key: cord-0899433-ceh0d8s8
authors: Emmerich, P.; von Possel, R.; Hemmer, C. J.; Fritzsche, C.; Geerdes-Fenge, H.; Menge, B.; Messing, C.; Borchardt-Lohoelter, V.; Deschermeier, C.; Steinhagen, K.
title: Longitudinal detection of SARS-CoV-2-specific antibody responses with different serological methods
date: 2021-04-16
journal: nan
DOI: 10.1101/2021.04.16.21255608
sha: 0cbcac7daeac50a4a6cbaa86744d62fd3d61b1b1
doc_id: 899433
cord_uid: ceh0d8s8

Serological testing for anti-SARS-CoV-2 antibodies is used to detect ongoing or past SARS-CoV-2 infections. To study the kinetics of anti-SARS-CoV-2 antibodies and to assess the diagnostic performances of eight serological assays, we used 129 serum samples collected on known days post symptom onset (dpso) from 42 patients with PCR-confirmed COVID-19 and 54 serum samples from healthy blood donors, and children infected with seasonal coronaviruses. The sera were analyzed for the presence of IgG, IgM and IgA antibodies using indirect immunofluorescence testing (IIFT) based on SARS-CoV-2-infected cells. They were further tested for antibodies against the S1 domain of the SARS-CoV-2 spike protein (IgG, IgA) and against the viral nucleocapsid protein (IgG, IgM) using ELISA. The assay specificities were 94.4%-100%. The sensitivities varied largely between assays, reflecting their respective purposes. The sensitivities of IgA and IgM assays were highest between 11 and 20 dpso, whereas the sensitivities of IgG assays peaked between 20 and 60 dpso. IIFT showed highest sensitivities due to the use of the whole SARS-CoV-2 as substrate and provided information whether or not the individual has been infected with SARS-CoV-2. ELISAs provided further information about both the prevalence and concentration of specific antibodies against selected antigens of SARS-CoV-2.

post symptom onset (dpso) from 42 patients with PCR-confirmed COVID-19 and 54 serum 23 samples from healthy blood donors, and children infected with seasonal coronaviruses. 24

The sera were analyzed for the presence of IgG, IgM and IgA antibodies using indirect 25 immunofluorescence testing (IIFT) based on SARS-CoV-2-infected cells. They were further 26 tested for antibodies against the S1 domain of the SARS-CoV-2 spike protein (IgG, IgA) and 27

against the viral nucleocapsid protein (IgG, IgM) using ELISA. 28

The assay specificities were 94.4%-100%. The sensitivities varied largely between assays, 29

reflecting their respective purposes. The sensitivities of IgA and IgM assays were highest 30 between 11 and 20 dpso, whereas the sensitivities of IgG assays peaked between 20 and 60 31 dpso.

IIFT showed highest sensitivities due to the use of the whole SARS-CoV-2 as substrate and 33

provided information whether or not the individual has been infected with SARS-CoV-2. ELISAs 34

provided further information about both the prevalence and concentration of specific antibodies 35

against selected antigens of SARS-CoV-2. 36

In the current pandemic, direct pathogen detection via reverse transcription and polymerase 38 chain reaction amplification as well as real-time detection (real-time RT-PCR) is the gold 39 standard for SARS-CoV-2 detection and enables early identification of acute SARS-CoV-2 40

infections. Serological testing for anti-SARS-CoV-2 antibodies is used to confirm ongoing or past 41

infections with SARS-CoV-2. The detection of antibodies enables confirmation of SARS-CoV-2 42 infection in patients with typical symptoms and in suspected (asymptomatic) cases. Analysis of 43

anti-SARS-CoV-2 antibodies is typically performed at an advanced stage of infection and thus 44 expands the time frame for COVID-19 diagnostics. 45

Seroconversion of anti-SARS-CoV-2 antibodies can occur at different points in time after virus 46 contact 1,2 . The features of immune responses to SARS-CoV-2 infections vary significantly 47 between individuals 3 , especially regarding the kinetics, immunoglobulin classes and antigen 48 specificity. In the majority of COVID-19 patients, anti-SARS-CoV-2 antibodies are detectable 49

within two weeks of infection 4-6 . Usually, specific IgM and IgA antibodies are detectable earlier 50 than specific IgG antibodies 5, 7, 8 . In individual cases, anti-SARS-CoV-2 antibodies are either only 51 detectable more than four weeks after onset of symptoms or not at all due to generally absent 52 antibody secretion 8-10 . 53

Anti-SARS-CoV-2 antibodies target different structural proteins of SARS-CoV-2. The main 54

immunogens are the spike and nucleocapsid proteins. The highly immunogenic S1 domain of the 55 spike protein of SARS-CoV-2 is a major target for neutralizing antibodies and is being used as the 56 antigen in many serological assays 11 . The immunologically relevant receptor binding domain 57

(RBD) represents another important target antigen for virus-neutralizing antibodies 12 . The 58 nucleocapsid protein (NCP) of SARS-CoV-2 is the antigen with the strongest immune dominance 59

among Coronaviridae 13 and contains diagnostically relevant epitopes of SARS-CoV-2. Previous 60 studies suggested heterogeneous binding antibody responses to S1/RBD and NCP viral 61 antigens 14 , and hence the presence of antibodies against one protein of SARS-CoV-2 does not 62

necessarily coincide with the presence of antibodies against another.

Current research is determined to illuminate kinetics of the humoral immune response against 64 SARS-CoV-2, potentially providing guidance on when to use serological tests effectively for 65 screening or monitoring of the infection. Results of serological tests can provide answers to 66 important epidemiological, clinical and virological questions concerning SARS-CoV-2, for 67

instance, on the traceability of infection chains and the role of asymptomatic or presymptomatic 68

transmission. Moreover, exact determination of the course of concentration of IgG antibodies 69

against SARS-CoV-2 before and after vaccination can provide valuable information on the 70 effectiveness of vaccination.

Currently, knowledge about SARS-CoV-2 antibody persistence is scarce, although it would help 72

to understand the possible role of humoral immunity in the protection against reinfection. The 73

aim of this research was to study the kinetics of antibodies against SARS-CoV-2 and to explore 74 the characteristic features of eight serological assays. 75

The sensitivities varied largely between assays ( Table 2 ). The IIFT revealed positive results for 123

anti-SARS-CoV-2 IgG, IgA and IgM antibodies in 94.6%, 72.9% and 65.9% of the patient samples, 124

respectively. The ELISAs detected specific antibodies against S1 IgG and IgA in 75.8% and 80.3% 125 of the patient samples, respectively. Anti-SARS-CoV-2 IgG and IgM antibodies against NCP were 126 detected in 82.0% and 19.8% of the patient samples, respectively. The specificity was 100% by 127

IIFT, Anti-SARS-CoV-2 ELISA (IgG, IgA) and Anti-SARS-CoV-2 NCP ELISA (IgM), while the four 128 remaining assays reached specificities between 92.9% and 97.6%. Cross-reactivities were not 129

observed.

The sensitivities of IgA and IgM assays were highest in the early phase of infection, while 132

positive results for IgG antibodies occurred most often in the intermediate phase (Table 3 , 133 Figure 1 ).

Positive results for anti-SARS-CoV-2 IgG antibodies against S1 reached a peak during the 135 intermediate phase of infection. In contrast, positive results for anti-SARS-CoV-2 IgA antibodies 136 as measured by IIFT showed an initial peak followed by a pronounced decrease after 60 dpso.

During the course of infection, the number of positive results for anti-SARS-CoV-2 IgM 138

antibodies dropped as measured both by ELISA and IIFT.

In the early phase of infection (11-20 dpso), IgG and IgA antibodies against S1 of SARS-CoV-2 140

were detected in 70.4% and 88.9% of the samples (n=29), respectively, while IgG and IgM 141

antibodies against NCP were detected in 86.2% and 50%, respectively. The IIFT detected SARS-142

CoV-2 specific IgG, IgA and IgM antibodies in 96.6%, 93.1% and 96.6% of the samples, 143

respectively. 144

In the intermediate phase of infection (21-60 dpso), IgG and IgA antibodies against S1 of SARS-145

CoV-2 were detected in 93.3% and 82.8% of the samples (n=31), respectively, while IgG and IgM 146

antibodies against NCP were detected in 96.8% and 12.9%, respectively. The IIFT detected 147 specific IgG, IgM and IgA antibodies in 100%, 87.1% and 100%, respectively. 148

In the late phase of infection (>60 dpso), IgG and IgA antibodies against S1 of SARS-CoV-2 were 149 detected in 85.1% and 80.5% of the samples (n=49), respectively, while IgG and IgM antibodies 150 against NCP were detected in 81.4% and 0%, respectively. The IIFT detected specific IgG, IgA 151

and IgM antibodies in 98%, 44.9% and 30.6%, respectively. 152

Overall, in samples taken later than 10 dpso, IgG and IgA antibodies against S1 of SARS-CoV-2 153

were detected in 83.7% and 83.5% of the samples (n=109), respectively, while IgG and IgM 154

antibodies against NCP were detected in 87.4% and 17%, respectively. The IIFT detected specific 155

IgG, IgA and IgM antibodies in 98.2%, 73.4% and 64.2%, respectively.

The Anti-SARS-CoV-2 QuantiVac ELISA (IgG) showed a high total agreement (98.9%, results with the Anti-SARS-CoV-2 ELISA (IgG) as well as a high correlation (r=0.98, p<0.001) 160

between (semi)quantitative results ( Figure 2 ). 161 . CC-BY-NC-ND 4.0 International license It is made available under a 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 16, 2021. ; https://doi.org/10.1101/2021.04.16.21255608 doi: medRxiv preprint

The aim of this research was to study long-term kinetics of antibodies against SARS-CoV-2 and to 163 assess the characteristic features of different serological methods. We present findings of the 164 temporal profiles of IgG, IgA and IgM antibody responses against SARS-CoV-2 determined in sera 165 from patients with mild to severe COVID-19 by means of eight serological assays. 166

The sensitivities varied between assays and phases of infection but corroborated that the 167 different substrates used in the assays serve different purposes. Due to the use of the whole 168

SARS-CoV-2 as substrate and the precharacterization by IIFT, the IgG IIFT showed, overall, the 169 highest sensitivity (94.6%) when testing all patient samples, independent of the infection phase 170 (Table 1) . 171

The ELISAs, in contrast, provide information about the prevalence of specific antibodies against 172 selected antigens of SARS-CoV-2. Hence, lower sensitivities of the S1-specific ELISAs compared 173

to the NCP-specific ELISA probably reflect the known fact that not all infected individuals 174

produce antibodies against the S1 domain of SARS-CoV-2 10 . Importantly, previous research 175

showed that responses of specific IgG against S1 and NCP may be heterogeneous between 176

individuals, time-delayed and do not always coincide with each other 8, 12, 14 . In the present panels, 177

the prevalence of specific IgG antibodies against NCP in the early phase of infection was higher 178 than that against S1 (Table 2) . However, the findings of the current study do not support 179

previous research by Herroelen et al who undertook a comparative evaluation of commercial 180 SARS-CoV-2 serological assays and observed no clear differences in the seroconversion kinetics 181

of antibodies targeting SARS-CoV-2 S and N protein epitopes between severe and milder SARS-182

CoV-2 infections. 183

Exclusively in the early phase of infection, the prevalence of specific IgA antibodies against S1 184 was higher than that of specific IgG antibodies against NCP as well as S1. This observation 185 reflects that of Okba et al 1 . However, it is contrary to a previous study that showed a higher 186 sensitivity of the Anti-SARS-CoV-2 IgG compared to the Anti-SARS-CoV-2 IgA ELISA in patient 187 samples taken later than fourteen dpso 19 , whereby the discrepancy might be due to 188

heterogeneous definitions regarding the early phase of infection. 189

The IgA IIFT showed a pronounced decrease in the antibody detection rate after 60 dpso, which 190

was not observed for the IgA ELISA (Table 2) . A possible explanation for this might be that the 191

IgA antibody response against the S1 protein largely remains constant, while the production of 192

IgA antibodies against other antigens of SARS-CoV-2 decreases. 193

More patients were seropositive for IgM by IIFT than by ELISA (Table 1, Table 2 ), which could be 194

accounted for by a low sensitivity of the NCP IgM ELISA, warranting further investigations.

However, the continuously low sensitivity of the NCP-specific IgM ELISA (Table 2 ) is in 196 accordance with previous results indicating a sensitivity of 55% at week three to four after 197 disease onset 20 . Liu et al also observed a higher sensitivity of an ELISA based on the spike 198 protein compared to an NCP-based ELISA for detection of IgM antibodies 8 . Two months after 199 symptom onset, we observed a decline in the sensitivity of both IgM-specific assays (Table 2) .

Independent of the serological method, the two IgM-specific assays reached maximal 201 sensitivities between 11-20 dpso ( Table 2 ) and could therefore especially be applied to detect 202 antibodies in samples taken during the early phase of infection. If patients develop specific IgM 203

against NCP, these antibodies seem to be present for only a short time during the early phase of 204 infection. A sharp decline in the IgM prevalence is to be expected because isotype switching of 205

virus-specific B-cells from IgM to IgG antibody production causes a decline in circulating IgM 21 .

The facts that SARS-CoV-2-specific IgM is detected mostly in the early infection phase but only in 207 . CC-BY-NC-ND 4.0 International license It is made available under a 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 16, 2021. ; https://doi.org/10.1101/2021.04.16.21255608 doi: medRxiv preprint rare cases 22 invites the question whether all isotypes should be measured during 208

serodiagnostics.

The agreement analysis revealed a very high correlation between results obtained with the Anti-211 SARS-CoV-2 ELISA (IgG) and the QuantiVac ELISA (IgG). The two samples that showed 212 inconsistent qualitative results between these assays (Table 3, Figure 2 ) were taken relatively 213 early and late (7 and 116 dpso) in the course of the disease. An explanation for these 214 inconsistencies might be that the assays were incubated using the same aliquot but on different 215 days, hence the experimental conditions might have differed slightly. Another reason might be 216 that the artificial division between positive and negative results does not match the natural 217 range of activity of some samples.

In general, the use of cells infected with the whole SARS-CoV-2 as substrate has the great 220 advantage of obtaining a high sensitivity due to presence of the complete antigenic spectrum, as 221 evident in the present IIFT results. This is, however, linked to the disadvantage that a positive 222

IIFT result does not allow for a conclusion on the molecular identity of the antigen(s) binding the 223 antibody. In contrast, recombinant cell substrates used in the ELISA technique are ideally suited 224

for the detection and precise identification of antibodies against selected proteins of SARS-CoV-2 225 such as S1/RBD and NCP. During the purification required for Anti-SARS-CoV-2 NCP ELISA 226 production, tertiary or quaternary structured epitopes are often destroyed or weakened. 227

Nevertheless, a selective loss of reactivity does have advantages, since undesired antibody 228

binding aside from the recombinant target protein can be suppressed. Thereby, the specificity of 229 the ELISA can be improved, which was evident in the present results. Moreover, the ELISA 230 technique has the advantage of yielding results in numeral form, which allows an objective 231 evaluation of results. The use of SARS-CoV-2 IIFT is (currently) reserved for specialized research 232 laboratories with high biosafety restrictions due to the handling of the full virus, and, compared 233

to other serological techniques, IIFT is less implemented in standard diagnostic laboratories.

The presence of anti-SARS-CoV-2 S1/RBD IgG antibodies seems to correlate with the 236 development of both virus neutralization and immunity 1,3,23 . Previous research found that titers 237 of neutralizing antibodies were significantly correlated with the levels of anti-RBD IgG 12 , and 238 RBD-specific IgG titers were suggested as a surrogate of neutralization potency against SARS-239

CoV-2 infection 24 . Nevertheless, it is possible that a patient does not develop antibodies against 240 S1 of SARS-CoV-2, but only against NCP. However, this would suggest that neutralizing 241 antibodies might not be present since binding antibodies against NCP seem to correlate to a 242 lesser degree with immunity than binding antibodies against S1/RBD 25 . The development of 243 immunity to SARS-CoV-2 is induced both by the humoral and the cellular immune response, 244

whereby especially IgG directed against the S1 subunit of the SARS-CoV-2 spike protein and 245 specific long-lived T cells are of great interest, as they are suspected to play the most relevant 246 roles in virus neutralization and sustained immunity. A combination of serological tests to 247 quantify both the interferon-gamma release by SARS-CoV-2-specific T cells, stimulated by SARS-248

CoV-2 specific antigens, and the presence of anti-S1/RBD IgG antibodies will enable 249 differentiated investigation of the immune response in the progression of infection and 250

vaccination. Especially the determination of relevant antibody concentrations will probably be 251 one of the most important instruments for determining the vaccination success, although it is yet 252 unknown how many antibodies against S1/RBD an individual must produce after vaccination to 253 be protected from COVID-19. Surrogate neutralization assays detect circulating neutralizing 254 . CC-BY-NC-ND 4.0 International license It is made available under a 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 16, 2021. ; https://doi.org/10.1101/2021.04.16.21255608 doi: medRxiv preprint antibodies against SARS-CoV-2 that block the interaction between the RBD of SARS-CoV-2 with 255 the ACE2-cell surface receptor of the human host cell, thus supporting a quick diagnostic 256 statement about the degree of immunity. In contrast to plaque-reduction neutralization tests, 257

which require handling of the virus, surrogate neutralization assays can easily be integrated in 258 the laboratory routine and do not require biosafety level 3 laboratories.

A detailed analysis of potential associations between antibody kinetics and disease severity was 261 not performed because symptoms were not systematically recorded and the disease severity 262 could therefore not be rated other than that patients in panel A had no or mild symptoms and 263 patients in panel B required hospitalization. Nevertheless, the assay sensitivities were reported 264 also for each panel separately (Table 1) . Analysis of temporal profiles was performed on samples 265

from both patient panels because the distribution of samples in the three infection phases was 266 unbalanced between panels A and B (Table 1) .

ELISA or immunoblot techniques might be used in future to differentiate between reactivities 268

against distinctive SARS-CoV-2 antigens, which might be useful for determination of biomarkers 269

indicative of early or late infection phases.

In summary, evidence of this study emphasizes that the assays have different advantages as well 272

as intended purposes. ELISAs provide an insight into the prevalences of specific antibodies 273

against selected antigens of SARS-CoV-2. Due to the heterogeneity of individual antibody 274 responses, ELISA may not yield positive results for all patients but a combination of ELISAs with 275 different antigens can reduce this diagnostic gap. The three Anti-SARS-CoV-2 ELISAs that detect 276

IgG antibodies can be used to confirm pathogen contact, starting from week two of the infection, 277

to monitor the humoral response following an acute infection confirmed by direct detection and 278

to detect past infections. The highly immunogenic S1 domain of the spike protein of SARS-CoV-2 279 is a major target for neutralizing antibodies and showed good correlation with different test 280 systems for the detection of neutralizing antibodies 19, 26, 27 . IgA-specific ELISAs might further be 281 used to monitor the immune response in COVID-19 patients. IIFT showed highest sensitivities 282

due to the use of the whole SARS-CoV-2 as substrate and provide information whether or not an 283

individual has been infected with SARS-CoV-2. 284 . CC-BY-NC-ND 4.0 International license It is made available under a 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 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Anti-SARS-CoV-2 QuantiVac ELISA (IgG). 437 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 

Severe Acute Respiratory Syndrome Coronavirus 2 -328

Specific Antibody Responses in Coronavirus Disease

Temporal profiles of viral load in posterior 333 oropharyngeal saliva samples and serum antibody responses during infection by SARS-334

CoV-2: an observational cohort study

Cross-Sectional Evaluation of Humoral 337 Responses against SARS-CoV-2 Spike

Longitudinal observation and decline of neutralizing 340 antibody responses in the three months following SARS-CoV-2 infection in humans

Assessment of immune response to SARS-CoV-2 343 with fully automated MAGLUMI 2019-nCoV IgG and IgM chemiluminescence 344 immunoassays

Antibody responses to SARS-CoV-2 in patients with 346 COVID-19

Evaluation of Nucleocapsid and Spike Protein-based ELISAs for 348 detecting antibodies against SARS-CoV-2

Profiling Early Humoral Response to Diagnose Novel 351

COVID-19)

Multi-center nationwide comparison of seven 353 serology assays reveals a SARS-CoV-2 non-responding seronegative subpopulation

The Role of Antibody 356 Testing for SARS-CoV-2: Is There One?

Orthogonal SARS-CoV-2 Serological Assays 359 Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral 360

Sensitive and Specific Monoclonal Antibody-Based Capture 362

Enzyme Immunoassay for Detection of Nucleocapsid Antigen in Sera from Patients with 363

Severe Acute Respiratory Syndrome

Heterogeneous antibodies against SARS-366

CoV-2 spike receptor binding domain and nucleocapsid with implications on COVID-19 367 immunity

Mütter-Screening in einem

Limited specificity of commercially available 376 SARS-CoV-2 IgG ELISAs in serum samples of African origin

Method agreement analysis: A review of correct methodology

Towards the next phase: evaluation of 381 serological assays for diagnostics and exposure assessment

Kinetics of SARS-CoV-2 384 specific antibodies (IgM, IgA, IgG) in non-hospitalized patients four months following 385 infection

Distinct features of SARS-CoV-2-specific IgA response in 387 COVID-19 patients

IgA dominates the early neutralizing antibody 389 response to SARS-CoV-2

Phenotype and kinetics of SARS-CoV-2-specific 392 T cells in COVID-19 patients with acute respiratory distress syndrome

Detection of SARS-CoV-2-Specific Humoral and Cellular 395 Immunity in COVID-19 Convalescent Individuals

Heterogeneous antibodies against SARS-398

CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 399 immunity

Infection fatality rate of SARS-CoV-2 infection in 401 a German community with a super-spreading event

Convalescent plasma treatment for SARS-CoV-2 404 infection: analysis of the first 436 donors in England

 12  12  12  12  12  12  12  12  Crossreactivity  none  none  none  none  none  none  none  none