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

İpekoğlu, Emre Mert
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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Vaccines are one of the most important public health interventions to combat the spread of infectious diseases. All currently approved COVID-19 vaccines target Spike antigen derived from the authentic Wuhan strain. However, with the emergence of new variants effectiveness of the current vaccines declined, commensurate to their ability to generate neutralizing antibodies against some variants of concern (VOC). Since new VOCs are predicted to emerge the next 2-4 years, variant specific vaccines are urgently n...
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COVID-19 is an infectious disease caused by SARS-CoV-2. The virus was first detected in Wuhan, China in late 2019, and the outbreak was declared a pandemic in January 2020 by WHO, and continues to spread worldwide. As of July 2022, more than 575 million confirmed cases have been detected all over the world, and more than 6 million people died from the disease. One of the most important public health measures in combating the spread of infectious diseases is vaccination. Despite the existence of rapidly deve...
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.