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.