key: cord-0824131-3hv4isqf authors: Rump, Airi; Risti, Robert; Kristal, Mai-Ly; Reut, Jekaterina; Syritski, Vitali; Lookene, Aivar; Boudinot, Sirje Ruutel title: Dual ELISA using SARS-CoV-2 nucleocapsid protein produced in E. coli and CHO cells reveals epitope masking by N-glycosylation date: 2020-11-20 journal: Biochem Biophys Res Commun DOI: 10.1016/j.bbrc.2020.11.060 sha: 9cee86b075ccec62c051538188bddc2ec4e133f5 doc_id: 824131 cord_uid: 3hv4isqf Spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-SP, SARS-CoV-2-NP) are the main immunogenic targets for antibodies. We herein demonstrate that the glycosylation of SARS-CoV-2-NP masks some of its antibody epitopes. In many cases, this can lead to false-negative serological tests. Deglycosylation of SARS-CoV-2-NP significantly increased the number of positive tests. The glycosylation pattern analysis of this protein revealed that the putative N-linked glycosylation sites, at the amino acid positions 48 and 270, co-located with two of the main immunodominant B cell epitopes. The most used SARS-CoV-2 proteins as antigens in the serological assays are nucleocapsid protein (SARS-CoV-2-NP), full trans-membrane spike protein (SARS-CoV-2-SP), and spike protein subunits RBD due to their ability to trigger a dominant and long-lasting immune response [1] . The NP is the most expressed of these immunodominant proteins. Antibodies (Abs) directed against the SARS-CoV-2-NP are longer-lived and occur in greater abundance in COVID-19 patients than antibodies targeting other viral proteins [2, 3] . Lack of anti-SARS-CoV-2 Abs has been reported from clinically confirmed cases, raising issues about sensitivity and reproducibility of serological tests. One reason for it may be the quality and composition of the antigen used in serological assays. Unfortunately, the nature of the antigens utilized for in vitro serological assays is generally not accurately documented. Several serological assays have been recently developed to detect serum immunoglobulins (Ig) directed against SARS-CoV-2, mainly based on enzymelinked immunosorbent assays (ELISA). Serological assays are critical to assess the proportion of the population that has been exposed to the virus, the heterogeneity of the Ab response, and to determine its duration. The correlation between the presence of Abs against the virus in the serum and the protection against secondary infection is also critical. In addition to the high proportion of false-negative RT-qPCR tests due to difficult swab sampling [4] , a surprising lack of anti-SARS-CoV-2 Abs has been reported, even from some clinically confirmed cases. These observations raised issues about sensitivity and reproducibility of serological tests, as well as about the quality of Ab response induced by the infection. Most persons infected with SARS-CoV-2 display an Ab response between day 10 and day 21 after infection [5, 6] . Although the duration of the Ab response to SARS-CoV-2 is still unknown, Ab levels to other coronaviruses wane over time in the range of 12 -52 weeks from the onset of symptoms [7] . It should be noted, the fact that Ab levels are falling does not mean that our immune system is not capable of inducing a protective immune response. Reinfections with seasonal coronaviruses (HCoV-OC43 and HCoV-HKU1) occur in nature, usually within three years [8] . Although the primary infection with SARS-CoV-2 was shown to protect Rhesus macaques from the subsequent challenge [9] , demonstration of long-term protection in humans from the same virus will require future studies. The SARS-CoV-2-NP is highly basic and forms a helical ribonucleoprotein complex with viral RNA [10] . It has a high degree of sequence homology between different beta coronavirus proteins [11] , regulates the replication and packaging of the viral genome that is essential for the viability of the virus. The SARS-CoV-2-NP is highly immunogenic and well expressed in antigen-presenting cells [15] , initiating a good T cell response. Using the conservation profile of the protein, Forcelloni et al, identified several conserved potentially immunogenic regions of SARS-CoV-2-SP and SARS-CoV-2-NP. The SARS-CoV-2 derived B cell and T cell epitopes of those two proteins were further studied, and the location of the main immunodominant epitopes was finally determined [12, 13] . In this work, we have developed a dual ELISA test against SARS-CoV-2-NP and showed the critical importance of epitope unmasking by deglycosylation when using a protein produced in a mammalian system. Indeed, the N glycosylation sites located at positions 48 and 270 coincide with important B cell epitopes. Our data also illustrates the potential importance of controls for contaminants in antigen preparation from the bacterial extract. Blood samples used in the current study were obtained from healthy donors in We tested 423 sera derived from blood donors and volunteers. Our set of samples does not represent well the whole population of Estonia. This sampling leads to a set of people aged from 19 to 95 years and about 90% of them live or work in Tallinn. Blood samples were collected from May to July 2020, possible infection time (when it was known) and clinical signs were noted, since there was a tight J o u r n a l P r e -p r o o f correlation between positivity of a serological test and time passed since Covid19 till blood sampling. Specifically, most serum samples collected after 2-3 months had lost the capacity to be detected positive in our ELISA test. The total Ab response against SARS-CoV-2 in plasma samples was tested using in-house ELISA. Briefly, the ELISA for total Ab detection was developed based on Deglycosylation under denaturing conditions was carried out by adding 1 μl of 0.2% SDS and 100 mM DTT mixture to 9 μl SARS-CoV-2-NP (1 mg/ml) in PBS (pH 7.4) buffer. The protein was denatured for 10 min at 99 °C, and the solution was subsequently allowed to cool to room temperature. Following, 1 μl of 15% Triton X-100 and 1 μl of PNGase F (500 U/ml, Sigma-Aldrich) were added. Deglycosylation was carried out for 2 hours at 37 °C, after which the solution was incubated at 99 °C for 5 min. For deglycosylation under native conditions, 2.5 μl of PNGase F was added to 10 μl SARS-CoV-2 NP and the mixture was incubated for 20 h at 37 °C. We tested 423 sera using ELISA based on 2 different recombinants and His- Next, we analysed the glycosylation pattern of SARS-CoV-2-NP using NetNGlyc However, approximately 60% of the human autoantigens are significantly homologous to the microbial proteins in their amino acid sequences. The homologous sequences between the humans and the microbes also suggest a probability that the autoantibodies in autoimmune diseases are derived from the host immune response to the commensal microbes [14] . This may partly explain the reason for the presence of anti-E. coli Abs in circulation in humans and the need to have respective controls when bacterially expressed proteins are used in serological tests. In this report, we have shown the interest of dual ELISA in serological analysis against SARS-CoV-2 proteins and addressed the possible mechanism leading to false-negative tests. Dual ELISA using in parallel two of the SARS-CoV-2-NPs produced in bacteria and eukaryotic cells can be useful for increasing the accuracy of the serological analyses of anti-SARS-CoV-2 Abs. However, epitope unmasking by N glycosylation has to be performed by proteins produced in eukaryotic cells. Mutation of N glycosylation can also be considered, but it may change the epitope. The results of this study also suggest that many Covid-19 infected people produce Abs whose epitopes are located at or near to the glycosylation sites of SARS-CoV-2-NP. We thank Dr. Ave Lellep, Director of Blood Centre, North Estonia Medical Centre, Dr Ülle Uustalu and Annely Rüütel for the donor blood samples and we would like to thank all our blood donors and volunteers. This work was supported by the Estonian Research Council grant COVSG34. 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