key: cord-0845616-70gzbcy4 authors: De Marco Verissimo, C.; O'Brien, C.; Lopez Corrales, J.; Dorey, A.; Cwiklinski, K.; Lalor, R.; Doyle, J. M.; Field, S.; Masterson, C.; Ribes Martinez, E.; Hughes, G.; Bergin, C.; Walshe, K.; McNicholas, B.; Laffey, J.; Dalton, J. P.; Kerr, C.; Doyle, S. title: Improved diagnosis of SARS-CoV-2 by using Nucleoprotein and Spike protein fragment 2 in quantitative dual ELISA tests date: 2021-04-09 journal: nan DOI: 10.1101/2021.04.07.21255024 sha: 3f80148829ac98d8d151ecd739c2808f92c18060 doc_id: 845616 cord_uid: 70gzbcy4 The novel Coronavirus, SARS-CoV-2, is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and Spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative ELISA assays that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive COVID-19 cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients that had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus. SARS-CoV-2 is a newly emerging member of the Coronaviridae (CoV) family, responsible opacities which aid in diagnosis. Part of the strategy to identify those exposed to infection 80 and with an established immune response includes serological tests to detect antibodies to 81 SARS-CoV-2. Furthermore, qRT-PCR and serological testing can be used in combination, 82 which was demonstrated to significantly increase the viral detection rates [8, 11] . 83 In general, it takes several days for individuals to build an immune response to the 84 virus. Antibodies to SARS-CoV-2 antigens are detectable in less than 40% of patients within 85 one week of the onset of symptoms, but rapidly increase in the following days [12, 13] . 86 Longitudinal studies are necessary to characterize the longevity of the antibodies in 87 convalescent individuals and to determine if these confer protective immunity [13, 14] , and 88 more specifically, to identify which antigen(s) this immunity is directed towards [15, 16] . 89 This knowledge is critical to assess the epidemiological context of the COVID-19 pandemic 90 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.21255024 doi: medRxiv preprint 4 and for the differentiation between exposed and non-exposed individuals to define the 91 locality and distribution of infection that can guide pandemic control measures such as social 92 distancing. It is also important for vaccine design and the evaluation of vaccine candidates. Selecting viral antigens 121 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In the present study, the full length SARS-CoV-2 Spike protein (Spro, ~135 kDa) and four 122 different sections, Spike protein fragment 1 (S1frag, 1-686, ~75 kDa), Spike protein fragment 123 2 (S2frag, 687-1273, ~54 kDa), the Spike protein fragment 2 prime region (S2Prime, 816-124 1273, ~38 kDa) and the receptor binding domain (RBD, 319-542, ~29 kDa) were selected for 125 recombinant expression (Fig 1A and B ) (see also [15] ). The entire Npro sequence (2-1269; 50 126 kDa) was synthesised for recombinant expression (Fig 1C) . supplemented with 50 µg/mL kanamycin, or 100 µg/mL ampicillin for Npro, was inoculated 137 from the glycerol stock and incubated shaking at 37°C overnight. The culture was then 138 diluted in fresh LB broth supplemented with the appropriate antibiotic, incubated at 37°C to 139 OD600 0.6 and protein expression induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside 140 (IPTG; ThermoFisher Scientific) for 4 hr at 30°C. For Npro the cultures were induced with 141 0.5 mM IPTG for 3 hr at 37°C. Following centrifugation at 10,000 x g for 10 min at 4°C, the 142 bacterial pellets were re-suspended in 10 mL ST buffer (10 mM Tris, 150 mM NaCl, pH 8.0) 143 and stored at -20 °C. DTT was added to the pellets, which were then sonicated twice for 2 min, 40% amplitude. The samples were centrifuged 15,000 x g at 4°C for 30 min and the resulting supernatant was 152 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.21255024 doi: medRxiv preprint 8 hospital research ethics committee). All participants provided written informed consent prior 215 to the study or assent followed by informed consent once able for patients admitted to the 216 ICU where informed consent was not possible. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Fig S1) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In order to assess the sensitivity of the Npro and S2Frag ELISA tests against a commercially 309 available test, serum samples were tested in parallel using the Abbott ARCHITECT ELISA 310 (ARCHITECT SARS-CoV-2) which employs Npro as its antigen (Fig 6) . Using the 42 qRT-311 PCR-confirmed positive serum samples, the data showed complete agreement between the 312 Abbott ARCHITECT and the Npro ELISA test developed in this study (i.e. 85.4% 313 sensitivity) (Fig 6A) . However, four patients that were negative and two that were positive by 314 both these tests showed a contrasting result when evaluated by the S2Frag-ELISA (Fig 6B) . 315 Combining the data for the Npro-ELISA and S2Frag-ELISA tests increased the sensitivity of 316 detection to 95.2% (Table 1) . When the ARCHITECT test was employed to screen plasma samples from the 98 318 healthcare workers suspected of exposure to SARS-CoV-2 only six (6.1%) of these samples 319 proved positive for SARS-CoV-2. In contrast, 14 (14.3%) individuals were identified as 320 positive using our in-house ELISA tests (Fig 6C and D antibodies against Npro and S2Frag (Fig 7) . The data shows that COVID-19 hospitalized Moreover, antibodies to Npro could be detected from day seven after onset of symptoms, 331 whilst antibodies to S2Frag were only detected from day 11 (Supplementary Table S1 ). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. the antibody responses to both Npro and S2frag begin to decline (Fig 7A and B) . Since the 338 S2Frag stimulates a weaker response, specific antibodies to this antigen dropped to levels Table S1 ). CoV-2 respond to the main viral antigens, six viral proteins were recombinantly expressed: 366 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.21255024 doi: medRxiv preprint 13 the full-length Spro and four different sub-segments, i.e. S1Frag, S2Frag, S2Prime and RBD 367 (Fig 1) , and the Npro. Through Western blot analysis, variability in the immune response to 368 each antigen between individuals was observed ( Supplementary Fig S2) . At least 85% of the 369 COVID-19 positive individuals tested in this study showed a consistent and strong antibody 370 response to Npro. However, our data shows that 7% of the COVID-19 non-hospitalized to remain elevated for variable periods, seven days to more than 48 days, and serve to protect 398 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.21255024 doi: medRxiv preprint the individual against reinfection [8, 24] . In our study we found that infected individuals did 399 not sustain high antibody levels to SARS-CoV-2 antigens for long periods, and even 400 individuals that developed severe disease and required intensive care exhibited antibody 401 declines, mainly those specific to S2Frag, after three weeks (Fig 7 A and Npro while 10% only recognised S2Frag alone (Fig 4A) . These antigen-selective immune 424 responses were confirmed using Western blot analysis (Fig 5) . A similar observation was (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.21255024 doi: medRxiv preprint When we analysed the antibody response of 15 patients that were hospitalized with 431 COVID-19 we found that the Npro OD/CO values for ICU and non-ICU patients were 10.34 432 and 6.82 (P < 0.05), respectively (Supplementary Table S1 ), while the values for S2Frag did 433 not vary significantly between each group (OD/CO = 1.93 and 2.22, respectively). Sun et al. [41] also found that anti-Npro IgG antibodies were significantly higher in ICU patients 435 compared to non-ICU patients. Therefore, anti-Npro antibodies could be an indicator of 436 disease severity, although we did not find a correlation between antibody levels and age of 437 the patients in our study (Supplementary Fig S3) . In the present study, we compared our ELISAs results with the commercially- (Fig 5 and Supplementary Fig S4) . A recent longitudinal seroprevalence study found found that their in-house Spro and Npro-based ELISAs performed with the highest sensitivity 458 and specificity. In all, our results indicate that our in-house quantitative ELISA performs 459 better than the non-quantitative ARCHITECT tests using a single Npro protein and can be 460 improved by running the dual ELISA assay with S2Frag. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.21255024 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.07.21255024 doi: medRxiv preprint The receptor binding domain of the viral spike protein is an 580 immunodominant and highly specific target of antibodies in SARS-CoV-2 patients Convergent antibody responses to SARS-CoV-2 in convalescent 583 individuals Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked 585 Immunosorbent Assays for Detecting Antibodies against SARS-CoV-2 Performance of nucleocapsid and spike-based SARS CoV-2 serologic assays Predicting infectious SARS-CoV-2 from diagnostic samples Antibody Status and Incidence of SARS-CoV-2 Infection in Health 592 Care Workers COVID-19 RT-PCR TEST SARS-CoV-2: Pathogenesis, and Advancements in Diagnostics and 596 Treatment Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 598 patients No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9 A: The amino acid sequence of 622 the Spike protein (1273 residues). Residues in bold and underlined represent the signal 623 peptide. Residues highlighted in black (319-542) represent the receptor-binding domain 624 (RBD) Residues in bold represent the transmembrane and endo-domain (1214-629 1273). B: Schematic representation of the Spike protein and its various portions 630 recombinantly expressed in the present study (see [15]). C: Nucleocapsid protein sequence 631 (Npro, residue 2-1269) used for recombinant expression in Echerichia coli A: Solubilisation of the S2Frag protein. P1, E. coli pellet after induction with IPTG 635 for 4 hr at 30°C; S1, supernatant containing soluble proteins after pellet digestion with 0 mg/mL of lysozyme; S2, supernatant containing insoluble proteins after pellet digestion with 637 lysis buffer containing 1% SDS. B: S2Frag purification over Ni-NTA beads column. ST, 638 supernatant total diluted Molecular weight marker in kDa. C. 4-20% SDS-PAGE analysis of recombinant SARS-CoV-640 2 nucleocapsid protein (Npro) following HisTrap HP columns No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9 C-F: The antibodies response to Npro and S2frag of different individuals positive 668 for SARS-CoV-2. Individual ELISA tests results are shown for each sample as positive or 669 negative for SARS Contrasting results obtained by the commercially available Abbott 672 ARCHITECT antibody test and the ELISA antibody test developed in the current 673 study. A and B, the agreement of SARS-CoV-2 diagnostic of 42 RT-PCR positive SARS CoV-2 individuals assessed using Npro and S2frag ELISA test or the commercially available CoV-2 diagnostic of 98 676 suspected SARS-CoV-2 individuals assessed using Npro and S2frag ELISA test or the 677 commercially available Abbott ARCHITECT antibody test. Samples were categorised 678 according to the positive or negative result of the commercially available Abbott 679 ARCHITECT test. Individual results for Npro and S2Frag ELISA test presented as optical 680 density (OD 450 nm) divided by the calculated cut-off (CO) (■ sera were negative for 681 antibodies by ELISA; • sera were positive for antibodies by ELISA). The cut-off value for 682 Nucleocapsid protein (Npro) and (B) Subunit 2 of spike protein 687 (S2Frag) in ELISA assays. The antibody response of each patient was assessed at two 688 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 9 No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity