Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment immunogen

Abstract

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 200mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100°C when lyophilized, and upto 70°C in solution. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design

“…In this case, the DMS data were obtained using yeast display of the RBD which, as opposed to other DMS strategies that measure viral fitness (79, 80), provided a direct readout of RBD expression and folding, making them highly relevant to recombinant forms of the antigen. The stabilizing mutations we identified were distinct from those reported in previous studies focusing on altering surfaceexposed positions to improve the expression and stability of the RBD, which were either similarly used to assist in multivalent display of the RBD (39), or alternatively to enhance production in yeast-based expression systems (27,(29)(30)(31). Our work instead prioritized stabilizing mutations in a buried, structurally suboptimal region of the RBD.…”

“…The SARS-CoV-2 RBD is more structurally homogeneous than metastable S ectodomains, with far higher expression yields (25,26). RBD-based immunogens in various oligomeric states have been investigated as genetic or protein-based vaccines for SARS-CoV-2, including monomers (27)(28)(29)(30)(31), dimers (32), trimers (33)(34)(35) and highly multivalent nanoparticles (25,(36)(37)(38)(39), several of which are now being evaluated in clinical trials. Although the RBD comprises only a minority of the total mass and antigenic surface of S and could in principle be more susceptible to escape mutations, this theoretical disadvantage may be mitigated by both functional constraints and the elicitation of antibodies targeting multiple distinct neutralizing epitopes in the RBD by infection or vaccination (25,(40)(41)(42)(43).…”

Prediction of residue-specific contributions to binding and thermal stability using yeast surface display

“…Buried residues were those with <10% side chain accessibility in chain C of PDB ID 7KMH [46]. ACE-2 binding (active-site) residues were assigned as those contacting ACE-2 [47]. To identify the active-site and buried residues from Sortseq data, we calculated the MrMFIcharged for each position.…”

“…The accessibility and depth was calculated using DEPTH server [76]. Active-site residues were identified from PDB ID 6M0J as explained earlier [47]. Criteria used to predict buried and active-site positions from MFI data were identical to those used for CcdB.…”