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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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Organic Electrochemical Transistors functionalized with Protein Minibinders for Sensitive and Specific Detection of SARS‐CoV‐2
Abstract There is a need for rapid, sensitive, specific, and low‐cost virus sensors. Recent work has demonstrated that organic electrochemical transistors (OECTs) can detect the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein. Here, a simple and low‐cost approach to the fabrication of OECT devices with excellent stability and unprecedented sensitivity and specificity for the detection of SARS‐CoV‐2 virus is demonstrated. The devices rely on the engineered protein minibinder LCB1, which binds strongly to SARS‐CoV‐2. The resulting devices exhibit excellent sensitivity for the detection of SARS‐CoV‐2 virus and SARS‐CoV‐2 spike protein receptor binding domain (RBD). These results demonstrate a simple, effective, and low‐cost biomolecular sensor applicable to the real‐time detection of SARS‐CoV‐2 virus and a general strategy for OECT device design that can be applied for the detection of other pathogenic viruses.
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- Award ID(s):
- 2029339
- PAR ID:
- 10416422
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Materials Interfaces
- Volume:
- 10
- Issue:
- 18
- ISSN:
- 2196-7350
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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