Development and immunological evaluation of SARS-CoV-2 virus-like particles (VLP) expressing the prefusion stabilized variants of the Spike protein

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2022-1-24

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İpekoğlu, Emre Mert

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Emergence of the COVID-19 pandemic necessitated rapid development of highly effective vaccines against SARS-CoV-2. One adaptable strategy to produce a vaccine candidate against SARS-CoV-2 is based on the exploitation of the self- assembly feature of the structural proteins of SARS-CoV-2: Spike, Membrane, Envelope and Nucleocapsid, forming Virus-like particles (VLP). Our early studies focused on laboratory-scale production of SARS-CoV-2 VLPs in a mammalian host cell system based on transient expression of viral proteins. Herein, we describe a process development framework for scalable production and purification of SARS- CoV-2 VLPs expressing either the prefusion stabilized (2p) or the thermostable six proline (6p; HexaPro) stabilized Spike proteins. Results showed that suspension culture-adapted HEK 293 cells were able to produce vesicular VLPs that were similar in size to the authentic SARS-CoV-2 virions. Combined use of multimodal chromatography, nucleic acid removal through Denarase treatment and tangential flow filtration,enabled large-scale production of the SARS-CoV-2 VLPs. v In this study, we also produced and affinity purified 6x histidine-tagged recombinant Spike (6p) protein of SARS-CoV-2 to conduct in-house serological assays to monitor VLP vaccine-induced anti-Spike immunoglobulin response in immunized mice. Our results demonstrated that a modified purification protocol was superior to conventional affinity purification technique for the production of the secreted recombinant protein in terms of reproducibility and standardization of ELISA experiments. Finally, we examined the immunogenicity of 2p and 6p Spike harbouring VLPs combined with vaccine adjuvants K3 CpG ODN and/or Alum. Collectively, our results suggest that Alum adsorbed, K3 CpG adjuvanted high dose 6p VLPs elicited robust humoral and cellular immune responses in vaccinated BALB/c mice as compared to non-adjuvanted, low dose or 2p VLP counterparts, proving the effectiveness of this formulation as a vaccine candidate against SARS-CoV-2.

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SARS-CoV-2, Virus-like particles, Vaccine, K3 CpG ODN, COVID-19

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https://hdl.handle.net/11511/96233

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

E. M. İpekoğlu, “Development and immunological evaluation of SARS-CoV-2 virus-like particles (VLP) expressing the prefusion stabilized variants of the Spike protein,” M.S. - Master of Science, Middle East Technical University, 2022.