An Efficient and Scalable Method for the Production of Immunogenic SARS-CoV-2 Virus-like Particles (VLP) from a Mammalian Suspension Cell Line

Abstract

The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.

Keywords: SARS-CoV-2; neutralizing antibodies; stable cell line; vaccine; virus-like particle (VLP).

Figure 1

Development of a cell line for the inducible production of SARS-CoV-2 VLPs containing all four major structural proteins. (A) Illustration of genetic constructs and fluorescent images of Expi293 cells. The presence of both fluorescent markers (EGFP and mCherry) indicates the presence of all four structural proteins after antibiotic selection. (B) Inverse relationship between cellular health and protein precipitated from daily samples of the cell culture supernatant of three individual batches after induction of VLP production with 1 µg/mL doxycycline. (C) Western blot of protein precipitated from daily samples after induction of VLP production with 1 µg/mL doxycycline. Human serum from a previously infected individual detected SARS-CoV-2 spike- and nucleoproteins from the third day after doxycycline stimulation (D,E) ELISA using monoclonal rabbit antibodies against the spike- or nucleoprotein. The harvested VLPs were coated at a concentration of 5 µg/mL to the solid phase of a microtiter plate. (D) From daily samples after doxycycline stimulation. (E) VLP production was induced with different concentrations of doxycycline and samples harvested on the 5th day after induction. (F) Quantification of VLP-diameter by electron microscopy. (G) Quantification of VLP-diameter by nanoparticle tracking analysis (NTA).

Figure 2

Comparison of VLPs produced by transient transfection and by the ExpiVLP_SMEN cell line. (A) Cell Diameter assessed by NTA. (B) VLP yield per ml. (C) Anti-spike protein and anti-nucleoprotein ELISA comparing the relative absorption generated by VLPs in a 2-Log dilution series of the coating concentration normalized to the absorption of 0.5 µg/mL spike- or nucleoprotein. *—p < 0.05 and ***—p < 0.001.

Figure 3

Cross-flow filtration followed by PEG precipitation improves SARS-CoV-2 VLP purity and specific protein content. (A) Workflow of VLP purification. (B) Example trace and UV quantification of analytical gel filtration of various samples from three individual batches during the purification workflow. VLP and unspecific peaks are highlighted. (C) Size distribution (additional graph in Figure S1) and (D) quantification of various samples during the purification workflow. (E) Western blot showing the presence of spike- and nucleoprotein in VLPs from three individual batches (complete membrane in Figure S2). (F) Spike- and nucleoprotein ELISA of the retentate and the precipitation from the retentate of three individual batches. (G) ELISA measuring the reactivity of the retentate and the precipitation from the retentate of three individual batches with convalescent and control serum. *— p < 0.05 and **—p < 0.01.

Figure 4

SARS-CoV-2 VLPs are immunogenic in mice. (A) Cartoon depicting the immunization protocol. (B) ELISA results. Antigens were coated to the microtiter plate at a concentration of 5 µg/mL VLP, 0.5 µg/mL spike protein, and 1 µg/mL nucleoprotein. Secondary antibody anti-mouse IgG (H+L). (C) Neutralization assay using pseudotyped lentiviral particles expressing firefly luciferase.

Figure 5

SARS-CoV-2 VLPs are stable at 4° and −80 °C in solution for at least 10 weeks. (A) Storage temperature monitored by a data logger. The quality of VLPs was assessed after 10 weeks by (B) NTA, (C) spike- and nucleoprotein ELISA and (D) by the reactivity of the VLPs with human convalescent (n = 16) and control serum (n = 16).