Transient production of receptor-binding domain of SARS-CoV-2 in Nicotiana benthamiana plants induces specific antibodies in immunized mice

Abstract

Background: The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has currently affected millions of people around the world. To combat the rapid spread of COVID-19 there is an urgent need to implement technological platforms for the production of vaccines, drugs and diagnostic systems by the scientific community and pharmaceutical companies. The SARS-CoV-2 virus enters the cells by the interaction between the receptor-binding domain (RBD) present in the viral surface spike protein and its human receptor ACE2. The RBD protein is therefore considered as the target for potential subunit-based vaccines.

Methods and results: We evaluate the use of Nicotiana benthamiana plants as the host to transiently-producing recombinant RBD (RBDr) protein. The identity of the plant-produced RBDr was confirmed by immune assays and mass spectrometry. Immunogenicity was confirmed through the specific antibodies generated in all of the immunized mice compared to the PBS treated group.

Conclusions: In conclusions, the immunogenicity of the RBDr produced in N. benthamiana was confirmed. These findings support the use of plants as an antigen expression system for the rapid development of vaccine candidates.

Keywords: Antibodies; Immunization; Mice; N. benthamiana; RBDr; SARS-CoV-2.

Fig. 1

Schematic representation of transitory transformation in plants of N. benthamiana. a Genetic construction of RBDr. 35 S: promoter of the Cauliflower Mosaic Virus; 5´ and 3´ UTR: non-translational regions of the Cowpea Mosaic Virus; sp: sweet potato sporamin signal peptide; 6his: sequence coding for 6 histidines; tNos: nopaline synthase terminator; RBD: sequence of the receptor binding domain contained in the spike protein of the SARS-CoV-2 virus; P19: gene coding for RNA silencing suppressor p19 from Tomato Bushy Stunt Virus; RB and LB: right and left border, respectively, flanking the transcriptional unit. b Diagram of the vacuum agroinfiltration protocol. c RBDr expression levels

Fig. 2

Purification of RBDr from N. benthamiana leaves agroinfiltrated. a Schematic representation of recombinant antigen purification process. b Stained 12% SDS-PAGE loaded with 10 µg in line 1,2,3,6 ; 200 ng in 4,7 and 1 µg in 5,8. Bottom, Western blot using the anti-histidine monoclonal antibody conjugated with peroxidase of the fractions obtained during purification; 1: TSP extract containing the RBDr; 2: matrix unbound fraction; 3: washing of the process at 70mM of imidazole; 4 and 5: Elution of the RBDr at 500mM; 6: + P19 leaves extract; 7: RBD produced and purified from E.coli; 8: eluate of concentrated RBDr in centricon (3.0 MWCO, Sartorius); MW: Prestained SDS-PAGE standards, Broad Range, cat # 161–0318 (BIO-RAD, USA); simple arrow indicates the band corresponding to the RBDr protein and the double arrow is E. coli his-RBD

Fig. 3

RBDr characterization through mass spectrometry. a ESI-MS spectrum through the digestion of the RBDr protein in polyacrylamide gel. The identified disulfide bonds between the cysteines (C₃₇₉-C₄₃₂ and C₄₈₀-C₄₈₈) are highlighted in red as -S-S-. The signs marked with blue arrows were the peptides that were analyzed with the MASCOT software as the criterion for the identity of the RBDr protein in the databases of the sequences. b Amino acid sequence of the RBDr protein. The residues highlighted in brown, correspond to the 4 amino acids contributed by the sub-cloning vector. The green colour highlights the six residues of the histidine for their purification by affinity chromatography using immobilized metal chelates. Those highlighted in blue are the residues of the spacer arm, and those in black correspond to the RBD theoretical sequence that includes the identified amino acids sequence in violet. Red letters are the cysteine residues. The red lines represent the disulfide bonds that link to cysteines, C₃₃₆-C₃₆₁, C₃₇₉-C₄₃₂, C₄₈₀-C₄₈₈ and C₃₉₁-C₅₂₅. The asterisks represent the identified bonds

Fig. 4

Titration of antibodies in mice immunized with RBDr produced in N. benthamiana. a Immunization schedule. From T0 to T42 we performed the administration of the recombinant antigen. The syringes (filled in red) throughout the entire period, show the time of the blood extraction of the inoculated animals. b Response in immunized mice in each trial period. The values represent the antibodies titters expressed as the base 10 logarithm. +RBDr: experimental group inoculated with the recombinant antigen produced in plants; –RBD: mice immunized with PBS; Graph show mean ± standard deviation (SD) for n = 9 mice/group. c Binding inhibition capacity of polyclonal antibodies RBDr to hACE2. +RBDr: sera of immunized mice with RBDr, C + sera of persons immunized with RBD produced in P. pastoris, that showed high percentages of inhibition and taken as the positive control; -RBD: mice immunized with PBS. For the evaluation, we chose all sera at time T57. We observed two sera with high inhibition capacity, similar to those used as the positive control. Data are mean ± SD. from n = 9 repetitions/groups. The different letters represent significant differences for P < 0.0001