Preclinical development of a molecular clamp‐stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2

Abstract

Objectives Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion‐stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 ‘MF59C.1’ (Seqirus, Parkville, Australia). Methods A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo‐electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. Results In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S‐specific CD4 + and cytotoxic CD8 + T cells in vivo . In the Syrian hamster challenge model ( n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. Conclusion The SARS‐CoV‐2 Sclamp vaccine candidate is compatible with large‐scale commercial manufacture, stable at 2–8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T‐cell responses and provides protection in animal challenge models.

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

“…To determine whether the ACE2 mutants affected binding to spike RBD, a flow cytometrybased binding assay was performed using the established cell lines and recombinant SARS-CoV-2 spike RBD-Fc protein purified by Protein-A affinity chromatography from RBD-Fc plasmid transfected Chinese Hamster Ovary (CHO) cells. SDS-PAGE followed by Coomassie Brilliant Blue staining illustrated the purity of the recombinant RBD-Fc (S1A Fig) , and ELISAs indicated that the RBD-Fc was correctly folded and reactive with an anti-SARS spike specific monoclonal antibody [42,43] Mock transduced cells showed negligible GFP expression or RBD binding, while all ACE2-transduced cell lines showed GFP expression levels in 67-82% of cells (S2 Fig) . The percentage of ACE2-transduced cells (GFP + ) that bound RBD-Fc was used as a measure of binding efficiency. WT hACE2 cells showed an 88.34% binding efficiency, while WT mACE2 cells showed a 0.26% binding efficiency (Figs 1E and S2).…”

Complete protection by a single dose skin patch delivered SARS-CoV-2 spike vaccine

“…Immunization provided protection in hamsters, though this immunity was not complete and required a dose of 100 µg, 50 times higher than required by HD-MAP immunization (66). Other work using a prefusion-stabilized spike protein (5 µg/dose, adjuvanted with MF59) showed reduced viral load in the lungs and throat swabs of hamsters on day 4 post-infection after one or two immunizations (7), with relatively high levels of replicating virus still recovered from these samples. Additionally, neither regime decreased the viral load in the nasal turbinates compared to naïve hamsters on day 4 or day 8 post-infection (7).…”

“…This construct has been modified to include a cleavage site substitution as well as six prolines for stability (35). Plasmids encoding heavy and light chains of SARS-CoV-2 S-specific antibodies, as well as spike receptor binding domain and N-terminal domain constructs were constructed inhouse as previously described (7,67,68).…”

“…The rapid spread of SARS-CoV-2 was accompanied by the rapid development of multiple vaccines. Many vaccine modalities were trialed including mRNA (2-4), DNA (5), protein subunit (6,7), viral vectored (8)(9)(10)(11), nanoparticle (12) and inactivated virus (13,14), and several have subsequently been granted emergency authorization for use in humans.…”

Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

“…VNT assays were conducted as described previously [26,27]. Brie y, overlay medium, block buffer, and polysorbate/phosphate saline (wash buffer) were prepared.…”