Recombinant virus construction
We based our studies on a previously described version of SARS-CoV-2 S protein sequence, stabilized in its prefusion conformation (Wrapp et al., 2020). Briefly, the expressed protein was expected to include the ectodomain (residues 19-1207) of the SARS-CoV-2 Wuhan-Hu-1 S protein (GenBank: QHD43416.1) without native signal peptide, where the furin cleavage site (residues 682–685, PRRA) was removed (GSAS mutations) and residues at positions 986 (K) and 987(V) were mutated to proline; additionally, a C-terminal T4 fibritin trimerization domain, a TEV protease cleavage site, and a histidine tag were included (Figure 1). For secretion, S protein was expressed under gp64 baculoviral signal peptide. The DNA sequence was codon-optimized for baculovirus expression and chemically synthesized (Genscript, Piscataway, NJ, USA). The S sequence and the enhanced green fluorescent protein (EGFP) cDNA were cloned into the pFastBacDual vector (Thermo Fisher Scientific, Waltham, USA) under the polyhedrin (polh) and p10 promoter, respectively, for expression in the baculovirus system. For this purpose, the EGFP (GenBank Accession No. NC_013179.1) was cloned into SmaI and Nco1 sites of the pFastBacDual vector (Targovnik et al., 2019)]. Then, the S cassette was subcloned into the pFastBacDual Vector into BamH1 and HindIII sites to generate the pFBD-p10-EGFP-polh-S construction.
The recombinant baculoviruses were obtained using the Bac to Bac® baculovirus expression system (Thermo Fisher Scientific, USA), following the manufacturer’s instructions. The pFBD-p10-EGFP-polh-S vector was transformed into a chemically competent E. coli DH10Bac™ strain (Thermo Fisher Scientific, USA) by heat shock to generate the recombinant bacmid by transposition. Then, the bacmids were purified and used to transfect 1 million Sf9 cells using Cellfectin II Reagent (Thermo Fisher Scientific, USA). After 4-day incubation at 27 °C, the cell culture supernatant was collected and centrifuged at 500 ×g for 10 min. The transfection efficiency was determined by measuring EGFP expression by fluorescence under UV light. The recombinantAutographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin-minus virus containing EGFP under the control of the p10 promoter and the S sequence under the control of the polyhedrin promoter was named rAcMNPV-S. Then, a round of amplification was performed in Sf9 cells seeded in T-25 flasks at 27 °C, at a low multiplicity of infection (MOI) of 0.02. The Sf900 II insect cell culture medium and the antibiotic and antimycotic solutions were from InvitrogenTM (Gaithersburg, MD, USA) and the fetal bovine serum (FBS) was from Nutrientes Naturales S.A. (Buenos Aires, Argentina). The amplified rAcMNPV–S was titrated by plaque assay (O´Reilly DR, Miller LK, 1994). This high-titer rAcMNPV–S was the viral stock used for protein production in insect larvae.