The study examines the production of a virosome-based COVID-19 vaccine candidate
The study examines the production of a virosome-based COVID-19 vaccine candidate

The study examines the production of a virosome-based COVID-19 vaccine candidate

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020.

By April 2022, an estimated global number of infections of 500 million and a total of over 6.1 million COVID-19-related deaths were recorded.

Although effective COVID-19 vaccinations were quickly produced and implemented, the number of new variants has increased the demand for vaccine formula updates.

Examination: Insect cells for high-yield production of SARS-CoV-2 tip protein: Building a virosome-based COVID-19 vaccine candidate. Image credit: Leonid Altman / Shutterstock

Background

The production of significant amounts of high quality stable SARS-CoV-2 S proteins is essential for the development of virosomal-based vaccines. Full-length S-protein production has been reported using a variety of expression systems, the majority of which are based on mammalian cells. The insect cell baculovirus expression vector system (IC-BEVS) is a viable option as it is widely considered as an inexpensive, scalable manufacturing platform.

The study

In a recent study published in Pharmaceuticalsvarious signal peptides, baculovirus transfer vectors, cell lines, infection techniques, and formulation buffers were studied with the aim of building a scalable bioprocess to generate high quality S protein for incorporation into a virosome-based COVID-19 vaccine candidate.

The stability, oligomeric state, and binding ability of the generated protein to the angiotensin-converting enzyme 2 (ACE2) receptor and selected neutralizing SARS-CoV-2 antibodies were all evaluated in depth. The S protein was also covalently linked to a clickemic lipid in the virosomal membrane through its polyhistidine (His) tag.

Study result

The most appropriate method of infection was identified via infection of sf-9 cells by cell concentration at infection (CCI) of 1 and 2 x 106 cell / ml with recombinant baculovirus rBac with a multiplicity of infection (MOI) of 0.1 and 1 pfu / cell and small-scale shake flasks (SF) were used to study the growth and S protein expression kinetics. Following infection, traditional profiles of insect cell viability and growth were seen. CCI = 2 x 106 cell / ml and MOI = 1 pfu / cell produced the highest S-protein titers and specific production rates.

The authors explored three different signal peptides that included insect honey bimelittin (BVM) (rBac 1), rBac gp67 (rBac 2), and the S protein signal peptide from the original SARS-CoV-2 strain (rBac 3). Insect Sf-9 cells were infected at CCI = 2 x 106 cell / ml with each rBac at MOI = 1 pfu / cell, and small-scale SF was used to study S protein expression kinetics and growth.

Following infection, the authors discovered traditional insect cell viability and growth profiles in which samples infected with rBac 1 were the only ones that had S protein detected via Western blot, therefore baculovirus constructs containing the BVM signal sequence were used in future assays.

For all N-linked glycan sites already identified in the current literature, purified S protein was analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine site-specific glycosylation and glycan composition. At glycosylation sites N 68_81, N172, N241 and N1081, a combination of high mannose and complex / paucimannose type glycans was detected; the remaining 15 sites were dominated by processed glycans of complex type.

High performance liquid chromatography size exclusion chromatography (HPLC-SEC) and differential scanning fluorimetry (DFS) were used to examine the intermediate shelf life of the isolated S protein. When maintained at 80 ° C and 4 ° C or after 5 freeze-thaw cycles, HPLC-SEC analysis showed a single peak under all conditions tested, which means that the S-protein trimmer structure is maintained for up to 90 days. The shelf life of S-protein was further confirmed by DSF data, which revealed a slight difference in S-protein melting temperatures across all circumstances examined.

Dibenzocyclooctyne (DBCO) azide clique chemistry was used to covalently bind virosomes to purified S protein, and an enzyme-linked immunosorbent assay (ELISA) was used to detect S protein on the virosomes through exposed epitopes and ACE2 binding. The S protein on the outside of the virosomes has the capacity to bind to the ACE2 receptor and is also recognized by CR3022 and all the tested neutralizing antibodies against different epitope clusters according to the results.

Implications

This research shows that an insect cell baculovirus expression vector system can be used to create high quality SARS-CoV-2 S protein for implementation in a virosome-based COVID-19 vaccine candidate. The authors claim that the bioprocess engineering approach used here allowed them to produce 4 mg / L full-length S protein, which is the largest value achieved to date using insect cells.

Furthermore, the S protein produced from insect Sf-9 cells showed glycan processing identical to mammalian cells and shelf life on medium storage. Furthermore, even after one month of storage at 4 ° C, the S protein on the outside of the virosomes had the capacity to bind to the ACE2 receptor and was recognized by a wide range of neutralizing antibodies. Immunogenicity and safety-toxicological studies in appropriate animal models should be performed to verify these particles as COVID-19 vaccine candidates.

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