key: cord-0963961-yvszj8af authors: Polvere, Immacolata; Voccola, Serena; Cardinale, Gaetano; Fumi, Maurizio; Aquila, Francesca; Parrella, Alfredina; Madera, Jessica Raffaella; Stilo, Romania; Vito, Pasquale; Zotti, Tiziana title: A peptide-based assay discriminates individual antibody response to SARS-CoV-2 date: 2021-02-05 journal: Genes Dis DOI: 10.1016/j.gendis.2021.01.008 sha: b10ebb1403044c3d4e385026a97de653ff0041d2 doc_id: 963961 cord_uid: yvszj8af SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. Understanding the antibody response to SARS-CoV-2 is crucial for the development of vaccines, therapeutics and public health interventions. However, lack of consistency in methods used to monitor antibody response to SARS-CoV-2 leaves some uncertainty in our fine understanding of the human antibody response mounted following SARS-CoV-2 infection. We developed a peptide-based enzyme-linked immunosorbent assay (ELISA) by selecting 7 synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of SARS-CoV-2, which effectively detects the antibody response mounted by all COVID-19 convalescent tested. Strikingly, the assay shows a profound difference in antibody response among individual subjects, which may have a significant impact on disease severity. Together, our results define an efficient and specific serological assay to consistently measure the antibody response following SARS-CoV-2 infection, as well as help the design of vaccine and therapeuticals for prevention and treatment of COVID-19. A peptide-based assay discriminates individual antibody response to 1 SARS-CoV-2 2 3 Immacolata Polvere 1,2 , Serena Voccola 1,2 , Gaetano Cardinale 2 , Maurizio Fumi 3 , 4 Francesca Aquila 3 , Alfredina Parrella 2 , Jessica Raffaella Madera 1 , Romania Stilo 1 , 5 Pasquale SARS-CoV-2 is crucial for the development of vaccines, therapeutics and public 20 health interventions. However, lack of consistency in methods used to monitor 21 antibody response to SARS-CoV-2 leaves some uncertainty in our fine 22 understanding of the human antibody response mounted following SARS-CoV-2 23 infection. We developed a peptide-based enzyme-linked immunosorbent assay 24 (ELISA) by selecting 7 synthetic peptides from the spike, membrane, and 25 nucleocapsid protein sequences of SARS-CoV-2, which effectively detects the 26 antibody response mounted by all COVID-19 convalescent tested. Strikingly, the 27 assay shows a profound difference in antibody response among individual subjects, 28 which may have a significant impact on disease severity. Together, our results 29 define an efficient and specific serological assay to consistently measure the 30 antibody response following SARS-CoV-2 infection, as well as help the design of 31 vaccine and therapeuticals for prevention and treatment of COVID-19. 32 Keywords: SARS-Cov-2, COVID-19, Antibodies, Assay, ELISA, Peptides. 33 34 The novel human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 36 is the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic (1, 37 2). Limited pre-existing immunity is supposed to account, at least in part, for the 38 extraordinary escalation of cases worldwide. Understanding of the human 39 antibody response to SARS-CoV-2 infection is relevant to vaccines development 40 and planning of vaccination strategies, and to guide appropriate design, 41 implementation and interpretation of serological assays for surveillance purposes. 42 As of today, more than 200 studies have been published that analyse the human 43 antibody response following SARS-CoV-2 infection. Indeed, the vast majority of 44 infected individuals mount a SARS-CoV-2-specific antibody response, with many 45 studies reporting 100% seroconversion (3-9). However, assays used to detect and 46 quantify antibody response were diverse, with target antigens including spike (S), 47 S1 and S2 subunits, receptor binding domain (RBD) and nucleocapsid (N). Indeed, 48 the lack of consistency in methods used leaves some uncertainty in our complete 49 understanding of the human antibody response mounted following SARS-CoV-2 50 infection. For example, mean or median time to seroconversion for IgG has been 51 reported to range from 12-15 days post symptom onset (3, 10, 11-16) with wide 52 variation in first to last detection from 4-73 days post symptom onset. Time to 53 seroconversion for IgA has also been measured in several studies, ranging from 54 4-24 days post symptom onset, although most were within 4-11 days (17-19), 55 including two reports of 24 days to first detection (8, 9). Therefore, a standardized 56 assay based on synthetic reagents would greatly help the uniformity of 57 measurement of serological responses. Here we present an ELISA for detection of 58 anti-SARS-Cov-2 antibodies that has synthetic peptide antigens as the 59 immunoabsorbent solid-phase. Strikingly, the assay finds significant differences in 60 antibody specificity in the sera of COVID-19 convalescents, indicating that each 61 antibody responses in SARS-CoV-2 infected patients has an individual repertoire 62 feature. Results and Discussion 65 10 peptides predicted to be immunodominant by the BebiPred 2.0 algorithm (6) 66 were synthesized as candidate antigens from the S, M and N proteins of 67 SARS-Cov-2 Wuhan-Hu-1 strain (GeneBank: MN908947) (Table I and 68 Supplementary Fig. 1 ). Candidate peptides were tested by ELISA procedure for 69 serologic reactivity to a panel of serum samples from 24 COVID-19 convalescents 70 (COVIDpos1-COVIDpos24) treated at San Pio Hospital in Benevento, Italy, in May 71 Table 1 ). For the peptide-based SARS-Cov-2 ELISA, wells 72 of microtiter plates were coated with 2 μg/mL of a single or a mixture of the S, M, 73 and N protein-derived peptides, and serologic reactivities were determined. Results 74 were scored on the basis of the signal/cutoff (S/C) ratio, where cutoff absorbance 75 was determined from the mean of four pre-2019 sera plus 3 standard deviations. No 76 reactivity was observed towards peptides n.1, n. 4 and n. 9, which were therefore 77 excluded from further analysis (data not shown). 78 The results shown Fig. 1 and summarized in Table II indicate that all convalescent 79 sera tested contain IgG that immuno-react with at least one of the 7 selected 80 J o u r n a l P r e -p r o o f peptides, individually or mixed. Only the serum of one convalescent (n. 15) 81 contained IgG for all 7 peptides. On the other hand, sera from 10 convalescents 82 immuno-react only with one peptide: sera n. 3, n. 22 and n. 23 with peptide n.7; 83 sera n. 9, n. 16 and n. 17 with peptide n. 10; sera n. 10, n. 14 and n. 21 with 84 peptide n. 3 and serum n. 19 with peptide n.1. Peptide n. 3 (M1-24), n. 7 (N153-85 172) and n. 10 (S524-598) are the ones most frequently seen by the tested sera. 86 Competition experiments, conducted using in soluble form the coated peptide or 87 irrelevant peptides confirmed the specificity of the sera response of sera to the 88 coated peptides (Supplementary Fig. 2) . 89 The same type of assay was subsequently conducted on a cohort of 23 additional 90 convalescent sera collected in October 2020 (COVIDpos101-COVIDpos123), with 91 consistent results (Supplementary Table 1 and Supplementary Fig. 3) . 92 The selected peptides were also tested for IgA immunoractivity (Fig. 2 and Table 95 III). Similarly to the IgG response, the IgA response also appears varied among Table 1 159 Clinical data of the convalescents analyzed for the study. 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 250 251 252 253 254 255 256 257 Table 3 . Summary of IgA sero-reactivities shown in Fig. 2 . HRP-conjugated goat anti-human immunoglobulins were added and 295 incubated for 1 h at RT in 2,5% BSA dissolved in TBST for 50 minutes at 296 room temperature with agitation. These secondary antibodies were 297 diluted 1:60000 for goat anti-Human IgG (MERK) and 1:50000 for goat 298 anti-human IgA (SIGMA). Unbound antibody was removed by washing a 299 six times with 300 µl/well PBS containing 0.05% Tween-20. After washing, 300 70 µl of freshly prepared TMB (Thermo Fisher) substrate diluted 1:3 in PBS 301 was added to every well and left for 15-30 min to allow colour to 302 develop. 0.3 M H2SO4 (70µl/well) was used to stop the reaction. 303 Absorbance readings at 450 nm were taken using a microplate reader 304 Seac-Sirio-S. Author Contributions: SV, GC, MF, FA, AP, PP and JRM performed the 307 experiments; TZ, IP and PP analysed the data; RS and PV wrote the 308 manuscript. 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