A batch-to-batch variation was observed, with two batches (batch#1 and batch#5) providing a signal to noise ratio of ~1

A batch-to-batch variation was observed, with two batches (batch#1 and batch#5) providing a signal to noise ratio of ~1.5 whereas all other tested batches provided ~2.5 (Figure 4C). Corona aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays. Keywords: virus-like particles (VLPs), SARS-CoV-2, insect cells, expression vector, antibodies, cellular assay 1. Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first described in December 2019, has caused a still ongoing worldwide pandemic. Biosafety restrictions limit the research with authentic SARS-CoV-2 virus. As a circumvention, scientists developed various pseudovirus systems, e.g., based on vesicular stomatitis virus (VSV) [1,2,3] or lentivirus [4,5,6]. However, the resulting pseudovirus particles typically only express one structural protein Benfotiamine of the authentic virus. In the case of SARS-CoV-2, this is typically the spike protein, which provides binding to the human virus receptor angiotensin II-converting enzyme 2 (ACE2). Therefore, the Benfotiamine functions of the other structural proteins of SARS-CoV-2 (envelope (E), nucleocapsid (N), and membrane (M) protein) might be overlooked [7]. Further, SARS-CoV-2-derived pseudoviruses still require Biosafety Level 2 (BSL2) safety-level laboratories. An alternative is virus-like particles (VLPs). VLPs can self-assemble upon co-expression of a subset or all viral structural proteins but do not contain genomic information [8]. Hence, they cannot replicate and are considered safe, allowing their production and use in BSL1 laboratories. Further, they can resemble all molecular and morphological features of an authentic virus, and several VLP-based vaccines are approved for clinical use [9]. The production of SARS-CoV-2 VLPs has been described in mammalian cells [10,11,12,13], plant cells [14], yeast [15], and insect cells using the Baculovirus expression vector system (BEVS) [16,17,18]. One of the first SARS-CoV-2 VLP studies was published by Xu et al. in 2020. Xu et al. produced VLP in mammalian cells, finding that E and M protein are critical for VLP formation [11], whereas the influence of N-protein is still debated [12,13]. Kumar et al. studied the VLP formation in Hela cells, allowing monitoring of the SARS-CoV-2 cell entry by a split-Luciferase-based assay [10]. In addition, the expression in plant cells was described [14] and Mazumder et al. produced SARS-CoV-2 VLP in yeast as a promising vaccine candidate [15]. Yet, the most common VLP production system so far is BEVS as it offers generally higher VLP yields than mammalian systems and in parallel can perform complex post-translational modifications [19,20]. Consequently, some Benfotiamine VLP produced by BEVS are already approved vaccines for humans, e.g., Cervarix against human papillomavirus [21]. The yield in the case of SARS-CoV-2 VLP by BEVS was reported to be 5.8 1011 particles per liter [17] while a yield was not reported for other expression systems. VLP produced by BEVS reacted with patient sera and elicited Benfotiamine a neutralizing IgG response in the Syrian hamster model of COVID-19, indicating a potential to be used as vaccine regardless of differences to mammalian glycosylation [18]. Despite these successes, baculovirus-based VLP production systems have some major drawbacks. First, adjustment of the ratios between the different proteins is challenging as co-infection by a mixture of baculovirus is not very efficient and generation of co-expressing baculovirus is not simple [22]. Second, baculoviruses are always produced in parallel to VLPs, requiring a tedious and difficult purification process [23]. Third, BEVS is a lytic system, which leads to the release of a large number of potential contaminants SFRP2 that could compromise the quality of the VLPs. In more recent studies, different plasmid-based expression systems in insect cells have been presented [24,25,26,27,28] that avoid the baculoviral limitations described above. The aim of this study was to establish such a plasmid-based VLP production system in insect cells and in parallel use these VLPs as a cell-based assay for a simple and fast screening of potential protection by sera and monoclonal antibodies. Hereto, we produced SARS-CoV-2 VLP in our baculovirus-free insect cell system [27]. We analyzed different expression vector designs and optimal ratios of the M Protein for our.