta, thus underscoring the importance of functionally tracking emerging variants. In summary, our results highlight the importance of measuring critical parameters such as ACE2 interaction, plasma recognition and neutralization from each emerging variant.

A Whole-Body Quantitative System Pharmacology Physiologically-Based Pharmacokinetic (QSP/PBPK) Model to Support Dose Selection of ADG20: An Extended Half-Life Monoclonal Antibody Being Developed for the Treatment of Coronavirus Disease (COVID-19)

Background. ADG20 is a fully human IgG1 monoclonal antibody engineered to have potent and broad neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like CoVs with pandemic potential and an extended half-life. ADG20 is administered intramuscularly (IM). A QSP/PBPK model was constructed to support dose selection for a Phase 2/3 trial of ambulatory patients with mild to moderate COVID-19 (STAMP: NCT04805671). Methods. A QSP/PBPK model was used to simulate receptor occupancy (RO) and drug exposure in the upper airway (nasopharyngeal/oropharyngeal epithelial lining fluid [ELF] compartment). RO was linked to an existing viral dynamic model to enable the prediction of the natural time course of viral load and the effect of ADG20 on viral clearance and infectivity rate. RO was calculated using: 1) in vitro ADG20-SARS-CoV-2 binding kinetics (association rate constant (kon) of 1.52E+06 M-1•s1 and dissociation rate constant (koff) of 2.81E-04 s-1 from a Biacore assay;2) time course of ADG20 concentrations in ELF;and 3) time course of viral load following ADG20 administration. Molar SARS-CoV-2 viral binding site capacity was calculated assuming 40 spike proteins per virion, 3 binding sites per spike, and an initial viral load of log 107 copies/mL for all patients. The QSP/PBPK model and a 2018 CDC reference body weight distribution (45-150 kg) were used to simulate 1000 concentration-time profiles for a range of candidate ADG20 regimens. ADG20 regimens were evaluated against 2 criteria: 1) ability to attain near complete ( >90%), and durable (28-day) SARS-CoV-2 RO in the ELF;and 2) ability to maintain ELF ADG20 concentrations relative to a concentration (0.5 mg/L) associated with 100% viral growth suppression in an in vitro post-infection assay. Results. A single 300 mg IM ADG20 dose met the dose selection criteria in terms of RO (Figure A) and viral growth suppression (Figure B). Conclusion. These data support the evaluation of an ADG20 300 mg IM dose for the treatment of mild to moderate COVID-19. ADG20 is forecasted to attain near complete ( >90%) SARS-CoV-2 RO in the ELF and maintain ELF ADG20 concentrations above that associated with 100% viral growth suppression in vitro.

Innovative FO-SPR Label-free Strategy for Detecting Anti-RBD Antibodies in COVID-19 Patient Serum and Whole Blood.

The ongoing COVID-19 pandemic has emphasized the urgent need for rapid, accurate, and large-scale diagnostic tools. Next to this, the significance of serological tests (i.e., detection of SARS-CoV-2 antibodies) also became apparent for studying patients' immune status and past viral infection. In this work, we present a novel approach for not only measuring antibody levels but also profiling of binding kinetics of the complete polyclonal antibody response against the receptor binding domain (RBD) of SARS-CoV-2 spike protein, an aspect not possible to achieve with traditional serological tests. This fiber optic surface plasmon resonance (FO-SPR)-based label-free method was successfully accomplished in COVID-19 patient serum and, for the first time, directly in undiluted whole blood, omitting the need for any sample preparation. Notably, this bioassay (1) was on par with FO-SPR sandwich bioassays (traditionally regarded as more sensitive) in distinguishing COVID-19 from control samples, irrespective of the type of sample matrix, and (2) had a significantly shorter time-to-result of only 30 min compared to >1 or 4 h for the FO-SPR sandwich bioassay and the conventional ELISA, respectively. Finally, the label-free approach revealed that no direct correlation was present between antibody levels and their kinetic profiling in different COVID-19 patients, as another evidence to support previous hypothesis that antibody-binding kinetics against the antigen in patient blood might play a role in the COVID-19 severity. Taking all this into account, the presented work positions the FO-SPR technology at the forefront of other COVID-19 serological tests, with a huge potential toward other applications in need for quantification and kinetic profiling of antibodies.

ACE2 Peptide Fragment Interaction with Different S1 Protein Sites.

We study the effect of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2), with the aim to characterize the interaction mechanism of the SARS-CoV2 virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 α1 domain, which marks the binding site for the S1 protein. The kinetics of S1-ACE2 complex formation was measured in the presence of various concentrations of the peptide using bio-layer interferometry. Formation of the S1-ACE2 complex was inhibited by the peptide in cases where it was preincubated with S1 protein before the binding experiment. The kinetic analysis of S1-ACE2 complex dissociation revealed that preincubation stabilized this complex, and this effect was dependent on the peptide concentration as well as the preincubation time. The results point to the formation of the ternary complex of S1 with ACE2 and the peptide. This is possible in the presence of another binding site for the S1 protein beside the receptor-binding domain for ACE2, which binds the peptide QAKTFLDKFNHEAEDLFYQ. Therefore, we conducted computational mapping of the S1 protein surface, revealing two additional binding sites located at some distance from the main receptor-binding domain on S1. We suggest the possibility to predict and test the short protein derived peptides for development of novel strategies in inhibiting virus infections. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10324-7.

Impact of the N501Y substitution of SARS-CoV-2 Spike on neutralizing monoclonal antibodies targeting diverse epitopes.

The emergence and rapid spread of the B.1.1.7 lineage (VOC-202012/01) SARS-CoV-2 variant has aroused global concern. The N501Y substitution is the only mutation in the interface between the RBD of B.1.1.7 and ACE2, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we assessed the neutralizing activity and binding affinity of a panel of 12 monoclonal antibodies against the wild type and N501Y mutant SARS-CoV-2 pseudovirus and RBD protein, respectively. We found that the neutralization activity and binding affinity of most detected antibodies (10 out of 12) were unaffected, although the N501Y substitution decreased the neutralizing and binding activities of CB6 and increased that of BD-23. These findings could be of value in the development of therapeutic antibodies.