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 Nālinked 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.