Development of a thermostable and broadly neutralizing pan-sarbecovirus vaccine candidate

Abstract

Zoonotic spillover of sarbecoviruses to humans resulted in theSARS-CoV-1 outbreak in 2003 and the current COVID-19 pandemic caused by SARS-CoV-2. In both cases, the viral spike protein (S) is the principal target of neutralising antibodies that prevent infection. Within spike, the immunodominant receptor-binding domain (RBD) is the primary target of neutralising antibodies in COVID-19 convalescent sera and vaccine recipients. We have constructed stabilized RBD derivatives of different sarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYN02 (Clade 2) and BtKY72 (Clade 3). Stabilization enhanced yield by an 3-23-fold. The RBD derivatives were conformationally intact as assayed by binding to multiple broadly neutralizing antibodies. The stabilized RBDs show significant enhancement in apparent Tm, exhibit resistance to a 2-hour incubation at temperatures up to 60 in PBS in contrast to corresponding WT RBDs, and show prolonged stability of over 15 days at 37 after lyophilization. In mice immunizations, both stabilization and trimerization significantly enhanced elicited neutralization titers by [~]100 fold. The stabilized RBD cocktail elicited high neutralizing titers against both homologous and heterologous pseudoviruses. The immunogenicity of the vaccine formulation was assessed in both naive and SARS-CoV-2 pre-immunized mice, revealing an absence of immune imprinting, thus indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics. Author summaryThe COVID-19 pandemic was caused by the sarbecovirus SARS-CoV-2. Phylogenetically, sarbecoviruses are divided into four clades: Clade 1a, Clade 1b, Clade 2 and Clade 3 and within these clades, are many other sarbecovirus strains with pandemic or epidemic potential. It is therefore important to develop a broadly protective, pan-sarbecovirus vaccine formulation that can be cheaply and rapidly produced. While mRNA vaccine formulations are efficacious, they have stringent low temperature storage requirements and there is limited manufacturing expertise in low and middle income countries for this modality. Neutralizing antibodies are important for protection and in the case of SARS-CoV-2 are primarily directed against the Receptor Binding Domain (RBD) of the surface spike protein. In this study, we have designed and developed an adjuvanted, protein subunit, pan sarbecovirus vaccine formulation using stabilized RBD derivatives from diverse sarbecoviruses as immunogens. We demonstrate that the stabilized RBD derivatives have considerably enhanced yield and thermal stability relative to corresponding WT proteins and that the formulation remains stable for up to two weeks at 37{degrees}C. The formulation was found to be highly immunogenic in both naive and pre-immunized mice, eliciting neutralizing titers well above the known protective threshold, indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics.