Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms

Abstract

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development.

Keywords: BY-2 cell culture; COVID-19 pandemic; RBD production; diagnostics; glycan profiles; plant-based biologics; serological tests; transient expression.

Figure 1

Protein structure of the receptor-binding domain (RBD) forms expressed in plants. Schematic representation of (A) RBD expressed transiently in N. benthamiana and (B) RBD expressed stably in BY-2. SP, signal peptide; HTAG, 6× histidine tag; KDEL, tetrapeptide sequence for ER retention; MTAG, cMyc tag; STAG, Strep tag.

Figure 2

NB-RBD time-course expression analysis. (A) Leaves detached from N. benthamiana agro-infiltrated leaves either with GV3101 A tumefaciens (N) or expressing receptor-binding domain (RBD) at different days post-infiltration 3–5 (d.p.i.). (B) Western blot analysis of the same plant samples using the polyclonal anti-RBD PA5-114529 antibody. (C) SDS-page Coomassie staining. N, negative control (leaves infiltrated with A tumefaciens GV3101); P, positive control (100 ng of a cytosolic RBD produced in E coli); M, molecular marker.

Figure 3

NB-receptor-binding domain (RBD) purification. (A) Western blot of anion exchange chromatography (top) and its SDS-PAGE Coomassie stain (bottom). (B) Western blot of affinity chromatography using the polyclonal anti-RBD PA5-114529 antibody (top) and its SDS-PAGE Coomassie stain (bottom). (C) RBD stability over-time from T0 to T4 (6 months). M, molecular marker; P, positive control (100 ng of a commercial RBD produced in HEK293, purity 90%); E, crude extract; L, load; FT, flow-through; W, wash; 1–3, elution fractions of DEAE; 1–9, elution with imidazole-raising concentrations. The red arrows indicate the monomeric RBD form.

Figure 4

NB-receptor-binding domain (RBD) and BY2-RBD production phases and timelines. In the x-axis, the weeks requested to reach each milestone indicated in the y-axis are reported. DSP, downstream processing.

Figure 5

Receptor-binding domain (RBD) screening in BY2 clones. Equal quantities of soluble crude extract were loaded into each lane. Soluble crude extract from wild-type BY-2 was used as negative control for all blots. The yellow labels mark the highest expressor clones per construct in each blot. (A) The pNGV010 set is portraying RBD in co-expression with ER-targeted GFP, and the pVNG011 set is portraying RBD without GFP. The highest estimated RBD accumulation for the latter (22) is indicated with a red arrow. RBD positive control, produced in HEK293, is present in all anti-RBD blots ranging from 25 to 200 ng; its apparent molecular weight is 35 kDa in reduced conditions. (B) Size and tag assessment of the highest-producing clones from the two sets. As denoted in the anti-myc and anti-strep blots, 011_22 is producing a full-sized RBD with both c-myc- and strep- tags.

Figure 6

Production of BY2-receptor-binding domain (RBD) in a 20-L bioreactor. (A) Fresh weight and dry weight throughout the 5-day cultivation. (B) DO% and CO₂% throughout the 5-day cultivation. (C) Biostat C, Sartorius AG 40L Bioreactor and lyophilized biomass resulting from the cultivation. Photos courtesy of Kaisa Rinta-Harri. (D) Anti-RBD Western blot of purified BY-2-produced RBD from the bioreactor cultivation. Equal volumes of the samples were loaded to each lane. S (start undiluted and 1:10 dil); FT (flow-through), F3–F7 (fractions 3 to 7); wild-type BY-2 extract as negative control. RBD positive control (SARS-CoV spike protein) is present in a gradient (25, 50, 100, and 200 ng). Its apparent molecular weight is 35 kDa in reduced conditions.

Figure 7

NB-receptor-binding domain (RBD) and BY-2 RBD biochemical comparison. (A) Non-reducing PAGE Western blot using the polyclonal anti-RBD PA5-114529 antibody—1: 500 ng, 2: 250 ng, and 3: 50 ng. hRBD: positive control, commercial HEK293-made RBD; BY2-RBD: BY-2-made RBD; NB-RBD: N. benthamiana-made RBD. M, molecular marker. (B) Output of size exclusion chromatography analysis of NB-RBD; the one of BY2-RBD was identical.

Figure 8

Preliminary N-glycan profiling of NB-receptor-binding domain (RBD) and BY2-RBD via affinodetection with concanavaline A. (A) Immunodetection with antibodies raised specifically against core β(1,2)- xylose (B) or core α(1,3)-fucose (C). Ribonuclease (Ribo B) and avidine produced in maize (avidine) are positive controls. Bovine serum albumin is a negative control.

Figure 9

N-glycan profiling of NB-receptor-binding domain (RBD) and BY2-RBD. MS/MS spectra of (A) the glycopeptide at mz 2,284.97 assigned to the peptide V₅₂FNATR₅₇ N-linked to Gn₂M₃XFGn₂ from NB-RBD and (B) the glycopeptide at mz 2,410.97 assigned to the peptide V₆₅FNATR₇₀ N-linked to Man₈GlcNAc₂ from BY2-RBD. GlcNAc, blue square; Man, green circle; Fuc, red triangle; Xyl, yellow star. (C) Ratio between oligomannosides and complex N-glycans present on the two specific N-glycosites of RBDs.

Figure 10

Diagnostic performance of the ELISA tests based on NB-receptor-binding domain (RBD) and BY2-RBD. Box plot distribution (box range, 25–75) of results from ELISA tests based on NB-RBD (A) and BY2-RBD (B). Dotted lines mark the medians. The cutoffs are shown in solid lines: the green ones represent the cutoff calculated as the mean plus the double value of SD, while the blue ones represent the cutoff calculated as the mean plus threefold the value of SD.

Figure 11

Correlation between ELISA tests based on plant-made receptor-binding domains (RBDs) and the golden standard serological test. Comparison of binding assays by linear regression. The black dots represent the antibody titer in every serum, and the red line represents the trend line. Pearson’s r values and relative p values are shown in the table on the bottom right of each graph. (A) A 20-sera comparison of Euroimmun QuantiVac (EI) with BY2-RBD test. (B) A 20-sera comparison of EI with the NB-RBD test. (C) A 20-sera comparison of BY2- with NB-RBD tests.