SARS-CoV-2 Antigens Expressed in Plants Detect Antibody Responses in COVID-19 Patients

Abstract

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year 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 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 6 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 optical density (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 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers.Conclusion: 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.

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

“…We expressed an RBD variant that was previously produced in mammalian or insect cells ( Stadlbauer et al, 2020 ; Klausberger et al, 2021 ) and observed low expression levels and high amounts of homodimers. Recombinant SARS-CoV-2 RBD variants have recently been produced in N. benthamiana using different transient expression systems ( Diego-Martin et al, 2020 ; Mamedov et al, 2020 ; Rattanapisit et al, 2020 ; Makatsa et al, 2021 ). Diego-Martin et al (2020) used a MagnICON-based expression vector to produce a His-tagged RBD variant carrying the same amino acid region (R319-F541) as present in our RBD.…”

The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests

“…Plants are a good alternative for the production of eukaryotic proteins, particularly using transient expression systems based on viral vectors (Gleba et al, 2007;Pogue et al, 2010). In fact, some SARS-CoV-2 proteins have already been made in plants (Burnett and Burnett, 2019;Sainsbury, 2020;Makatsa et al, 2021;Siriwattananon et al, 2021), including N (Diego-Martin et al, 2020, although the suitability of plant-made N for detection and diagnosis of antibodies in samples of human sera has not been tested yet. In addition to these technical advantages, plant production of recombinant proteins presents an important economic advantage in terms of production costs, which becomes an important aspect when massive production is required.…”

“…Very recently, another study has been published showing the usefulness of other plant-produced SARS-CoV-2-derived antigens, the S (or "spike") protein and its Receptor-Binding Domain (RBD) (Makatsa et al, 2021). Apart from the antigens used, the main difference with our study is that Makatsa et al used a lower number of samples in the validation (77 positive, 58 negative), that IgA and IgM were also tested, and that IgA/IgG in saliva were also evaluated.…”

Performance of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies in South Africa

“…This ELISA method was adapted from an FDA-approved protocol [ 42 ], with the modification that S1 was cloned, expressed and purified from Nicotiana benthamiana plants. Following coating of 96-well plates (Nunc MaxiSorp, Thermo Fisher, Waltham, Massachusetts, United States) with purified recombinant S1, individual patient samples were diluted 1:50 in PBS and added for 2 h at room temperature; detection was via a goat anti-human IgG (Fc-specific) peroxidase conjugate, as described [ 41 ] Thresholds for true positive signal were determined using the mean plus 2 standard deviations of the data from pre-pandemic controls (n = 58).…”

“…Enzyme-linked immunosorbent assay (ELISA). ELISA assays were carried out to detect IgG binding to the S1 domain of the spike protein, as previously described [41]. This ELISA method was adapted from an FDA-approved protocol [42], with the modification that S1 was cloned, expressed and purified from Nicotiana benthamiana plants.…”