Rapid and cost‐effective process based on insect larvae for scale‐up production of SARS‐COV‐2 spike protein for serological COVID‐19 testing

Abstract

Serology testing for COVID‐19 is important in evaluating active immune response against SARS‐CoV‐2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus‐exposed individuals and vaccine‐mediated immunity. In this study, recombinant S protein of SARS‐CoV‐2 was expressed in Rachiplusia nu , an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS‐CoV‐2 S protein that showed immunoreactivity for anti‐SARS‐CoV‐2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost‐effective platform for large‐scale S protein production, and the scale‐up is linear, thus avoiding the use of complex equipment like bioreactors.

Novel bridge multi-species ELISA for detection of SARS-CoV-2 antibodies

“…The gp64 signal peptide was effective to target S protein to the secretory pathway and its localization to hemolymph. This strategy facilitates its recovery from the larvae extract as a high-quality trimeric version as previously described by Smith et al (2021) . The one-step purification allowed to obtain recombinant trimeric S protein with a sufficient degree of purity for its subsequent labelling with biotin and to use it as an antigen in the immunoassay coating.…”

“…S protein was expressed as a fusion protein with a histidine tag in insect larvae as previously described by our group ( Smith et al, 2021 ). Briefly, batches of 500 fifth-instar Rachiplusia nu larvae (AgIdea, Pergamino, Argentina) were injected with 50 μL of the recombinant baculovirus stock (diluted to 1 × 10 7 PFU/mL) which was obtained using the Bac to Bac® baculovirus expression system (ThermoFisher Scientific, USA) in our laboratory.…”

“…Larvae were selected by fluorescence under UV light, harvested at day 4 post-infection and frozen immediately at −80 °C until they were processed for analysis. The recombinant S protein was obtained with high purity in one step using Immobilized Metal-Ion Affinity Chromatography (IMAC) as reported previously by Smith et al (2021) .…”

“…The most characteristic feature is the spike (S) glycoprotein on its surface that mediates entry into host cells by interacting with angiotensin converting enzyme 2 (ACE2) ( Ke et al, 2020 ). That is why S protein immediately became the main target in the development of antibody tests kits, as well as the focus on therapeutic vaccines ( Salvatori et al, 2020 ; Smith et al, 2021 ).…”

Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review

“…As a result, Sf9 cells are employed to make recombinant baculovirus while Tnao 38 or High Five cells are used for large-scale protein production. For low-cost production, the large-scale SARS-CoV-2 spike protein is also produced in insect larvae such as silkworms and Rachiplusia nu [ 28 ]. A commercial ELISA test has been developed from RBD antigen which can be performed manually using basic laboratory equipment [ 31 ].…”