Citation
- Authors: Yilmaz IC. et al.
- Year: 2021
- Journal: Allergy
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293
Description: Human embryonic kidney Fibroblast
Known as: HEK293, 293
Method
- HEK293 suspension adapted cells were grown in serum free Orchid293 CD transfection medium supplemented with 400 mg/L L-glutamine.
- Cells were transiently transfected with 1 μg each of pVitro1 + pVitro2 and PEIpro at a ratio of 2:1 (v/w).
- 96-120 hours of post-transfection, VLP containing culture supernatants were harvested and filtered through a 0.22 μm filter membrane.
- To eliminate host cell-derived nucleic acids, the harvest was treated with 200 U/ml of Denarase for 2 hours at 37°C.
- VLPs were purified on a Hi-Screen Capto Core 400 column using AKTA-GO fast protein liquid chromatography system.
- Flow-through fractions containing VLPs were pooled and subjected to ultrafiltration/dialfiltration on a Sartocon® Slice 200 Hydrosart® 100kDA cassette
Abstract
Background: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2.
Methods: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine.
Results: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals.
Conclusion: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.
