Abstract
BackgroundThe fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms.MethodsWe expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test.ResultsThe performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05–98.44) and diagnostic specificity of 96.37 (95% CI: 93.05–98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892–0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA).ConclusionThis study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.
Highlights
The present SARS-CoV-2 coronavirus pandemic is the most important global sanitary crisis humankind has faced in the last 100 years
Production of Infectious RNA The DNA sequence of SARSCoV-2 N (Gen Bank YP_009724397.2, aa 231–419), designed with His-tag C-terminal and optimized to achieve its correct expression in N. benthamiana plants was successfully cloned into TMVPSN vector and the recombinant Tobacco mosaic virus (TMV)-PSN-NCP2 clone was confirmed by sequencing
In vitro 6 kb RNA transcripts produced from the recombinant TMV-PSN-NCP2 clone were used to infect plants by mechanical inoculation (Figure 2A)
Summary
The present SARS-CoV-2 coronavirus pandemic is the most important global sanitary crisis humankind has faced in the last 100 years. Serological tests are essential tools for the detection of antibody responses to SARS-CoV-2 infection They play an essential role in diagnosis and in the evaluation of the seroprevalence in exposed populations and in research studies aimed to provide answers to still open key questions regarding the complete characterization of the course of the infection such as, for instance, the duration of antibodies. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms
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