Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant.

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8-92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses

Combined point of care nucleic acid and antibody testing for SARS-CoV-2 following emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in hospital is essential, though complicated by 30-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant now dominates the pandemic and it is unclear how serological tests designed to detect anti-Spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95CI 57.8-92.9%) by rapid NAAT alone. Combined point of care antibody test and rapid NAAT is not impacted by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Structural studies of SARS-CoV-2 spike protein and vesicular coats

The first part of this thesis concerns SARS-CoV-2, the causative agent of the coronavirus disease 2019 (COVID-19). The COVID-19 pandemic has raised an immediate need for vaccine and therapeutic development. The SARS-CoV-2 S protein, forming a crown on the surface of the virus envelope, is responsible for the receptor interaction and facilitating entry into host cells. It is one of the main antigens and a promising therapeutic target. In my work, I expressed and purified stabilised constructs of SARS-CoV-2 S ectodomain, used in multiple avenues of research. We produced cysteine stabilised mutants, which exhibit increased stability and are trapped in a closed conformation, hindering receptor engagement. In a collaborative project, I investigated the use of such stabilised construct as an antigen in immunising mice and showed that the protein induced potent neutralising responses. I also investigated structural characteristics of synthetic antibodies binding the S protein, to identify promising therapeutics. Lastly, in another collaboration, I used cryo-electron microscopy and single particle analysis to characterise a series of antiviral peptides and their interaction with the S protein in solution as well as on intact virions. In the second part, I discuss my work on vesicular coat proteins. Protein coated vesicles facilitate transport between multiple cell organelles in the secretory and endocytic pathways. The trafficking is mediated by various protein coats, which select appropriate cargo through recognition of sorting signals, mould, and structurally support a vesicle. The four archetypal protein coats are formed by clathrin and its adaptor proteins, COPI, COPII and retromer. COPI facilitates transport within the Golgi apparatus, as well as from the Golgi towards the Endoplasmic Reticulum (ER). GOLPH3 is a Golgi resident protein acting as a gatekeeper at the late Golgi to retain other Golgi-residents. It is known to interact with the COPI coat to modulate its cargo binding capabilities, but the interaction and cargo recognition by the two proteins remains elusive. In my PhD work I studied the COPI and GOLPH3 complex using an in vitro reconstituted system and cryo-electron tomography. I obtained a 14 A resolution structure of the complex on vesicles by subtomogram averaging, which provides insights into the sites of interaction between GOLPH3 and COPI subunits, as well as their possible cargo interaction

SARS-CoV-2 Spike Protein Stabilized in the Closed State Induces Potent Neutralizing Responses

The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as prefusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here, we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with the receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern P.1 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. We suggest that closed spikes, together with their improved stability and storage properties, may be a valuable component of refined, next-generation vaccines. IMPORTANCE Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next -generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next-generation vaccines

A thermostable, closed SARS-CoV-2 spike protein trimer.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. It exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor-binding site and, subsequently, from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S-protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S that allow the production of thermostable, disulfide-bonded S-protein trimers that are trapped in the closed, prefusion state. Structures of the disulfide-stabilized and non-disulfide-stabilized proteins reveal distinct closed and locked conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen

SARS-CoV-2 Delta and Omicron variants evade population antibody response by mutations in a single spike epitope

10.1038/s41564-022-01235-4NATURE MICROBIOLOGY7101635-164

Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

Acknowledgements: This work was supported by the MRC (UK; U105181010), a Wellcome Trust Investigator Award (223054/Z/21/Z), a Wellcome Trust Collaborator Award (214344/A/18/Z) and Innovate-UK (UKRI Ideas to Address COVID-19 – Innovate UK Article 25 funding strand). AO and JPS acknowledge funding from EPSRC, Unitaid, Wellcome Trust and MRC for funding which supported development of preclinical models for SARS-CoV-2 infection. We would like to thank Viroclinics Xplore for in vivo studies, Evotec for contract research services, Rachel Dods and Gustavo Arruda Bezerra for structural analysis, Radu Aricescu for design of lentiviral S protein expression systems, Andrew Carter for cloning of S-pseudotyped vectors, Dean Clift for Incucyte analysis, Tyler Rhinesmith and Jakub Laptuk for cell culture and assay support and the Bicycle team and James lab for general scientific support.COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses

Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses