Researchers in South Africa have developed a serological assay that readily detects antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using recombinant viral proteins produced in plants.
The assay enabled the detection of antibodies directed at antigens of the SARS-CoV-2 spike protein that were expressed in and derived from the Nicotiana benthamiana plant – colloquially known as benth or benthi and a close relative of tobacco and species of Nicotiana indigenous to Australia. N. benthamiana is also a common plant used for "pharming" of monoclonal antibodies and other recombinant proteins.
Nicotiana benthamiana. Image Credit: Hanjo Hellmann / Shutterstock
The spike protein is the membrane structure SARS-CoV-2 uses to bind the human host cell receptor angiotensin-converting enzyme 2 (ACE2) and gain viral entry.
The indirect enzyme-linked immunosorbent assay (ELISA) developed here enabled robust detection of antibodies against portions of this spike protein in samples taken from patients who had previously been infected with SARS-CoV-2.
Serological assays using such plant-based expression systems could represent convenient, cost-effective tools for measuring seroprevalence and protective immunity against SARS-CoV-2 in less-resourced settings, say Wendy Burgers (University of Cape Town, South Africa) and colleagues.
A pre-print version of the paper is available on the server medRxiv*, while the article undergoes peer review.
Serological tests are urgently needed to track the pandemic’s spread
The COVID-19 pandemic - caused by SARS-CoV-2 - has rapidly swept the globe since it began in Wuhan, China, late last year. The virus, which has now infected more than 18.65 million people and caused more than 703,000 deaths, continues to pose significant global health and socioeconomic threat.
“There is a critical need for the development of serological tests to detect SARS-CoV-2 antibodies,” write Burgers and team. “Population seroprevalence studies to estimate the extent of pandemic spread in communities, and studies defining protective immunity to SARS-CoV-2, all depend on reliable serological tests.”
Ideally, these tests also need to be cost-effective, so that their application in low-income settings is feasible, adds the team.
Generating recombinant viral proteins
The purified recombinant proteins required for serological assays can be generated using various different protein expression systems, including bacterial, mammalian, yeast, insect, and plant-based systems.
Plant-based systems offer several advantages over the more widely used conventional mammalian- and insect-based systems. In particular, they are rapid and cost-effective since they do not require expensive media or growth conditions, which is especially desirable in low-income settings. They also offer advantages over bacterial or yeast-based systems, since they support post-translational modifications similar to those of mammalian cell systems.
Historically, the main disadvantage associated with plant-based protein expression systems has been low yield, but advances in plant technology have helped to overcome this challenge.
The authors say Nicotiana benthamiana is preferred as a protein expression system due to its rapid generation of biomass, defective post-transcriptional gene silencing system, and a wide range of engineering strategies that all help to overcome the problem of low yield.
New ELISA assay uses viral proteins expressed in Nicotiana benthamiana
The S1 subunit of the viral spike protein is highly immunogenic, and its RBD portion is the main target of neutralizing antibodies following infection, meaning these spike protein antigens have become an important focus of many serological studies.
Now, Burgers and colleagues have developed an indirect ELISA assay using spike S1, and RBD antigens from SARS-CoV-2 expressed in Nicotiana benthamiana.
The team collected serum samples from 77 South African convalescent patients who had previously been infected with SARS-CoV-2, as confirmed by polymerase chain reaction (PCR) testing. Samples were taken a median of six weeks following diagnosis, and most patients had experienced mild-to-moderate COVID-19.
Virus-specific antibodies were readily detected
Using the newly developed ELISA with plant-derived viral antigens, the researchers showed that SARS-CoV-2-specific immunoglobulins (Igs) were readily detectable in the samples.
Among the 77 participants, S1-specific IgG reactivity was detected in 51 (66%) cases, and RBD-specific IgG reactivity was detected in 48 (62%) cases.
Notably, the results were highly concordant with a validated, high-sensitivity commercial ELISA assay, says the team.
“Robust detection of SARS-CoV-2 specific antibodies”
“Our study demonstrates that recombinant SARS-CoV-2 proteins produced in plants enable the robust detection of SARS-CoV-2-specific antibodies,” write Burgers and colleagues.
The authors say one of the main aims was to develop a cost-effective assay that could be used for both large-scale seroepidemiology and research into SARS-CoV-2 humoral immunity.
“We achieved this by making use of plants for the production of viral antigens, which has the benefit of rapid scale-up, and sourcing reagents that were available locally and thus available at a lower cost,” they write.
“Serological studies in a setting like ours, in South Africa, where comorbidities such as HIV and TB are highly prevalent, are underexplored and can benefit from this assay,” concludes the team.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.