50?L of the biotinylated ACE2-Fc:sample mixture was transferred to the RBD-SpyVLP-coated plates and incubated for 1?h at RT

50?L of the biotinylated ACE2-Fc:sample mixture was transferred to the RBD-SpyVLP-coated plates and incubated for 1?h at RT. with this paper. Abstract There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Subject terms: Viral infection, Protein vaccines, SARS-CoV-2, Preclinical research Vaccines for SARS-COV-2 are needed in the ongoing pandemic. Rabbit Polyclonal to ARNT Here the authors characterize a vaccine candidate that presents the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on a synthetic VLP platform using SpyTag/SpyCatcher technology and show immunogenicity of a prime-boost regimen in mice and pigs. Introduction Coronavirus disease 2019 (COVID-19), caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China in December 20191. Since then COVID-19 has spread across the world and was declared a pandemic by the World Health Organisation (WHO) in March 2020. As of August 2020, there have been over 20 million confirmed COVID-19 cases worldwide and around 800,000 deaths2. There are no vaccines or effective treatments for COVID-19 to date; however, as of August 2020, there are 48 vaccine candidates in clinical evaluation and around 160 are in pre-clinical testing3. Vaccine candidates in current clinical evaluation include inactivated, viral vector (replicating and non-replicating), protein subunit, nucleic acid (DNA and RNA) and virus-like particle (VLP) vaccines with the majority of them focusing on using the full-length SARS-CoV-2 spike glycoprotein (S) as an immunogen. SARS-CoV-2 is an enveloped virus carrying a single-stranded positive-sense RNA genome (~30?kb), belonging to the genus from the family4. The virus RNA encodes four structural proteins including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, 16 non-structural proteins, and nine accessory proteins5. The S glycoprotein consists of an ectodomain (that can be processed into S1 and S2 subunits), a transmembrane tCFA15 domain, and an intracellular domain6. Similar to the SARS-CoV, SARS-CoV-2 binds the human angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) within the S1 subunit to facilitate tCFA15 entry into host cells, followed by membrane fusion mediated by the S2 subunit7C9 Of the many vaccine platforms, protein subunit vaccines generally have good safety profiles and their production is rapid and easily scalable10. Recombinant RBD proteins of SARS-CoV and MERS-CoV have been shown to be immunogenic and induce protective neutralising antibodies in animal models and are therefore considered promising vaccine candidates (reviewed in refs. 11,12). RBD from SARS-CoV-2 has recently been confirmed to be inducing neutralising antibodies13,14. Recently published studies, including one from our group, found that the majority of the potent neutralising antibodies isolated from SARS-CoV-2-infected patients bound to the RBD15C17. We therefore chose to study the immunogenicity of RBD. To improve tCFA15 immunogenicity, we conjugated the RBD onto a VLP. VLP display of protein antigen has been shown to further enhance immunogenicity by facilitating antigen drainage to lymph nodes, enhancing uptake by antigen-presenting cells and increasing B cell receptor crosslinking10,18. Moreover, we recently showed that influenza antigens (haemagglutinin (HA) or neuraminidase (NA)) tCFA15 displayed on VLPs (the same VLP used in this study) were highly immunogenic at a low dose (0.1?g) in mice18. In the present study, we used the SpyTag/SpyCatcher technology for the assembly of SARS-CoV-2 RBD on a protein nanoparticle platform, SpyCatcher003-mi318. The VLP platform, based on an engineered aldolase from thermophilic bacteria, spontaneously assembles into a hollow 36-nanometre dodecahedral cage with 60 subunits19,20. SpyCatcher003 is a variant of SpyCatcher tCFA15 which was engineered for accelerated reaction with SpyTag21. The SpyCatcher003-mi3 VLP can be expressed to high yields (100?mg/L of culture media) in and purified using scalable ammonium sulfate precipitation followed by size exclusion chromatography18. Furthermore, the VLP platform has high thermal stability and good colloidal properties18. Each VLP subunit is genetically fused to SpyCatcher003-protein, allowing efficient decoration of the VLP with SpyTag-fused antigens through covalent isopeptide bonds (Fig.?1a). Previously, SpyTag-mediated VLP decoration has been successfully used for the display of diverse antigens from, e.g. spp., influenza A virus, HIV and cancer cells (PD-L1)18,19,22C24. Here we show that the RBD-SpyVLP vaccine candidate is highly immunogenic in mice and pigs, inducing robust.