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Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04–0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72–144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

 

Abstract Eukaryotes express a multi-component fatty acid elongase to produce very long chain fatty acids (VLCFAs), which are building blocks of diverse lipids. Elongation is achieved by cyclical iteration of four reactions, the first of which generates a new carbon–carbon bond, elongating the acyl-chain. This reaction is catalyzed by either ELONGATION DEFECTIVE LIKE (ELO) or 3-ketoacyl-CoA synthase (KCS) enzymes. Whereas plants express both ELO and KCS enzymes, other eukaryotes express only ELOs. We explored the Zea mays KCS enzymatic redundancies by expressing each of the 26 isozymes in yeast strains that lacked endogenous ELO isozymes. Expression of the 26 maize KCS isozymes in wild-type, scelo2 or scelo3 single mutants did not affect VLCFA profiles. However, a complementation screen of each of the 26 KCS isozymes revealed five that were capable of complementing the synthetically lethal scelo2; scelo3 double mutant. These rescued strains express novel VLCFA profiles reflecting the different catalytic capabilities of the KCS isozymes. These novel strains offer a platform to explore the relationship between VLCFA profiles and cellular physiology.