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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.
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This content will become publicly available on December 1, 2025
Analyzing atomic force microscopy images of virus-like particles by expectation-maximization
Abstract Analysis of virus-like particles (VLPs) is an essential task in optimizing their implementation as vaccine antigens for virus-initiated diseases. Interrogating VLP collections for elasticity by probing with a rigid atomic force microscopy (AFM) tip is a potential method for determining VLP morphological changes. During VLP morphological change, it is not expected that all VLPs would be in the same state. This leads to the open question of whether VLPs may change in a continuous or stepwise fashion. For continuous change, the statistical distribution of observed VLP properties would be expected as a single distribution, while stepwise change would lead to a multimodal distribution of properties. This study presents the application of a Gaussian mixture model (GMM), fit by the Expectation-Maximization (EM) algorithm, to identify different states of VLP morphological change observed by AFM imaging.
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- Award ID(s):
- 2003839
- PAR ID:
- 10520290
- Publisher / Repository:
- Nature
- Date Published:
- Journal Name:
- npj Vaccines
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2059-0105
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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