Introduction
The outbreak of a new virus, SARS-CoV-2, in December 2019 has had a serious impact on human health (Zhu et al., 2020). The severe acute respiratory syndrome related to this virus, officially designated COVID-19, has rapidly spread all over the world, progressing into a pandemic. This situation has urgently impelled many companies and public research institutes to concentrate their efforts on the search for effective vaccines, therapeutics, and diagnostic tests.
SARS-CoV-2 is a single-stranded RNA-enveloped virus. The coronavirus spike (S) glycoprotein is surface-exposed in a large number of copies, and it mediates entry into host cells by interacting with angiotensin-converting enzyme 2 (ACE2) (Ke et al., 2020). For these reasons, S protein rapidly became the main target of neutralizing antibodies and the focus of therapeutic and vaccine design (Salvatori et al., 2020). In virions, the S protein exists as a large (over 500 kDa), highly glycosylated homotrimer, each monomer consisting of a globular head, the S1 subunit with its receptor-binding domain (RBD), and the S2 subunit, containing the protein-machinery that mediates viral-cell membrane fusion. The S protein normally exists in a metastable, prefusion conformation, but once the virus interacts with the host cell, an extensive rearrangement occurs, allowing the virus to fuse with the host cell membrane. The spikes are coated with polysaccharide molecules to camouflage themselves, evading surveillance by the host immune system during entry (Watanabe et al., 2020).
Studies conducted in COVID 19 patients have reported that S and nucleocapsid (NCP) proteins are the main SARS-CoV-2 antibody targets (Burbelo et al., 2020). These antibodies are detectable from approximately 6 days after PCR confirmation of infection. It was demonstrated that those antibodies directed against RBD into S show a neutralizing capacity and, hence, can prevent infection (Seydoux et al., 2020; Suthar et al., 2020).
Serological tests for COVID-19 are based on the detection of multiple targets of the virus, including S, RBD, NCP, and non-structural proteins, and are extensively used to identify whether people have been exposed to SARS-CoV-2 by looking at their immune response (Ghaffari et al., 2020). During the pandemic, many efforts have been directed towards detecting, tracking, and better understanding human humoral responses to SARS-CoV-2 infection. It is crucial to develop robust and reliable serological assays to study the antibody kinetics and neutralization efficiency and monitor reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. Currently, the RBD and S proteins are the most reliable antigens for measuring the abundance of neutralizing antibodies (Galipeau et al., 2020).
Different strategies were described to obtain the S protein by biotechnological methods. Because of the structural complexity and post-translational modifications of the S protein, major efforts were directed to mammalian cell culture as a suitable productive system (Stuible et al., 2021). As already described, the S protein is a large homotrimer with 22 N‐linked glycosylation sites per monomer [4]. As a consequence of its structural complexity, it is not surprising systems like Chinese hamster ovary (CHO) and human embryonic kidney (HEK) render low yields in mammalian (Esposito et al., 2020)(Walls et al., 2020). Moreover, the great volume of culture media needed for S expression in mammalian cultures at a high scale is too expensive, especially to obtain this antigen for serological assays.
We previously exhaustively studied the use of insect larvae such asRachiplusia nu , an important agronomic plague in America, as a platform to produce different proteins in a short time and at a low cost (M. Targovnik et al., 2016). Also, we previously identified and described the chromatographic behavior of the main contaminant proteins present in the host to facilitate the downstream processing of any recombinant protein produced in this system (Mc Callum et al., 2019).
Here we established a rapid and cost-effective process for the expression and purification of a high-quality trimeric version of SARS-CoV-2 S protein by using the baculovirus-insect larvae system. This novel recombinant protein was used for developing a new serological ELISA test for COVID-19, showing high sensitivity and specificity with low operational complexity and cost.