SARS-CoV-2 antigens expressed in plants detect antibody responses in COVID-19 patients

Abstract

Background: The SARS-CoV-2 pandemic has swept the world and poses a significant global threat to lives and livelihoods, with over 16 million confirmed cases and at least 650 000 deaths from COVID-19 in the first 7 months of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings. Methods: We established an indirect enzyme-linked immunosorbent assay (ELISA) using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n=77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of six weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n=58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva. Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and OD values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers. Conclusions: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

Rapid expression of COVID-19 proteins by transient expression in tobacco

“…Other recent reports (Diego-Martin et al, 2020;Makatsa et al, 2020;Margolin et al, 2020;Rattanapisit et al, 2020) indicate similar results. The rapid and high level of expression makes this an attractive system for larger scale production for research, or development of tests or of vaccines.…”

“…We successfully purified recombinant spike and Nucleocapsid SARS-CoV-2 proteins from N. benthamiana at a concentration comparable to other studies (Diego-Martin et al, 2020;Makatsa et al, 2020;Margolin et al, 2020;Rattanapisit et al, 2020). The spike ectodomain was purified in both monomeric and trimeric forms, suggesting that it was folding properly.…”

“…Together, our data support the idea that plants are a suitable eukaryotic system for recombinant production of SARS-CoV-2 proteins. Indeed, other studies have used plant-produced SARS-CoV2 antigens in ELISA assays to detect human antibodies, as well as monoclonal antibodies produced in planta, demonstrating their efficacy in diagnostics (Diego-Martin et al, 2020;Makatsa et al, 2020;Rattanapisit et al, 2020).…”

Validation of Roche immunoassay for severe acute respiratory coronavirus 2 in South Africa

“…To address the potential misclassification of samples as false negatives, that were in fact true negatives in patients with positive RT-PCR that did not produce antibodies, a comparison was performed against a composite serology platform consisting of a Western blot and immunofluorescence, that utilised insect cell-expressed recombinant full length N and S antigens and an in-house serology ELISA assay utilising plant-based recombinant spike 1 (S1) and receptor binding domain (RBD) antigens, in collaboration with another large university. 21 After completion on the Roche platform, all samples were sent for analysis on the composite serology platform.…”

Contributions of the international plant science community to the fight against human infectious diseases – part 1: epidemic and pandemic diseases

“…The S protein has been produced in mammalian cells with a yield of ˜2 mg/ml whereas the much smaller RBD is expressed at higher levels, up to 25 mg/ml (Amanat et al ., 2020 ; Zhao et al., 2020 ). Many groups are now producing variants of this heavily glycosylated protein in plant cell suspension cultures or whole plants by transient expression or in transgenic lines (Makatsa et al., 2020 ; authors’ unpublished data). Differences in the glycan profiles of S protein variants produced in plants may affect the immunogenicity of different vaccines, contributing to differences in efficacy.…”