Background:
The emergence of new variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has consistently been a challenge for vaccine development progress. While the leading vaccines such as BNT162b2 and mRNA-1273 make use of mRNA-based technology, the unmodified self-amplifying mRNA (SAmRNA) vaccine platform uses low doses and has self-replicative properties conferred by the alphavirus replicase genes. The aim of our study is to increase the neutralisation breadth conferred by our RNA vaccines by expressing three different linked-receptor binding domains (RBDs) using either a typical modified mRNA or an alphavirus SAmRNA.
Methods:
The 3RBD antigen we designed tethered together the RBDs of Beta, Delta and BA.1 variants with a short flexible linker and incorporating a C-terminal transmembrane domain. A plasmid template was optimized for cap, codons, poly(A) and pseudouridine for maximum mRNA expression. We compared the same antigen expressed from native structured RNA in a Venezuelan equine encephalitis virus-derived (VEEV) SAmRNA expression vector. A 2kb mRNA and 9kb SAmRNA were produced by in vitro transcription with T7 RNA polymerase using 5’ N-1-m7G cap and polyadenylated at 3’ end. Our vaccine RNAs were encapsulated into lipid nanoparticles (LNPs) and particle quality controls were assessed by dynamic light scattering.
Results:
The 3RBD vaccine antigen was robustly expressed in vitro and readily detected by Western blot from either a typical mRNA or VEEV SAmRNA. The binding of our antigen to human ACE2 receptor was visualised via protein modelling and was confirmed in vitro by binding to hACE2 monomers. Flow cytometry-based binding assays demonstrated the recognition of human monoclonal antibodies to neutralising epitopes across the 3RBD antigen surface. Expression of single RBDs from both types of RNA platform were used as a comparison. Encapsulated RNA LNPs were made to optimal size, polydispersity and encapsulation efficiency.
Conclusion:
We were successful in producing two alternate versions of a booster RNA COVID vaccine and have validated the antigenicity of our novel polyvalent 3RBD antigen. Our research shows promising results for the development of a high neutralisation breadth yet RBD focused SARS-CoV-2 vaccine antigen which has now proceeded to preclinical mice study to investigate protective efficacy in vivo.