Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at -80 and 4 °C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 °C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate

A low dose of RBD and TLR7/8 agonist displayed on influenza virosome particles protects rhesus macaque against SARS-CoV-2 challenge

Abstract Influenza virosomes serve as antigen delivery vehicles and pre-existing immunity toward influenza improves the immune responses toward antigens. Here, vaccine efficacy was evaluated in non-human primates with a COVID-19 virosome-based vaccine containing a low dose of RBD protein (15 µg) and the adjuvant 3M-052 (1 µg), displayed together on virosomes. Vaccinated animals (n = 6) received two intramuscular administrations at week 0 and 4 and challenged with SARS-CoV-2 at week 8, together with unvaccinated control animals (n = 4). The vaccine was safe and well tolerated and serum RBD IgG antibodies were induced in all animals and in the nasal washes and bronchoalveolar lavages in the three youngest animals. All control animals became strongly sgRNA positive in BAL, while all vaccinated animals were protected, although the oldest vaccinated animal (V1) was transiently weakly positive. The three youngest animals had also no detectable sgRNA in nasal wash and throat. Cross-strain serum neutralizing antibodies toward Wuhan-like, Alpha, Beta, and Delta viruses were observed in animals with the highest serum titers. Pro-inflammatory cytokines IL-8, CXCL-10 and IL-6 were increased in BALs of infected control animals but not in vaccinated animals. Virosomes-RBD/3M-052 prevented severe SARS-CoV-2, as shown by a lower total lung inflammatory pathology score than control animals