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Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

ABSTRACT: 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.

SUBMITTER: Zak AJ

PROVIDER: S-EPMC10572262 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Zak Andrew J AJ Hoang Trang T Yee Christine M CM Rizvi Syed M SM Prabhu Ponnandy P Wen Fei F

International journal of molecular sciences 20230927 19

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 t ...[more]

PMID: 37834067

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.

Dataset Information

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Publications

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

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