Analysis of Humoral Immune Responses in Patients with Severe

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The Journal of Infectious Diseases Major Article Downloaded from https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaa680/5944723 by Instituut Voor Tropische Geneeskunde, Bibliotheek user on 29 November 2020 Analysis of Humoral Immune Responses in Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection Lisa Henss,1 Tatjana Scholz,1 Christine von Rhein,1 Imke Wieters,3 Frauke Borgans,3 Fabian J. Eberhardt,3 Kai Zacharowski,4 Sandra Ciesek,5 Gernot Rohde,6 Maria Vehreschild,3 Christoph Stephan,3 Timo Wolf,3, Heike Hofmann-Winkler,7 Heinrich Scheiblauer,2 and Barbara S. Schnierle1 1Paul-Ehrlich-Institut, Department of Virology, Langen, Germany, 2Paul-Ehrlich-Institut, IVD, Langen, Germany, 3Zentrum für Innere Medizin, Infektiologie, Universitätsklinikum Frankfurt, Goethe Universität Frankfurt, Frankfurt am Main, Germany, 4Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany, 5Institut für Medizinische Virologie, Universitätsklinikum Frankfurt am Main, Goethe Universität Frankfurt, Frankfurt am Main, Germany, 6Medizinische Klinik 1, Pneumologie/Allergologie, Universitätsklinikum Frankfurt–Goethe-Universität, Frankfurt am Main, Germany, and 7Deutsches Primatenzentrum GmbH, Leibniz-Institut für Primatenforschung, Göttingen, Germany

Background. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused a pandemic with tens of millions of cases and hundreds of thousands of deaths. The infection causes coronavirus disease 2019 (COVID-19), a disease of the respiratory system of divergent severity. In the current study, humoral immune responses were characterized in a cohort of 143 patients with COVID-19 from the University Hospital Frankfurt am Main, Germany. Methods. SARS-CoV-2-specific–antibodies were detected by enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 and human coronavirus NL63 neutralization activity was analyzed with pseudotyped lentiviral vectors. Results. The severity of COVID-19 increased with age, and male patients encountered more serious symptoms than female patients. Disease severity was correlated with the amount of SARS-CoV-2–specific immunoglobulin (Ig) G and IgA and the neutralization activity of the antibodies. The amount of SARS-CoV-2–specific IgG antibodies decreased with time after polymerase chain reaction conformation of the infection, and antibodies directed against the nucleoprotein waned faster than spike protein-directed antibodies. In contrast, for the common flu coronavirus NL63, COVID-19 disease severity seemed to be correlated with low NL63- neutralizing activities, suggesting the possibility of cross-reactive protection. Conclusion. The results describe the humoral immune responses against SARS-CoV-2 and might aid the identification of correlates of protection needed for vaccine development. Keywords. coronavirus; antibody; neutralization; SARS-CoV-2; COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS- assay (ELISA) kits are available. The coronavirus structural pro- CoV-2) belongs to the Coronaviridae family and is the caus- teins, the surface glycoprotein termed spike (S) and the more ative agent of pneumonia, defined as coronavirus disease abundant nucleocapsid (N) are the principle immunogens used 2019 (COVID-19), which first emerged in the Hubei province for detection of anti–SARS-CoV-2–specific antibodies [3]. in China [1]. The virus rapidly spread worldwide, and the The spike protein consists of 2 subunits, S1 and S2. S1 medi- SARS-CoV-2 pandemic was declared by the World Health ates the attachment of the virus to human cells via its receptor- Organization (WHO) on 11 March 2020. Coronaviruses can binding domain (RBD), and S2 mediates the fusion of the viral cause different diseases in humans. Four endemic human cor- and cellular membranes. Antibodies that bind to the spike onaviruses, OC43, 229E, HKU1, and NL63, are the causative protein, and in particular to the RBD, can neutralize corona- agents of common colds. Two other coronaviruses, the severe viruses. Recombinant RBD or S1 protein facilitates detection of acute respiratory syndrome virus coronavirus and the Middle coronavirus-specific antibodies with ELISA [4]. East respiratory syndrome virus, have a high pathogenic poten- In the current pandemic, the urgent need for effective thera- tial, with 15%–30% mortality rates in humans and have caused peutic measures requires a profound understanding of the path- small epidemics of severe pneumonia [2]. ogenesis of SARS-CoV-2. In the current study, we analyzed the Coronavirus serology has developed rapidly in the last few humoral immune response of German patients with COVID- month, and several commercial enzyme-linked immunosorbent 19 to characterize the disease and support the identification of correlates of protection needed for the development of vaccines

and therapeutic antibodies. Received 8 September 2020; editorial decision 20 October 2020; accepted 26 October 2020; published online October 31, 2020. Correspondence: Barbara S. Schnierle, Paul-Ehrlich-Institut, Department of Virology, Paul- METHODS Ehrlich Strass 51–59, 63225 Langen, Germany ([email protected]). The Journal of Infectious Diseases® 2020;XX:0–0 Cell Culture © The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: [email protected]. HEK293T-hACE2 [5] and HEK293T (American Type Culture DOI: 10.1093/infdis/jiaa680 Collection CRL-3216) cells were cultured at 37°C under 5%

Serological Findings in Patients With COVID-19 • jid 2020:XX (XX XXXX) • 1 carbon dioxide and grown in Dulbecco modified Eagle medium Pseudotyped vectors and serially diluted human serum (1:60 to (Sigma), supplemented with 10% fetal bovine serum (Sigma) 1:14 580) were incubated in triplicate for 30 minutes at 37°C and 5% L-glutamine (200 mmol/L; Lonza) and 1% penicillin/ and used to transduce HEK293T-hACE2 cells. After 48 hours, Downloaded from https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaa680/5944723 by Instituut Voor Tropische Geneeskunde, Bibliotheek user on 29 November 2020 streptavidin (Fisher Scientific). luciferase substrate was added to measure luciferase activity. The reciprocal area under the curve (AUC) value calculated for Patient Serum Samples each sample corresponds to the neutralization activity. Human naive serum was obtained from the German Red Cross from volunteer blood donors and was collected be- ELISA Protocols fore the introduction of SARS-CoV-2 into Germany. Human The following CE-marked ELISA kits were used: Liaison SARS- serum samples from patients positive for SARS-CoV-2 by CoV-2 S1/S2 IgG (Diasorin) and Architect SARS-CoV-2 IgG polymerase chain reaction (PCR) were obtained from the (Abbott). The tests were carried out and interpreted according University Hospital, Frankfurt am Main, under the ethical to the manufacturers’ instructions, as stated in the package approval for the COVID CAPNETZ cohort (no. 11/17). The inserts. study was reviewed and approved by Frankfurt University’s An ELISA using the SARS-CoV-2 RBD as antigen was es- ethics committee. The clinical symptoms of the patients were tablished in house, following a protocol from Stadlbauer et al documented by the medical staff of the University Hospital [9]. The RBD was transiently expressed from HEK293T cells Frankfurt. and purified by nickel affinity chromatography. The expression The score for classification of the severity of symptoms was plasmid was generously provided by Florian Krammer, Icahn determined as recommended by the Clinical Characterisation School of Medicine at Mount Sinai, New York, and is described and Management Working Group of the WHO Research and elsewhere [9]. RDB was used to coat 96-well microtiter ELISA Development Blueprint Programme, with some modification plates at a concentration of 2 μg/mL, and antibodies directed THAT resulted in the following scoring system [6]. Score 1 indi- against either human immunoglobulin (Ig) G (Merck; catalog cated outpatient, SARS-CoV-2 positive, no impairments in mo- no. 6029) or IgA (Thermo Fisher Scientific; catalog no. A18781) bility; score 2, outpatient, SARS-CoV-2 positive, impairments (horseradish-peroxidase coupled) were used to detect binding in mobility; score 3, hospitalization, SARS-CoV-2 positive, no antibodies. The cutoff was the median value for 5 samples from oxygen therapy; score 4, hospitalization, SARS-CoV-2 positive, naive individuals plus 2 standard deviations thereof. oxygen (mask/nasal cannula); score 5, hospitalization, SARS- CoV-2 positive, none-invasive ventilation or high-flow oxygen; Statistical Analysis of the Neutralization Experiments and Software score 6, hospitalization, SARS-CoV-2 positive, intubation and AUC values were determined using GraphPad Prism 7.04 soft- mechanical ventilation; and score 7, hospitalization, SARS- ware (GraphPad). Mean values and standard deviations were CoV-2 positive, ventilation plus additional support (pressors, calculated using Excel software (Excel 2016). registered respiratory therapist, extracorporeal membrane RESULTS oxygenation). The samples were single donations with 3 exceptions, in 2 pa- Characterization of Patients With COVID-19 tients with score 7 and 1 with initial score 5. The score 7 patients From 143 patients with PCR-confirmed SARS-CoV-2 infec- donated blood twice, and at the second donation they improved tions with various clinical symptoms, serum samples were col- their clinical score to score 6. The patient with initial score 5 lected between 9 March and 25 June 2020 in Frankfurt am Main, showed more clinical signs at the second blood donation and Germany. The patients ranged in age from 18 to 81 years, and had a score of 6. Blood samples were collected between 7 and samples were collected at different intervals after confirmation 130 days after PCR confirmation of the infection. of infection by PCR (day 7–130). Patients were scored for clinical symptoms, with a score of 1–2 indicating mild symptoms Pseudotype-Based Neutralization Assay without the need for hospitalization. Scores of 3–4 indicated Lentiviral vectors were prepared in HEK293T cells by moderate disease with hospitalization and, for score 4, the need cotransfection using Lipofectamine 2000 (Thermo Fisher for oxygen. Scores of 5–7 indicated severe disease, with patients Scientific), as described elsewhere [7]. Plasmids encoding needing oxygen, artificial respiration, and intensive care. human immunodeficiency virus type 1 gag/pol, rev, the Most patients (77.39 %) had mild disease, 14.38 % had mod- luciferase-encoding lentiviral vector genome, and the SARS- erate disease, and only 8.21 % had severe disease. The mean age CoV-2 delta 19 spike gene (no. MN908947) or the NL63 delta increased with disease severity, from 40.5 to 62.2 years in female 19 spike gene (no. AFV53148.1) were transfected. The coro- and from 45.7 to 53.3 years in male patients (Figure 1). There navirus genes were synthesized (Eurofins) and cloned into was a significant difference in age between patients with mild the vector pIRES-GFP, as described elsewhere [8]. Vectors to moderate disease and those with severe disease (Figure 1). were concentrated by ultracentrifugation and stored at –80°C. Among the patients with clinical scores of 1–3, female patients

2 • jid 2020:XX (XX XXXX) • Henss et al were significantly younger than male patients (mean age, 40.5 antibodies than female patients, which corresponds to the vs 45.7 years) (Figure 1). This sex difference was not significant higher number of male patients with severe disease (Figure 1B). among patients with severe disease. However, only 8% of the Downloaded from https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaa680/5944723 by Instituut Voor Tropische Geneeskunde, Bibliotheek user on 29 November 2020 patients had severe disease, which might not be representative. SARS-CoV-2 Spike Protein–Binding Antibodies A total of 71 male and 75 female patients were analyzed, and, The presence of spike protein–binding antibodies in patient with one exception, all patients with severe disease (score 5–7) serum samples was analyzed by an in-house ELISA coated with were male (Figure 1). the SARS-CoV-2 S1 RBD, according to a previously developed protocol [4, 9]. The amount of RBD-binding IgG (Figure 3A) SARS-CoV-2 Neutralization Activity of Patient Serum Samples and IgA (Figure 3B) increased with the severity of disease and First, the humoral immune responses against SARS-CoV-2 showed a significant increase between patients with a clinical were studied by the ability of serum to neutralize SARS-CoV-2. score of 1 and those with a score of 4–7. me were only 2 pa- Neutralization activity was determined using lentiviral vectors tients with a score of 7, so the data might not be representative. pseudotyped with the SARS-CoV-2 spike protein. These vectors The clinical score was also correlated with the amount of S1/ acquire the host spectrum of SARS-CoV-2, and a rapid readout S2-binding IgG, determined by the quantitative ELISA from of the results is made possible by the transfer of the luciferase Liaison (Diasorin) (Figure 3C). Compared with mildly af- gene, serving as a surrogate measure of infection. Addition of fected patients, a significant increase in S1/S2-binding IgG was neutralizing antibodies directed against SARS-CoV-2 reduces detected in serum samples from patients with a score of 4 or the luciferase activity. The neutralization activity of serum higher. This increase in IgG binding was less obvious when the was determined using the AUC generated by serum dilutions. Architect nucleocapsid protein (N) ELISA (Abbott) was used Neutralization activity, depicted as the reciprocal AUC, in- (Figure 3D), with a less significant difference between patients creased with the severity of disease and was highest in patients with a score of 6 and those with a score of 1. with a disease score of 7 (Figure 2A). Overall, the neutralization activity of patient-derived antibodies was quite diverse, with Analysis of Patients With a Clinical Score of 2 occasional high-level neutralization activity in serum samples Most patients had a clinical score of 2 (n = 96); however, these from patients with mild disease. However, when patients with a single-patient serum samples were collected at different time clinical score of 2 were analyzed for the sex distribution of neu- points after PCR-based diagnosis. A decrease in antibody titers, tralizing antibody levels, male patients had more neutralizing especially for antibodies directed against the N protein, has been described elsewhere [10]. Therefore, patients with a score of 2 were investigated for the presence of antibodies directed

P < .001 against the viral N protein (Figure 4A), and a significant de- ‡ crease in antibody binding to N was observed in samples from P < .001 81 to 130 days after diagnosis. A similar decrease was observed ‡ for IgG directed against RBD (Figure 4B) and is suggestive of P = .03 * NS short-lasting antibody responses. However, for IgA responses, no significant decrease was detectable (Figure 4C), although the 45.7 62.2 53.3 mean optical density values decrease from 0.34 for samples at 80 40.5 days 32–45 to 0.20, 0.18, and 0.099 for samples collected at later time points. Overall, the IgA detection was not very sensitive 60 and might underrate the decrease in SARS-CoV-2–specific IgA. , y 40 Human Coronavirus NL63 Neutralizing Activity Age Preexisting immune responses against the common cold 20 causing human coronaviruses have been described elsewhere [11, 12]; consequently, neutralization of one human 0 coronavirus, NL63, by the patient material was studied. Score Score Score Score Neutralization activity was determined; lentiviral vectors 1–3 F 1–3 M 4–7 F 4–7 M were pseudotyped with the NL63 spike protein and, as before, the result is depicted as the reciprocal AUC (Figure 5). Figure 1. Distribution of clinical scores of patients with coronavirus disease 2019 (COVID-19) stratified by age and sex. The ages of female (F) and male (M) patients Once again, the neutralization activity of patient-derived with COVID-19 are indicated for patients with mild to moderate (score, 1–3) or se- antibodies was quite diverse, with high activity observed in a vere (score, 4–7) disease. Mean ages are indicated above the columns. P values in- small number of patients. However, high NL63 neutralizing dicate significant differences between samples and were calculated using Student t tests and GraphPad Prism 7.04 software. *P ≤ .05; ‡P ≤ .001. Abbreviation: NS, activity was not detected in samples from patients with se- not significant P( > .05). vere COVID-19 (score, 5–7) patients. However, the overall

Serological Findings in Patients With COVID-19 • jid 2020:XX (XX XXXX) • 3 A P < .001 B ‡ P = .005 P = .02 † Downloaded from https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaa680/5944723 by Instituut Voor Tropische Geneeskunde, Bibliotheek user on 29 November 2020

* UC P = .01 P < .001 * ‡ 6 UC 10 NS NS NS , ln 1/A

5 2 Neutralization, 1/A

0 0 Neutralizing Antibodies 1234567Naive Male Female Patients Patients Score

Figure 2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–neutralizing activity in serum samples from patients with coronavirus disease 2019. The SARS- CoV-2–neutralizing activity was determined with SARS-CoV-2–pseudotyped lentiviral vectors. A, Reciprocal area under the curve (AUC) plotted against the corresponding clinical score. P values indicate significant differences compared with 7 naive samples and were calculated using Student t tests and GraphPad Prism 7.04 software. B, SARS-CoV-2–neutralizing activity in serum samples from patients with a clinical score of 2 plotted, against patient sex. *P ≤ .05; †P ≤ .01; ‡P ≤ .001. Abbreviation: NS, not significant P( > .05). difference in neutralization activities between patients with levels of neutralizing antibodies but also had more severe dis- clinical scores of 1 or 2 and those with scores of 5–7 was sig- ease. In addition, IgG or IgA directed against the SARS-CoV-2 nificant (Figure 5). spike protein or RBD and, less significantly, against the N protein, was correlated with disease severity. The less significant DISCUSSION correlation shown by antibodies directed against the N protein The analysis of 143 patients with COVID-19 from the University might reflect different sensitivities in the detection assays or the Hospital Frankfurt am Main revealed that most patients had transient nature of the antibody responses. Similar correlations mild disease (78%). However, severe cases were more frequent have been observed by others [13–16]. in older patients, particularly male patients. The clinical score Among the patients with a clinical score of 2, a decrease in was positively correlated with SARS-CoV-2–neutralizing ac- S- and N-directed IgG antibodies was observed in blood sam- tivity in serum samples from the patients. This was reflected ples drawn at later time points after infection. This might in- by the observation that male patients not only had increased dicate the waning of SARS-CoV-2–specific antibodies that has

ABCDP < .001 ‡ P =.004 P < .001 † P = .001 ‡ P < .001 P <.001 P =.006 ‡ ‡ ‡ NS P =.006 NS NS P =.01 2.5 P =.005 P = .004 10 † † 400 P =.04 P = .02 e Units ‡ † U/mL

450 † 1.0 P < .001 450 P = .02 * *

‡ , A 2.0 NS NS * 8

P =.01 elativ , OD NS P = .004 * ‡ 1.5 NS , R 6 0.5 otal IgG 200 1.0 4 otal IgG 0.5 2 RBD-Binding IgG 0.0 RBD-Binding IgA, OD 0.0 0 0 S1/S2-Binding T

1234567 1234567 1234567 N-Binding T 1234567 Score Score Score Score RBD-binding IgG RBD-binding IgA S1/S2-binding IgG N-binding IgG

Figure 3. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–binding antibodies. A, SARS-CoV-2–binding antibodies were determined by different enzyme- linked immunosorbent assays (ELISAs). P values indicate significant differences in binding compared with serum samples from patients with a score of 1 and were calculated using Student t tests and GraphPad Prism 7.04 software. A, In-house ELISA with receptor-binding domain (RBD) as antigen and detection of immunoglobulin (Ig) G as optical density values at 450 nm (OD450). Naive serum samples had a mean OD450 value of 0.16. B, In-house ELISA with RBD as antigen and detection of IgA as OD450 values. Naive serum samples had a mean OD450 value of 0.31. C, Liaison SARS-CoV-2 S1/S2 IgG ELISA for quantitative detection of anti–spike protein antibodies. Values <15 Arbitrary units (AU) are considered negative. D, Architect SARS-CoV-2 IgG ELISA for the detection of N-directed antibodies. Values <1.4 relative units are considered negative. *P ≤ .05; †P ≤ .01; ‡P ≤ .001. Abbreviation: NS, not significant P( > .05).

4 • jid 2020:XX (XX XXXX) • Henss et al A B C NS NS P = .03 NS NS P = .02 NS NS NS * 0.8 * 10 1.5 Downloaded from https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaa680/5944723 by Instituut Voor Tropische Geneeskunde, Bibliotheek user on 29 November 2020 450 e Units 450 8 0.6 elativ , OD 1.0

, R 6 0.4 4 otal IgG 0.5 0.2 2 RBD-Binding IgA, OD RBD-Binding IgG 0 0.0 0.0 N-Binding T 32–45 46–59 60–77 81–130 32–45 46–59 60–77 81–130 32–45 46–59 60–77 81–130 Time After Positive PCR Result, d Time After Positive PCR Result, d Time After Positive PCR Result, d N-binding IgG RBD-binding IgG RBD-binding IgA

Figure 4. Antibody titers of score 2 patient samples at different time points after a positive polymerase chain reaction (PCR) test. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–binding antibodies were determined using different enzyme-linked immunosorbent assays (ELISAs). P values indicate significant differences in binding compared with initial samples obtained at the earliest time points and were calculated using Student t tests and GraphPad Prism 7.04 software. A, Architect SARS- CoV-2 IgG ELISA (Abbott) for the detection of N-directed antibodies. B, In-house ELISA with receptor-binding domain (RBD) as antigen and detection of immunoglobulin (Ig) G as optical density values at 450 nm (OD450). C, In-house ELISA with RBD as antigen and detection of IgA as OD450 values. *P ≤ .05. Abbreviation: NS, not significant P( > .05). been described elsewhere [17, 18]. Longitudinal analysis of to high neutralizing activity and was reached by only 5.4% of individual patients is needed to fully confirm a decline in an- the samples. Thereby we confirm previous observations that tibody titer. Similar observations of declining antibody levels most convalescent plasma samples obtained from individuals have been described for human coronaviruses. A study of indi- who recover from COVID-19 do not contain high levels of viduals experimentally infected with human CoV-229E showed neutralizing activity [13, 20]. Transfusion of convalescent that levels of IgG and IgA antibodies directed against the virus plasma is being considered and evaluated as a therapeutic op- waned to background levels within 11 weeks to 1 year [19]. tion for the treatment of COVID-19, but the identification of Overall, the neutralizing titers of patients with mild disease suitable plasma donors is hampered by this substantial var- were very low, and higher titers were only detected in patients iability in levels of neutralizing antibodies in convalescent with severe disease. A reciprocal AUC value >8 corresponds patients. Interestingly, patients with severe disease (scores, 5–7) had no high-level NL63-neutralizing antibodies, which might be P = .005 related to the small number of patients that were analyzed. However, the small number of SARS-CoV-2–naive individuals NS analyzed, had high NL63-neutralizing antibodies and a high se- roprevalence. Serological data about NL63 infections is mainly available from children and indicate that human CoV-NL63 in-

UC 10 fections are common during childhood [21]. It is tempting to speculate that preexisting immunity to NL63 or other common cold coronaviruses might reduce the risk of severe disease. 5 Neutralizing antibodies might only be a surrogate for this immunity, since preexisting SARS-CoV-2 T-cell immunity has

Neutralization, 1/A been described in naive individuals [11, 12]. Conserved pep- tides, with low homology among β-coronaviruses have been 0 1–2 3–4 5–7 Naive recognized before, and infection with human coronaviruses Score might induce a pancoronavirus T-cell immunity [11]. Currently, vaccine development relies mainly on the assump- Figure 5. Human coronavirus NL63-neutralizing activity of serum samples from tion that antibodies will be essential for protection. Protection patients with coronavirus disease 2019. The NL63-neutralizing activity of serum of individuals with anti–SARS-CoV-2 antibodies has recently samples was determined with NL63-pseudotyped lentiviral vectors, and the recip- been described [22]. However, the minimal threshold level rocal area under the curve (AUC) was plotted against the indicated clinical scores. Significant differences were detected using Studentt tests and calculated using for protection is currently unknown. In the current study, GraphPad Prism 7.04 software. Abbreviation: NS, not significant P( > .05). we characterized the humoral immune responses of patients

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