Plant material and growing conditions
N. benthamiana seeds were grown on the TerraPlantR 2 substrate, in floating trays soaked with water under controlled conditions (25 oC with photoperiods of 16h light/8h darkness) to obtain plants of approximately 7 weeks of age. Every 15 days we sprinkled the foliar fertilizer Bayfolan® S- Bayer.
RBD recombinant variants as model antigens
For western blot analysis his-RBD variant produced in E. coli (8) was used as positive control. Human ACE2 receptor (hACE2) and chimeric protein hFc-RBD-HRP were supplied by the Center of Molecular Immunology, Havana, Cuba. RBD produced in Pichia pastoris (9) was used as a positive control in the hACE2 inhibition assay.
Construction of the expression vector of the RBD
The nucleotide sequence (amino acids 331-530 of the Spike protein from SARS-CoV-2) coding the RBD region that carried six histidine amino acids at the N-terminal end was extracted from the PET-28 plasmid (8). It was inserted in a plant expression vector, pCambiaHT, using the BamHI restriction sites, and flanked by the 5´ and 3´ non-translatable (UTR) sequences of the Cowpea Mosaic Virus. The 35S promoter of the Cauliflower Mosaic Virus and the tNos nopaline synthase terminator were the signals used for starting and ending the transcription, respectively. The genetic design considered the fusion of the peptide signal of the sporamin from sweet potato (Ipomoea batatas L.), at the N-terminal end of the gene of interest. As a result, we obtained the binary vector pCambiahis-RBDapo (Fig. 1a), used in N.benthamiana transient transformation assays.
Transitory expression of the N. benthamiana leaves
Agrobacterium tumefaciens strain GV3101 was individually transformed with the expression vector pCambiahis-RBDapo and pCambiaP19, by heat shock (Fig. 1a). The resulting strains were checked by PCR and cultivated in the YEB medium (lab-lemco 4g/L, sacarose 5g/L, lacto-pectone 5g/L, yeast extract 1g/L, MgSO4 2 mM, pH 7.2 ) supplemented with 50 mg/mL of rifampicin and kanamycin, while stirring at 200 rpm for 16h at 28 oC. We later transferred them to a YEB medium without antibiotics, under the same conditions where they reached the optical density (OD) of 0.6 to 0.7 at 600 nm. Cell were harvested by centrifugation at 3000 rpm for 30 min and each bacterial pellet was resuspended in the Murashige-Skoog liquid medium (Sigma, USA), with 30 mg/mL of acetosyringone (Sigma-Aldrich, USA) and then incubated at room temperature while slowly stirring in the dark for 4h.
The vacuum infiltration protocol was performed following the process shown in Fig. 1b, using a final solution, resulting from the mixture of the two bacterial solutions (1:1 proportion). At 7 weeks of growth, we took the plants from the floating tray and submerged them, with their roots upward, in the mixture of the Agrobacterium, within a vacuum chamber. The agro-infiltration procedure had 2 cycles consisting of 1 min of vacuum and 1 min of decompression each. Afterwards, we rinsed the treated plants with water to detach the excess bacteria, and they were incubated in water for 5 days at 23 oC with photoperiods of 16h of light and 8h of darkness in glass jars. Agro-infiltrated plants with pCambiaP19 were used as negative control (+P19).
RBDr expression and extraction
The 5 day-post-infiltrated leaves (5dpi) were harvested and total soluble proteins were extracted (10). To determine the levels of expression of the RBDr we used an enzyme-linked immunosorbent assay (ELISA). Plates (Nunc MaxiSorp™) were coated with 10 µg/mL of the monoclonal antibody anti-RBD (AcM-RBD) produced at the CIGB, Sancti Spíritus, Cuba, and incubated for 16h at 4 ᵒC. The plate was blocked with 5% skim milk in PBS-T, for 2h at 37 ᵒC. In order to quantify the RBDr expression, (serial dilutions starting from 1:100) of N. benthamiana protein extracts containing RBDr were added to plate and kept it for 2h at 37 ᵒC. As a second antibody was used monoclonal anti-poly-histidine, produced in mice and conjugated to the horseradish peroxidase (mAb-his-HRP, 1:2000, catalog A7058 Sigma-Aldrich), incubated for 1h at 37 ᵒC. Intermediate washing were established between each step with PBS-T. In the quantification were used a standard curve of RBD produced in Pichia pastoris. As negatives controls were utilized crude extract +P19 and PBS. The reaction was detected after the addition of 3,3-5,5-tetramethylbenzidine and quantified using a microplate reader at 450 nm using a spectrophotometer (Thermo Scientific UV-Vis, USA).
RBDr antigen purification
Before starting the purification, TSP was clarified by centrifugation at 9000 rpm for 20 min and the supernatant was filtrated with a membrane of 0.22µm (Sartorius Minisart, Germany). Recombinant His-tagged proteins were then purified by Immobilized Metal Affinity Chromatography (IMAC) using the procedure (9) with modifications described in the caption of Fig. 2. This fraction was concentrated through centricom 3 kDa (Millipore. USA). The protein concentration of the all fractions were determined by the Bradford method (11).
Samples from the fractions of the RBDr purification process were assayed in a 12% acrylamide SDS-PAGE stained with coomassie Blue G25 (Applichem, Germany). For Western blotting, the mAb-his-HRP was used to detect the his-tag of RBDr. his-RBD, a variant of RBD produced in E.coli, was used as a positive control. The substrate used for the colorimetric detection of the assay was diaminobenzidine (Sigma, USA).
Electrospray ionization mass spectrometry (ESI-MS) analysis
The RBDr purified by IMAC was concentrated and separated using SDS-PAGE in polyacrylamide gel at 12.5% under reducing conditions. The bands of protein corresponding to RBDr were cut from the gel and divided into small cubes of approximately 1 mm3. The gel cubes were treated to obtain tryptic peptides. The peptide mixture was analyzed with a QToF-2 hybrid tandem mass spectrometer (Micromass, UK) according to (12)
Immunogenicity of the RBDr in mice
According to the Institutional Committee for Animal Care and Use of the CIGB, Cuba, the regulations for the use of laboratory animals were followed to study the immunogenicity of RBDr in mice. Balb/c mice 6 weeks old were used in the experiment. They were maintained under controlled temperature and lighting, with feed and water ad libitum. Two experimental groups were designed, which consisted of nine animals per group for the subcutaneous injection. The first one was the group of interest in which we inoculated 5 μg RBDr. A second group identified as the negative control (-RBD) to which we administered the same volume of PBS. The inoculum was mixed with aluminum hydroxide in a 6:1 ratio (antigen/adjuvant). Three doses were assayed, corresponding to 0, 21 and 42 days, while mice bleeding were developed at 0, 21, 42 and 57 days in order to evaluate specific antibody production and their ability to inhibit the binding of RBD to hACE2.
Anti RBDr titers of immunized mice
To evaluate the immune response against RBDr, the described protocol (9) was followed with the adaptations described below and only up to the plate reading step. Coating with 5 µg/mL of RBDr produced in Pichia pastoris and only the SS-1 antibody was used. In addition, the RBDr and -RBD sera used in the assay were diluted in PBS-T (dilutions from 1:25 to 1:10000).
RBD‐hACE2 Inhibition Assay
The capacity of the polyclonal antibodies generated by RBDr to inhibit the binding of the hACE2 receptor was evaluated through a competitive ELISA previously reported (9). Mice sera after the last immunization with RBDr (T57) were used for the analysis. As positive control was considered the sera of nine volunteers (90% inhibition) immunized with RBD of P. pastoris and mice sera immunized with PBS at T57 were used as a negative control. The percentage of inhibition was calculated according to the following formula:
Inhibition %=(1-(OD450nm samples preincubated)/(OD450nm AB))*100
The mean of the experimental values was represented and the standard deviation indicated as error bars. In the analysis of the data, we used the GraphPad Prism software version 8.0.2, with a Kruskal-Wallis test and Dunn’s test.