A cell based assay using virus-like particles to screen AM type mimics for SARS-CoV-2 neutralisation.

A number of small molecule and protein therapeutic candidates have been developed in the last four years against SARS-CoV-2 spike. However, there are hardly a few molecules that have advanced through the subsequent discovery steps to eventually work as a therapeutic agent. This is majorly because of the hurdles in determining the affinity of potential therapeutics with live SARS-CoV-2 virus. Furthermore, affinity determined for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, at times, fails to mimic physiological conditions of the host-virus interaction. To bridge this gap between in vitro and in vivo methods of therapeutic agent screening, we report an improved screening protocol for therapeutic candidates using SARS-CoV-2 virus like particles (VLPs). To minimise the interference from the bulkier reporters like GPF in the affinity studies, a smaller hemagglutinin (HA) tag has been fused to one of the proteins of VLP. This HA tag serves as readout, when probed with fluorescent anti-HA antibodies. Outcome of this study sheds light on the lesser known virus neutralisation capabilities of AM type miniprotein mimics. Further, to assess the stability of SARS-CoV-2 spike - miniprotein complex, we have performed molecular dynamic simulations on the membrane embedded protein complex. Simulation results reveal extremely stable intermolecular interactions between RBD and one of the AM type miniproteins, AM1. Furthermore, we discovered a robust network of intramolecular interactions that help stabilise AM1. Findings from our in vitro and in silico experiments concurrently highlight advantages and capabilities of mimic based miniprotein therapeutics.

Assessment of the Biological Impact of SARS-CoV-2 Genetic Variation Using an Authentic Virus Neutralisation Assay with Convalescent Plasma, Vaccinee Sera, and Standard Reagents.

In the summer of 2020, it became clear that the genetic composition of SARS-CoV-2 was changing rapidly. This was highlighted by the rapid emergence of the D614G mutation at that time. In the autumn of 2020, the project entitled "Agility" was initiated with funding from the Coalition for Epidemic Preparedness Innovations (CEPI) to assess new variants of SARS-CoV-2. The project was designed to reach out and intercept swabs containing live variant viruses in order to generate highly characterised master and working stocks, and to assess the biological consequences of the rapid genetic changes using both in vitro and in vivo approaches. Since November 2020, a total of 21 variants have been acquired and tested against either a panel of convalescent sera from early in the pandemic, and/or a panel of plasma from triple-vaccinated participants. A pattern of continuous evolution of SARS-CoV-2 has been revealed. Sequential characterisation of the most globally significant variants available to us, generated in real-time, indicated that the most recent Omicron variants appear to have evolved in a manner that avoids immunological recognition by convalescent plasma from the era of the ancestral virus when analysed in an authentic virus neutralisation assay.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.