ARTICLE OPEN ACCESS Humoral and Cellular Responses to SARS-CoV-2 in Convalescent COVID-19 Patients With Multiple Sclerosis Ana Zabalza, MD, Georgina Arrambide, MD, PhD, Paula Tagliani, MD, Simón Cárdenas-Robledo, MD, Correspondence Dr. Zabalza Susana Otero-Romero, MD, PhD, Juliana Esperalba, MD, PhD, Candela Fernandez-Naval, azabalza@cem-cat.org Jesus Trocoli Campuzano, Mónica Martı́nez Gallo, MD, PhD, Mireia Castillo, Mercè Bonastre, Mireia Resina Sallés, Jordina Beltran, Pere Carbonell-Mirabent, Marta Rodrı́guez-Barranco, Samuel López-Maza, MD, Pedro José Melgarejo Otálora, MD, Mariano Ruiz-Ortiz, MD, Agustin Pappolla, MD, Breogán Rodrı́guez Acevedo, MD, Luciana Midaglia, MD, Angela Vidal-Jordana, MD, Alvaro Cobo-Calvo, MD, PhD, Carmen Tur, MD, PhD, Ingrid Galán, MD, Joaquı́n Castilló, MD, PhD, Jordi Rı́o, MD, PhD, Carmen Espejo, MD, PhD, Manuel Comabella, MD, PhD, Carlos Nos, MD, Jaume Sastre-Garriga, MD, PhD, Mar Tintore, MD, PhD,* and Xavier Montalban, MD, PhD* Neurol Neuroimmunol Neuroinflamm 2022;9:e1143. doi:10.1212/NXI.0000000000001143 Abstract MORE ONLINE Background and Objectives COVID-19 Resources Information about humoral and cellular responses to severe acute respiratory syndrome For the latest articles, invited commentaries, and coronavirus 2 (SARS-CoV-2) and antibody persistence in convalescent (COVID-19) patients blogs from physicians with multiple sclerosis (PwMS) is scarce. The objectives of this study were to investigate factors around the world influencing humoral and cellular responses to SARS-CoV-2 and its persistence in convalescent COVID-19 PwMS. NPub.org/COVID19 Methods This is a retrospective study of confirmed COVID-19 convalescent PwMS identified between February 2020 and May 2021 by SARS-CoV-2 antibody testing. We examined relationships between demographics, MS characteristics, disease-modifying therapy (DMT), and humoral (immunoglobulin G against spike and nucleocapsid proteins) and cellular (interferon-gamma [IFN-γ]) responses to SARS-CoV-2. Results A total of 121 (83.45%) of 145 PwMS were seropositive, and 25/42 (59.5%) presented a cellular response up to 13.1 months after COVID-19. Anti–CD20-treated patients had lower antibody titers than those under other DMTs (p < 0.001), but severe COVID-19 and a longer time from last infusion increased the likelihood of producing a humoral response. IFN-γ levels did not differ among DMT. Five of 7 (71.4%) anti-–CD20-treated seronegative patients had a cellular response. The humoral response persisted for more than 6 months in 41/56(81.13%) PwMS. In multivariate analysis, seropositivity decreased due to anti-CD20 therapy (OR 0.08 [95% CI 0.01–0.55]) and increased in males (OR 3.59 [1.02–12.68]), whereas the cellular response decreased in those with progressive disease (OR 0.04 [0.001–0.88]). No factors were associated with antibody persistence. *These authors contributed equally to this work (co–senior authors). From the Servei de Neurologia-Neuroimmunologia (A.Z., G.A., P.T., S.C.-R., S.O.-R., M. Castillo, M.B., M.R.S., J.B., P.C.-M., M.R.-B., S.L.-M., A.P., B.R.A., L.M., A.V.-J., A.C.-C., C.T., I.G., J.C., J.R., C.E., M. Comabella, C.N., J.S.-G., M.T., X.M.), and Preventive Medicine and Epidemiology Department (S.O.-R.), Centre d’Esclerosi Múltiple de Catalunya (Cemcat), Vall d’Hebron Institut de Recerca, Hospital Universitari Vall d’Hebron, Departament de Medicina, Universitat Autònoma de Barcelona; Microbiology Department (J.E., C.F.-N., J.T.C.), Vall d’Hebron University Hospital, Vall d’Hebron Barcelona Hospital Campus, Universitat Autònoma de Barcelona; Immunology Division (M.M.G.), Hospital Universitari Vall d’Hebron and Diagnostic Immunology Research Group, Vall d’Hebron Research Institute (VHIR), Barcelona; Department of Neurology (P.J.M.O.), Hospital General Universitario Gregorio Marañón, Madrid; and Department of Neurology (M.R.-O.), Hospital Universitario 12 de Octubre, Madrid, Spain. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CIS = clinically isolated syndrome; CLIA = chemiluminescence immunoassay; COVID-19 = coronavirus disease 2019; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; IFN = interferon; Ig = immunoglobulin; IGRA = interferon-gamma release immunoassay; IQR = interquartile range; MS = multiple sclerosis; PwMS = patients with MS; RRMS = relapsing-remitting MS; RT-PCR = reverse transcription–PCR; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2. Discussion Humoral and cellular responses to SARS-CoV-2 are present in COVID-19 convalescent PwMS up to 13.10 months after COVID-19. The humoral response decreases under anti-CD20 treatment, although the cellular response can be detected in anti–CD20-treated patients, even in the absence of antibodies. Coronavirus disease 2019 (COVID-19) is a pandemic in- convalescence, and a serologic study performed at any time fection caused by severe acute respiratory syndrome corona- point during the observation period. Following the European virus 2 (SARS-CoV-2) and has caused almost 5 million deaths Centre for Disease Prevention and Control guidelines,14 worldwide.1 Although patients with multiple sclerosis (MS) COVID-19-confirmed cases were defined as a positive SARS- do not have an increased risk of COVID-19 compared with CoV-2 reverse transcription–PCR (RT-PCR) or a positive the general population, risk factors for severe COVID-19 in antibody test. Data were collected using a REDCap-based patients with MS include older age, male sex, comorbidities, electronic case report form. progressive forms, and higher disability.2-5 In relation to disease-modifying therapy (DMT), only anti-CD20 therapies Demographic and Clinical Data appear to increase the risk of COVID-19 severity, and in- Demographic, clinical, laboratory, and COVID-19 data were terferon (IFN) may play a protective role.2-4 retrieved from hospital electronic health records. De- mographic data included age, sex, and ethnicity. Clinical data Emerging evidence shows that DMTs alter immunologic re- included comorbidities (obesity, lung disease, cardiovascular sponses to both SARS-CoV-2 infection and vaccination against disease, diabetes, hypertension, hematologic disease, chronic the disease. Some DMTs may induce immunomodulation, kidney disease, liver disease, other autoimmune disease, HIV, whereas others deplete T cells, B cells, or both. In this regard, or malignancy), MS phenotype (clinically isolated syndrome some studies have shown a decreased humoral response to [CIS], relapsing-remitting MS [RRMS], secondary pro- SARS-CoV-2 infection in patients treated with anti-CD20 gressive MS, and primary progressive MS), MS disease du- therapies.6-9 For SARS-CoV-2 vaccines, recent studies demon- ration, Expanded Disability Status Scale (EDSS), DMT at the strate that the humoral response is blunted not only in patients time of COVID-19, treatment duration, and, for patients on on anti-CD20 therapies but also in those on anti-SP1 receptor anti-CD20 therapy, cladribine, or alemtuzumab, time since treatment.10,11 Encouragingly, vaccinated patients with MS on last administration. The absolute lymphocyte count (cell/m3) anti-CD20 therapies seem to present a specific cellular re- was retrieved for all patients. In anti–CD20-treated patients, sponse, even in the absence of a humoral response.12,13 How- immunoglobulins (IgM, IgG, and IgA; mg/dL) and flow ever, whether these treatments may affect cellular or long-term cytometry lymphocyte phenotypes (total lymphocytes: humoral responses against SARS-CoV-2 natural infection is still CD3+, CD4 T cells: CD4+, CD8 T cells: CD8+, B cells: unknown. Therefore, the aims of this study were to investigate CD19+) were also collected. COVID-19 data included the humoral and cellular responses to SARS-CoV-2 in COVID-19 presence or absence of symptoms and SARS-CoV-2 RT-PCR. convalescent patients with MS (PwMS), to identify factors for COVID-19 severity was categorized as mild-moderate disease developing humoral and cellular responses, and to evaluate or severe-critical disease, as previously described.6 factors for humoral response persistence. Humoral and Cellular Response Studies Humoral and cellular responses were analyzed in the clinical Methods microbiology and immunology laboratories of Vall d’He- This is a retrospective study involving a cohort of PwMS con- bron’s hospital. The qualitative humoral response was ana- ducted at the Multiple Sclerosis Centre of Catalonia (Cemcat) lyzed using different commercial chemiluminescence in Barcelona between February 1, 2020, and May 22, 2021. immunoassays (CLIAs) targeting specific SARS-CoV-2 anti- bodies against spike and nucleocapsid, as per clinical practice. Study Population Qualitative and quantitative CLIA studies were performed in We included PwMS with all of the following criteria: older than a group of selected patients: SARS-CoV-2 antibodies (IgG, 18 years, not vaccinated against SARS-CoV-2, COVID-19 IgM, and IgA) against the nucleocapsid protein (Ig-N) were 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 Neurology.org/NN detected by the Elecsys Anti-SARS-CoV-2 test (Roche Di- response were those with positive results for antigen mix 1 agnostics, Mannheim, Germany) with a cutoff of 1.0 index and/or 2. performed using a Cobas 8800 system autoanalyzer (Roche Diagnostics, Basel, Switzerland); IgG antibodies against the Humoral response persistence was analyzed only in patients spike protein (IgG-S) were measured by the LIAISON Tri- with at least 2 serologic determinations: 1 performed within mericS IgG SARS-CoV-2 IgG test (DiaSorin, Stillwater, MN) the first 6 months of COVID-19 diagnosis and another at least with a cutoff point of 13.0 AU/mL performed using an XL 6 months thereafter. The humoral response was considered Analyzer (DiaSorin, Saluggia, Italy). persistent when serologic determinations were positive both before and after 6 months or when presenting a positive de- The cellular response was assessed in a group of selected termination after 6 months. Unless otherwise specified, sta- patients according to DMT with consecutive sampling, pri- tistical tests were performed at the 0.05 level of significance oritizing those on anti-CD20 therapy. The cellular response using Stata version 14.0 (Stata Statistical Software, College was assessed using IFN-γ release immunoassay (IGRA) Station, TX) and GraphPad Prism version 9.0 (GraphPad methodology with 2 QuantiFERON SARS-CoV-2 RUO Software, San Diego, CA). tubes from Qiagen (Hilden, Germany), proprietary mixes of SARS-CoV-2 S protein (Ag1 and Ag2) selected to activate Standard Protocol Approvals, Registrations, both CD4 and CD8 T cells, as per the manufacturer’s in- and Patient Consents structions. IFN-γ (IU/mL) was measured in these plasma The study was approved by the ethics committee of Vall samples using ELISA (QuantiFERON-TB Gold Plus; Qia- d’Hebron University Hospital [EOM(AG)003/2021(5768)]. gen) tests. According to the manufacturers, a test was con- Patient consent was obtained. sidered positive if IFN-γ was higher than 0.15 IU/mL. Data Availability Statistical Analysis Anonymized data that support the findings of this study are For the purpose of this study, data capture was locked on May available on request from the corresponding author. The data 22, 2021. Descriptive statistics were used to compare de- are not publicly available due to privacy or ethical restrictions. mographics and disease characteristics according to classifi- cation of the presence or absence of humoral and cellular responses. Results Patient Identification Univariable logistic regression models were performed on Until May 22, 2021, 256 patients with suspected or confirmed identified variables to assess their association with the pres- COVID-19 were identified in our center. Of those, 243 (94.92%) ence of humoral and cellular responses and antibody persis- had MS; the other 13 had inflammatory-demyelinating diseases tence. Age was treated as a continuous variable, and the EDSS of the central and peripheral nervous systems. Of the 243 with was categorized into 2 bins (EDSS <3.0 and ≥3.0). DMTs MS, 187 had confirmed COVID-19; 56 had clinically suspected were categorized into 3 categories (untreated, anti-CD20 COVID-19. Only those with confirmed COVID-19 and at least 1 therapies and other DMTs). The Pearson χ 2, Fisher exact test, serologic determination (n = 145; 77.54%) were included in this Student t test, and Mann-Whitney U tests were used for study (eFigure 1, links.lww.com/NXI/A693). The distribution of comparisons, as appropriate. ORs with 95% CIs were esti- patient DMTs is specified in Table 1. The median follow-up time mated using a logistic regression model. after COVID-19 of these patients was 10.5 months (interquartile range [IQR] 8.2 months). Multivariate logistic regression was applied to determine variables independently associated with presenting a humoral SARS-CoV-2 Humoral and Cellular Responses response, a cellular response, and humoral response persis- One hundred twenty-one (83.44%) of the 145 PwMS in- tence over 6 months. The model included age, sex, presenting cluded in the study had a positive serologic determination at any comorbidity, MS phenotype (CIS/RRMS vs progressive some time point. Positive humoral responses were detected at forms), EDSS, DMTs, and COVID-19 severity. Other vari- 0–13.10 months after COVID-19 diagnosis. Demographic, ables, such as months after COVID-19 of serologic de- clinical, and laboratory characteristics of patients according to termination or SARS-CoV-2 RT-PCR, were included in the positive or negative antibody results are summarized in models if considered clinically relevant or if a p value <0.1 was Table 1. Demographic, MS, and COVID-19 characteristics obtained by univariate analysis. Disease duration was excluded and previous laboratory findings were similar in those with from the model because collinearity between age and disease positive and negative antibodies. SARS-CoV-2 RT-PCR was duration was detected. performed for 106 (73.1%) of the patients, which was positive in 72 (59.50%) seropositive patients and in all seronegative For the purpose of this analysis, seropositive patients were patients. Nineteen (13.1%) PwMS presented severe or critical those with positive serologic testing for IgG-S and/or Ig-N at COVID-19; 11 (7.6%) had asymptomatic disease. Patients any time point during follow-up. Patients with a cellular with severe disease were mostly on anti-CD20 therapies Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 3 Table 1 Clinical and Demographic Characteristics of the Cohort in Relation to SARS-CoV-2 Serostatus Total Negative Positive (N = 145) serology (n = 24) serology (n = 121) OR (95% CI)a p Valuea Age, y, mean (SD) 46.87 (11.25) 46.39 (12.90) 46.97 (10.97) 1.00 (0.97–1.04) 0.817 Male sex, n (%) 52 (35.86) 4 (16.67) 48 (39.67) 3.29 (1.03–10.45) 0.032 Any comorbidity, n (%)b 69 (75.59) 15 (62.50) 54 (44.63) 0.48 (0.19–1.20) 0.111 Obesity, n (%) 27 (18.62) 3 (12.50) 24 (19.83) 1.73 (0.47–6.34) 0.401 c Progressive MS, n (%) 27 (18.62) 9 (37.50) 18 (14.88) 0.29 (0.11–0.78) 0.010 EDSS ≥3.0, n (%) 54 (37.24) 13 (54.17) 41 (33.88) 0.43 (0.17–1.07) 0.061 Disease duration, y, median (IQR) 14 (11.0) 12.55 (11.1) 14.7 (11.1) 1.01 (0.95–1.06) 0.793 Corticosteroids last 3 mo, n (%) 2 (1.38) 1 (4.17) 1 (0.83) 0.19 (0.01–3.25) 0.203 DMTs, n (% of the row) No treatment 30 (20.69) 2 (6.67) 28 (93.33) Ref Ref Interferon-β 19 (13.10) 3 (15.79) 16 (84.21) 0.38 (0.06–2.63) 0.309 Glatiramer acetate 13 (8.97) 1 (7.69) 12 (92.31) 0.86 (0.07–10.69) 0.905 Dimethyl fumarate 18 (12.41) 2 (11.11) 16 (88.89) 0.57 (0.07–4.58) 0.594 Teriflunomide 12 (8.28) — 12 (100.0) — 0.365 Fingolimod 6 (4.14) — 6 (100.0) — 0.521 Natalizumab 4 (2.76) — 4 (100.0) — 0.600 Alemtuzumab 7 (4.83) 2 (28.57) 5 (71.43) 0.18 (0.02–1.79) 0.097 Cladribine 2 (1.38) — 2 (100.0) — 0.711 Ocrelizumab 7 (4.83) 2 (28.57) 5 (71.43) 0.18 (0.18–1.78) 0.097 Rituximab 22 (15.17) 10 (45.45) 12 (54.55) 0.086 (0.01–0.56) 0.001 Other anti-CD20 4 (2.76) 2 (50.0) 2 (50.0) 0.07 (0.00–1.10) 0.013 Other DMTs 1 (0.69) — 1 (100.0) — 0.793 Anti-CD20 33 (22.76) 14 (42.42) 19 (57.58) 0.13 (0.05–0.37) 0.000 Treatment duration, y, median (IQR) 2.7 (4.9) 1.85 (3.0) 3.0 (5.0) 1.04 (0.94–1.16) 0.405 Time of COVID-19 since last infusion, 4.0 (5.2) 3.15 (5.10) 5.0 (8.7) 1.08 (0.96–1.22) 0.203 mo, median (IQR)1 Time of serology since last infusion, 4.47 (6.83) 5.42 (6.64) 4.47 (9.36) 1.06 (0.96–1.16) 0.269 mo, median (IQR)1 Previous lymphocyte count, median (IQR)2 1,700 (1,000) 1,400 (900) 1,730 (1,000) 1.00 (0.99–1.00) 0.381 3 Previous IgG count, median (IQR) 914 (287) 942.5 (338) 869 (317.5) 1.00 (0.99–1.00) 0.440 3 Previous IgM count, median (IQR) 88 (50) 78.5 (52.5) 90.5 (50.5) 1.02 (0.98–1.04) 0.264 Previous IgA count, median (IQR)3 192 (109) 182 (41) 221 (76) 1.01 (0.99–1.02) 0.094 Negative RT-PCR, n (%) 10 (6.90) — 10 (8.26) Ref Ref Positive RT-PCR, n (%) 96 (66.21) 24 (100.0) 72 (59.50) 2.10 (0.71–6.21) 0.170 RT-PCR not performed, n (%) 39 (26.90) — 39 (32.23) 0.83 (0.28–2.43) 0.730 COVID-19 symptoms, n (%) 134 (90.34) 20 (83.33) 111 (91.74) 2.22 (0.63–7.86) 0.205 Continued 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 Neurology.org/NN Table 1 Clinical and Demographic Characteristics of the Cohort in Relation to SARS-CoV-2 Serostatus (continued) Total Negative Positive (N = 145) serology (n = 24) serology (n = 121) OR (95% CI)a p Valuea COVID-19 severe-critical course, n (%)d 19 (13.10) 3 (12.50) 16 (13.22) 1.07 (0.28–4.01) 0.924 Time of serologies after COVID-19 diagnosis, 3.19 (2.40) 3.38 (4.42) 3.15 (2.3) 0.98 (0.85–1.12) 0.747 months, mean (IQR) Abbreviations: COVID-19 = coronavirus disease 2019; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; IgG, IgM, IgA = immunoglobulin G, M, or A; IQR = interquartile range; MS = multiple sclerosis; RT-PCR = reverse transcription-PCR; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2. Percentage is the proportion of patients of the column with that variable if not otherwise specified. Bold indicates statistically significant p value <0.05. Count of total cases of variables with missing information: n1 = 73, n2 = 129, and n3 = 28. a Statistical analysis was performed using a not adjusted logistic regression model. b Any comorbidity includes obesity, lung disease, cardiovascular disease, diabetes, hypertension, hematologic benign disease, chronic kidney disease, liver disease, HIV, or malignancy. c Progressive MS includes secondary progressive multiple sclerosis and primary progressive multiple sclerosis. d COVID-19 severity is categorized as (1) mild-moderate disease if patients had no signs or symptoms of pneumonia or a mild pneumonia and (2) severe-critical disease if they presented dyspnea, or a respiratory rate of ≥30 breaths per minute or a blood oxygen saturation of ≤93%, or a ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen of <300 mm Hg, or infiltrates in >50% of the lung field within 24–48 hours from the onset of symptoms and/or organ or multiple organ failure. (42.1%) or untreated (47.4%) (data not shown, eTable 5, cladribine and alemtuzumab, the association of antibody titer links.lww.com/NXI/A693). Multivariable analysis revealed that with time since last treatment administration or treatment males were more likely to become seropositive (OR 3.59, 95% CI duration could not be analyzed due to the low number of 1.02–12.68, p < 0.05), whereas PwMS under anti-CD20 therapy cases (eFigures 2 and 3, links.lww.com/NXI/A693). had a higher risk of remaining seronegative than untreated patients (OR 0.08, 95% CI 0.01–0.55, p = 0.01) (Figure 1A). The cellular response was analyzed in 42 patients selected according to DMT: 22 on anti-CD20 therapy, 5 without When exploring the 33 patients on anti-CD20 therapy, 19 treatment, and 15 on other DMTs. Twenty-five (59.5%) of (57.6%) had a positive humoral response. In multivariable these 42 patients presented a cellular response, which was analysis, only severe COVID-19 infection (OR 14.06, 95% CI detected 0.6–13.0 months after COVID-19, with a median 1.02–192.68, p = 0.048) and a longer time between the last time of 7.0 months (IQR 7.2 months). No differences were treatment infusion and COVID-19 disease (OR per month found in demographic and MS variables between positive 1.51, 95% CI 1.01–2.24, p = 0.042) were significantly asso- and negative responders (eTable 2, links.lww.com/NXI/ ciated with a higher probability of developing a humoral re- A693). Nonetheless, all patients with severe COVID-19 sponse after COVID-19 (eTable 1, links.lww.com/NXI/ presented a cellular response (p = 0.018). Five of these A693). patients were on anti-CD20 therapy, 1 was on dimethyl fumarate, and 1 was untreated. In multivariable analysis, the Antibody titers were measured in 124 patients with de- occurrence of a cellular response was decreased with pro- mographic and MS characteristics similar to those of the gressive MS forms (OR 0.04, 95% CI 0.001–0.88, p < 0.05) total cohort (data not shown). Anti–CD20-treated pa- (Figure 2A). tients presented lower IgG-S (15.4 [IQR 60.0]) and Ig-N median (0.08 [IQR 0.13]) titers than those on other A cellular response was detected in patients with all types of DMTs (37.8 [IQR 68.3], p > 0.05, and 19.55 [IQR 42.92], DMT, except for glatiramer acetate (n = 0/2). IFN-γ titers p < 0.001) or untreated patients (74.3 [IQR 182.4], p < against antigen 1 or 2 did not differ between anti–CD20- 0.05, and 34.3 [IQR 128.8], p < 0.001). Patients on fin- treated patients, untreated patients, or those with other DMTs, golimod presented lower median titers of IgG-S (17.0 although the individual proportions of positive determinations [IQR 13.5]) and Ig-N (2.16 [IQR 2.21]) than those on varied among DMTs. However, no significant differences were other DMTs, although no significant differences were observed due to the low number of cases per DMT (Figure 2, B found due to the small number of cases. A higher pro- and C). portion of seropositivity according to each DMT was ob- served for antibodies against nucleocapsid compared with Twenty (47.6%) of the 42 patients analyzed presented both those against spike, especially in patients treated with IFN humoral and cellular responses. In severe cases, the pro- (61.5% vs 38.5%) (Figure 1, B and C). portion of those with both responses increased up to 85.7% (6 of 7) (eTable 3, links.lww.com/NXI/A693). In seronegative In patients treated with anti-CD20 therapy, the number of patients, a cellular response was observed in 5 of 7 (71.4%) months since the last infusion of COVID-19 correlated with anti–CD20-treated patients and in none of the patients IgG-S titer (r = 0.50 [95% CI 0.13–0.75]; p < 0.01). However, treated with other DMTs or untreated patients. Mean titers of no correlation was found with Ig-N antibodies or with anti- IFN-γ in anti–CD20-treated patients were similar between body titers and treatment duration. In patients treated with seropositive and seronegative PwMS (IFN-γ against Ag.1: 1.4 Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 5 Figure 1 Humoral Response to SARS-CoV-2 (A) Forest plot depicting adjusted ORs for presenting positive antibodies against SARS-CoV-2 (n = 145). De- mographic and clinical characteristics, comorbidities, and laboratory data are represented with OR, 95% CI, and p values. In dichotomous variables, the reference is not specified. Statistical analysis was performed us- ing a logistic regression model adjusted for age, sex, presenting any comorbidity, MS phenotype EDSS, DMTs, COVID-19 severity, and months of the serology after COVID-19. (B and C) Median titers of IgG against SARS-CoV-2 spike (IgG-S; A) antibody and total Igs against SARS-CoV-2 nucleocapsid (Ig-N; B), each dot represents a different subject. Statistical analysis was performed using a Mann-Whitney test analysis; only statistically significant differences are indicated. Cutoff values for antibody positivity are indicated by a dotted line. Spot’s color indicates previous lymphocyte count >1,000 U/μL (gray), between 500 and 1,000 U/μL (or- ange), and <500 cells/mm3 U/μL. Any comorbidity in- cludes obesity, lung disease, cardiovascular disease, diabetes, hypertension, hematologic benign disease, chronic kidney disease, liver disease, HIV, or malig- nancy. *Statistically significant, p value <0.05. Ab = SARS-CoV-2 antibody; ALZ = alemtuzumab; CLA = cla- dribine; COVID-19 = coronavirus disease 2019; DMF = dimethyl fumarate; EDSS = Expanded Disability Status Scale; FTY = fingolimod; GA = glatiramer acetate; Ig = immunoglobulin; IFN = interferon; NTZ = natalizumab; OCR = ocrelizumab; other anti-CD20 = other anti-CD20 therapies; other DMTs = patients with disease-modifying treatment different from anti-CD20 therapies; progressive MS = secondary progressive MS and primary progressive MS; RTX = rituximab; SARS-CoV-2 = severe acute re- spiratory syndrome coronavirus 2; TFN = teriflunomide. [SD: 2.0] vs 2.4 [SD: 2.8]; p > 0.05; Ag.2: 1.1 [SD: 1.8] vs 1.9 SARS-CoV-2 Humoral Response Persistence [SD: 2.6]; p > 0.05) (Figure 3). In anti–CD20-treated pa- To analyze humoral response persistence, a sensitivity analysis tients, mean titers of IFN-γ did not correlate with months was performed in patients with 1 serology result within the first since the last infusion or test or treatment duration (eFigures 6 months after COVID-19 and another after 6 months. Fifty- 2 and 3, links.lww.com/NXI/A693). three PwMS were included, with a median follow-up after COVID-19 of 14.2 months (IQR 0.36). Of those PwMS, 41 No correlation was found between antibody titers (Ig-N and (81.13%) were persistently positive or became positive during IgG-S) and IFN-γ titers. Moreover, previous Ig levels, follow-up. Furthermore, a persistent humoral response to total lymphocyte counts, CD19+ cells, CD4+ cells, or CD8+ cells SARS-CoV-2 after more than 12 months after COVID-19 was did not correlate with antibody or IFN-γ titers (data not shown). found in patients treated with anti-CD20 therapy, other DMTs, 6 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 Neurology.org/NN Figure 2 Cellular Response to SARS-CoV-2 (A) Forest plot depicting adjusted ORs for present- ing cellular response against SARS-CoV-2 (n = 42). Demographic and clinical characteristics, comor- bidities, and laboratory data are represented with OR, 95% CI, and p values. In dichotomous variables, the reference is not specified. Statistical analysis was performed using a logistic regression model adjusted for age, sex, presenting any comorbidity, MS phenotype EDSS, DMTs, COVID-19 severity, and positive SARS-CoV-2 antibody. (B and C) Mean titers of interferon-gamma produced by T-cell against SARS-CoV-2 antigen mix 1 (A) and SARS-CoV-2 an- tigen mix 2 (B); each dot represents a different subject. Statistical analysis was performed using a Mann-Whitney test analysis, no statistical differ- ences were found. Cutoff values for positive cellu- lar response are indicated by a dotted line. Values over the lower line of the gray area are considered positive. Spot’s color indicates previous lympho- cyte count >1,000 U/μL (gray), between 500 and 1,000 U/μL (orange), and <500 cells/mm3 U/μL. Any comorbidity includes obesity, lung disease, car- diovascular disease, diabetes, hypertension, he- matologic benign disease, chronic kidney disease, liver disease, HIV, or malignancy. *Statistically sig- nificant, p value <0.05. Ab = SARS-CoV-2 antibody; ALZ = alemtuzumab; CLA = cladribine; COVID-19 = coronavirus disease 2019; DMF = dimethyl fuma- rate; EDSS = Expanded Disability Status Scale; FTY = fingolimod; GA = glatiramer acetate; IFN = in- terferon; ns = not significant; NTZ = natalizumab; OCR = ocrelizumab; other anti-CD20 = other anti- CD20 therapies; other DMTs = patients with dis- ease-modifying treatment different from anti- CD20 therapies; progressive MS = secondary pro- gressive MS and primary progressive MS; RTX = rituximab; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; TFN = teriflunomide. and untreated patients. Nevertheless, the percentage of positive Discussion serology in anti–CD20-treated patients was lower than that in patients on other DMTs and untreated patients (Figure 4A). In In this study, we found that humoral and cellular responses to univariable analysis, patients with humoral persistence over 6 SARS-CoV-2 in convalescent COVID-19 PwMS are present months presented a higher median lymphocyte count before for up to a year after COVID-19 diagnosis and that a cellular COVID-19 than those without persistence (1,715 [IQR 685] response can be present in anti–CD20-treated patients, even vs 1,200 [IQR 100], p < 0.05) (eTable 4, links.lww.com/NXI/ in the absence of a humoral response. There are 2 main pillars A693). Regardless, no factors were independently associated of an effective antiviral response. One is cellular immunity, with humoral response persistence over 6 months in multi- specifically T-cytotoxic cells (CD8+), which eliminate infec- variable analysis (Figure 4B). ted cells. The other is humoral immunity with plasma cells Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 7 Figure 3 Cellular Response to SARS-CoV-2 according to Serostatus and Treatment (A and B) Mean IFN-γ titers produced by T cells against SARS-CoV-2 antigen mix 1 (A) and antigen mix 2 (B) according to SARS-CoV-2 antibody positivity (Ab+; Ab−) and treatment. Each dot represents a different subject. Cutoff values for positive cellular response are indicated by a dotted line. Statistical analysis was performed using a Mann-Whitney test analysis comparing differences between groups and between antibody positive and negative cases, no statistical differences were found. Ab = antibody; other DMTs = patients with disease-modifying treatment different from anti-CD20s therapies; IFN-γ = interferon- gamma; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2. that secrete neutralizing antibodies and prevent viruses from Lymphodepleting therapies such as cladribine, alemtuzumab, infecting cells. After the initial response, T helper cells co- or fingolimod might also modify immunologic responses to ordinate the long-term immune reaction, collaborating in the SARS-CoV-2 by reducing peripheral B-cell counts. We found creation of long-lived plasma cells.15 an acceptable positive rate among all 3 treatments, as seen in previous studies.8,9 Although there is growing evidence The presence of neutralizing antibodies against SARS-CoV-2 showing that the response to SARS-CoV-2 vaccination is provides the best current indication for protection against re- blunted in patients on fingolimod,10,11 controversy remains infection.16 We assessed the humoral response to SARS-CoV-2 regarding the response after COVID-19.8,9,21 Similar to other using a commercially available assay with a demonstrated corre- studies,9,21 our results suggest that antibody titers are lower in lation between IgG and antibody neutralization titers of 94.4%.17 patients on fingolimod than in those on other DMTs. Alto- In our cohort, 83.4% of patients presented antibodies against gether, confirmation of these data in larger cohorts is needed. SARS-CoV-2. In line with previous reports in PwMS, anti-CD20 Our results also suggest that male sex increases the probability therapy decreases both the probability of presenting a serologic of seroconversion, which is consistent with some of the data response and the median titers of antibodies against SARS-CoV- published for the general population, in which higher anti- 2.6-9 On the other hand, patients treated with other DMTs and body titers were found in male patients.22,23 untreated patients present an antibody positivity rate of more than 70%. Of interest, we found lower titers of anti-S IgG than Ig- The magnitude and profile of the T-cell response against SARS- N, especially in IFN-β–treated patients. These differences may be CoV-2 is heterogeneous and may be a reflection of individual due to the glycosylation state of the spike protein, which makes it immunologic responses during acute infection.24 We assessed less immunogenic.18 However, because a recent publication the specific T-cell response using a commercially available IGRA found higher IgG-S antibody titers in convalescent COVID-19 kit25 and found cellular responses in 59.5% of patients. As pre- PwMS treated with IFN and glatiramer acetate compared with viously described in the general population,26,27 the cellular re- other DMTs,9 our results should be interpreted with caution. sponse was associated with severe COVID-19 in univariable analysis. In fact, all patients with severe COVID-19 had detect- Anti-CD20 therapy affects the B-cell lineage, impairing differen- able cellular responses. This suggests an increased immune re- tiation into memory B cells or plasma cells. It is not surprising sponse with higher viral loads and inflammatory mediators then that PwMS on such treatment fails to develop a humoral during acute infection.28 response. Among these PwMS, we found a higher humoral re- sponse in those with severe COVID-19.19 Similarly, the longer it Nevertheless, we detected a specific cellular response despite was since SARS-CoV-2 infection after the last anti-CD20 infusion, the absence of a humoral response in 5 patients given anti- the higher was the serum anti-S IgG titer and proportion of CD20 therapy but not in patients on other DMTs. Some humoral response positivity. This is probably due to an increasing studies of SARS-CoV-2 infection and vaccination have already repopulation of memory B cells over the months. These results described specific cellular responses in the absence of humoral are consistent with previous findings in patients treated with anti- responses.12,13,29 In fact, a study of COVID-19 patients with CD20 therapy after COVID-199 and after vaccination,20 but given hematologic cancer treated with rituximab showed that those our cohort’s small sample size, further confirmation is needed. with a higher proportion of T cells had a better outcome.30 8 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 Neurology.org/NN Figure 4 SARS-CoV-2 Humoral Response Persistence (A) Proportion of positive SARS-CoV-2 serologies according to the months since COVID-19 diagnosis and treatment. (B) Forest plot depicting adjusted ORs for presenting antibody response persistence over 6 months (n = 53). Only patients with 2 or more serologic determinations are included. Demographic and clinical characteristics are represented with OR, 95% CI and p values. In dichotomous variables, the reference is not specified. Statistical analysis was performed using a logistic regression model ad- justed for age, sex, presenting any comorbidity, MS phenotype EDSS, DMTs, COVID-19 severity, lympho- cyte count, and RT-PCR results (not done, negative, or positive). Patient’s treatments are classified as un- treated, anti-CD20 therapies (rituximab, ocrelizu- mab, and other anti-CD20s), and other DMTs (interferon, glatiramer acetate, teriflunomide, di- methyl fumarate, cladribine, fingolimod, or alemtu- zumab). Any comorbidity includes obesity, lung disease, cardiovascular disease, diabetes, hyperten- sion, hematologic benign disease, chronic kidney disease, liver disease, HIV, or malignancy. COVID-19 = coronavirus disease 2019; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; MS = multiple sclerosis; progressive MS = secondary progressive MS and primary progressive MS; RT-PCR = reverse transcription-PCR; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2. Therefore, the cellular response might play an important role Limitations of our study are as follows. During the first wave in COVID-19 recovery when humoral immunity is impaired. of the pandemic, RT-PCR testing in our province was re- stricted to hospitalized patients. Therefore, our cohort’s first In our cohort, progressive phenotypes were less likely to pre- cases were either severe cases or patients with positive se- sent humoral and cellular responses. In both cases, the de- rology performed in the convalescence phase. This might creased response might be justified by the older age of these have led to an increased estimation of the positive serologic patients or premature immunosenescence associated with rate, as there might be an overrepresentation of patients with progressive forms,31 leading to a weakened immune response. severe COVID-19 infection. In addition, all tests were per- However, potential confounders such as anti-CD20 therapy formed according to clinical practice and not to established should be ruled out in future analyzes with larger cohorts. time points after COVID-19 diagnosis, which has led to var- iability in the time and frequency of testing after COVID-19, Humoral and cellular responses against SARS-CoV-2 are detec- increasing the heterogeneity of the sample. Another limitation ted within a few days of COVID-19 onset to up to 12 is the relatively small sample size of the study, probably leading months.32,33 We were able to detect both of them up to 13.10 to an overestimation of associations and a high degree of un- months after COVID-19 disease. The humoral response per- certainty. In the cellular response substudy in particular, there sisted for more than 6 months in 81.1% of patients with 2 de- were few cases for each DMT, which prevented us from per- terminations. In the general population, increased severity of forming group comparisons. Our strengths include the deeply COVID-19 and younger age have been associated with longer phenotyped cohort with valuable information on previous SARS-CoV-2 humoral persistence,34,35 although we did not find laboratory data and a long-term follow-up of PwMS and any association in this regard, probably because of the small COVID-19. Moreover, to detect the cellular response, we used cohort. Nonetheless, further information about long-term im- a commercially available test suitable for clinical laboratories munity in PwMS after SARS-CoV-2 vaccination is needed. and amenable to automation, making it potentially useful to Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 9 evaluate cellular response after COVID-19 or SARS-CoV-2 JR19/00007. C. Tur is currently being funded by a Junior Leader vaccination. However, it should be considered that previous La Caixa Fellowship; the fellowship code is LCF/BQ/PI20/ studies on the SARS-CoV-2 cellular response in MS have used 11760008; she has also received the 2021 Fundación Merck other methods, such as intracellular cytokine staining or other Salud Award for the Investigation in Multiple Sclerosis (Spain); in IGRAs, which may limit the reproducibility of our results. 2015, she received an ECTRIMS Post-doctoral Research Fel- lowship and has received funding from the UK MS Society. I. In conclusion, convalescent COVID-19 patients with MS Galán and J. Castilló report no disclosures relevant to the man- have preserved specific humoral and cellular responses to uscript. J. Rı́o has received speaking honoraria and personal SARS-CoV-2 up to 13 months after COVID-19, although the compensation for participating on Advisory Boards from Almirall, humoral response is reduced in patients on anti-CD20 ther- Bayer Schering Healthcare, Biogen Idec, and Genzyme. C. Espejo apy. Overall, these data provide valuable information about reports no disclosures relevant to the manuscript. M. Comabella the immune response in convalescent COVID-19 MS pa- has received compensation for consulting services and speaking tients and can be used for clinical guidance. honoraria from Bayer Schering Pharma, Merck Serono, Biogen Idec, Teva Pharmaceuticals, Sanofi-Aventis, and Novartis. C. Nos Acknowledgment has received funding for travel from Biogen Idec and F. The authors thank Dr. Moises Labrador and Dr. Manuel Hoffmann-La Roche, Ltd., and speaker honoraria from Novartis. Hernandez from the Immunology service of Vall d’Hebron service J. Sastre-Garriga has received compensation for consulting ser- for suggesting the IGRA technique and his immunologic support. vices and speaking honoraria from Almirall, Bayer, Biogen, Cel- gene, Sanofi, Merck, Novartis, Roche, Bial, Biopass, and Teva and Study Funding is member of the editorial committee of Multiple Sclerosis Journal No targeted funding reported. and director of Revista de Neurologı́a. M. Tintore has received compensation for consulting services and speaking honoraria Disclosure from Almirall, Bayer Schering Pharma, Biogen Idec, Genzyme, A. Zabalza has received travel expenses for scientific meetings Merck Serono, Novartis, Roche, Sanofi-Aventis Viela Bio, and from Biogen Idec, Merck Serono, and Novartis, speaking hono- Teva Pharmaceuticals and is coeditor of Multiple Sclerosis raria from Eisai, and a study grant from Novartis. G. Arrambide Journal–Experimental, Translational and Clinical. X. Montalban has received compensation for consulting services or participation has received speaking honoraria and travel expenses for scientific in advisory boards from Sanofi, Merck, and Roche, research meetings, has been a steering committee member of clinical trials, support from Novartis, travel expenses for scientific meetings or participated in advisory boards of clinical trials in the past 3 from Novartis, Roche, Stendhal, and ECTRIMS, and speaking years with Actelion, Alexion, Bayer, Biogen, Celgene, EMD honoraria from Sanofi and Merck and is a member of the Serono, Genzyme, Immunic, MedDay, Merck, Mylan, NervGen, International Women in Multiple Sclerosis (iWiMS) network Novartis, Roche, Sanofi-Genzyme, Teva Pharmaceutical, TG executive committee. P. Tagliani was an ECTRIMS clinical Therapeutics, Excemed, MSIF, and NMSS. Go to Neurology. fellowship awardee in 2019–2020 and has received travel ex- org/NN for full disclosures. penses for scientific meetings from Roche. S. Cárdena-Robledo was an ECTRIMS clinical fellowship awardee 2019-2020 and has Publication History received travel expenses for scientific meetings from Biogen Idec Received by Neurology: Neuroimmunology & Neuroinflammation and Genzyme, compensation for consulting services or partici- August 20, 2021. Accepted in final form December 21, 2021. pation in advisory boards from Roche and Novartis, and speaking honoraria from Novartis. S. Otero-Robledo has received com- Appendix Authors pensation for consulting services from Biogen Idec and Genzyme and research support from Novartis. J. Esperalba; C. Fernandez- Name Location Contribution Naval, J. Trocoli Campuzano, M. Martı́nez Gallo, M. Castillo, M. Ana Zabalza, Servei de Neurologia- Drafting/revision of the Bonastre, M. Resina Sallés, and J. Beltran report no disclosures MD Neuroimmunologia, Centre manuscript for content, relevant to the manuscript. P. Carbonell-Mirabent has received d’Esclerosi Múltiple de including medical writing for Catalunya (Cemcat), Vall content; major role in the travel expenses from Biogen. P.C.-M’s yearly salary is supported d’Hebron Institut de Recerca, acquisition of data; study by a grant from Biogen to Fundació Privada Cemcat toward Hospital Universitari Vall concept or design; and d’Hebron, Departament de analysis or interpretation of statistical analysis. M. Rodrı́guez-Barranco, S. López-Maza, P.J. Medicina, Universitat data Melgarejo Otálora, and M. Ruiz-Ortiz report no disclosures rel- Autònoma de Barcelona, Spain evant to the manuscript. A. Papolla is an ECTRIMS clinical Georgina Servei de Neurologia- Drafting/revision of the fellowship awardee. B. Rodrı́guez Acevedo has received honoraria Arrambide, Neuroimmunologia, Centre manuscript for content, MD, PhD d’Esclerosi Múltiple de including medical writing for for consulting services from Wellspect. L. Midaglia has received Catalunya (Cemcat), Vall content; major role in the honoraria for consulting services and speaking honoraria from d’Hebron Institut de Recerca, acquisition of data; study Hospital Universitari Vall concept or design; analysis Roche and Novartis. A. Vidal-Jordana has received compensation d’Hebron, Departament de or interpretation of data; for consulting services and speaking honoraria from Novartis, Medicina, Universitat and additional Autònoma de Barcelona, Spain contributions: study Roche, Teva, Mylan, Biogen, and Genzyme-Sanofi. A. Cobo- supervision Calvo has received grant from Instituto de Salud Carlos III, Spain; 10 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 Neurology.org/NN Appendix (continued) Appendix (continued) Name Location Contribution Name Location Contribution Paula Servei de Neurologia- Drafting/revision of the Mercè Servei de Neurologia- Major role in the acquisition Tagliani, MD Neuroimmunologia, Centre manuscript for content, Bonastre Neuroimmunologia, Centre of data d’Esclerosi Múltiple de including medical writing for d’Esclerosi Múltiple de Catalunya (Cemcat), Vall content, and major role in Catalunya (Cemcat), Vall d’Hebron Institut de Recerca, the acquisition of data d’Hebron Institut de Recerca, Hospital Universitari Vall Hospital Universitari Vall d’Hebron, Departament de d’Hebron, Departament de Medicina, Universitat Medicina, Universitat Autònoma de Barcelona, Spain Autònoma de Barcelona, Spain Simón Servei de Neurologia- Drafting/revision of the Mireia Servei de Neurologia- Major role in the acquisition Cárdenas- Neuroimmunologia, Centre manuscript for content, Resina Sallés Neuroimmunologia, Centre of data Robledo, MD d’Esclerosi Múltiple de including medical writing for d’Esclerosi Múltiple de Catalunya (Cemcat), Vall content, and major role in Catalunya (Cemcat), Vall d’Hebron Institut de Recerca, the acquisition of data d’Hebron Institut de Recerca, Hospital Universitari Vall Hospital Universitari Vall d’Hebron, Departament de d’Hebron, Departament de Medicina, Universitat Medicina, Universitat Autònoma de Barcelona, Spain Autònoma de Barcelona, Spain Susana Servei de Neurologia- Drafting/revision of the Otero- Neuroimmunologia, Centre manuscript for content, Jordina Servei de Neurologia- Major role in the acquisition Romero, MD, d’Esclerosi Múltiple de including medical writing for Beltran Neuroimmunologia, Centre of data PhD Catalunya (Cemcat), Vall content; study concept or d’Esclerosi Múltiple de d’Hebron Institut de Recerca, design; and analysis or Catalunya (Cemcat), Vall Hospital Universitari Vall interpretation of data d’Hebron Institut de Recerca, d’Hebron, Departament de Hospital Universitari Vall Medicina, Universitat d’Hebron, Departament de Autònoma de Barcelona, Spain Medicina, Universitat Autònoma de Barcelona, Juliana Microbiology Department, Drafting/revision of the Spain Esperalba, Vall d’Hebron University manuscript for content, MD, PhD Hospital, Vall d’Hebron including medical writing for Pere Servei de Neurologia- Drafting/revision of the Barcelona Hospital Campus, content; major role in the Carbonell- Neuroimmunologia, Centre manuscript for content, Universitat Autònoma de acquisition of data; study Mirabent d’Esclerosi Múltiple de including medical writing for Barcelona, Spain concept or design; analysis Catalunya (Cemcat), Vall content; major role in the or interpretation of data; d’Hebron Institut de Recerca, acquisition of data; and and additional Hospital Universitari Vall analysis or interpretation of contributions: contribution d’Hebron, Departament de data of vital reagents/tools/ Medicina, Universitat patients Autònoma de Barcelona, Spain Candela Microbiology Department, Major role in the acquisition Fernandez- Vall d’Hebron University of data; analysis or Marta Servei de Neurologia- Major role in the acquisition Naval Hospital, Vall d’Hebron interpretation of data; and Rodrı́guez- Neuroimmunologia, Centre of data Barcelona Hospital Campus, additional contributions: Barranco d’Esclerosi Múltiple de Universitat Autònoma de contribution of vital Catalunya (Cemcat), Vall Barcelona, Spain reagents/tools/patients d’Hebron Institut de Recerca, Hospital Universitari Vall Jesus Trocoli Microbiology Department, Major role in the acquisition d’Hebron, Departament de Campuzano Vall d’Hebron University of data; analysis or Medicina, Universitat Hospital, Vall d’Hebron interpretation of data; and Autònoma de Barcelona, Barcelona Hospital Campus, additional contributions: Spain Universitat Autònoma de contribution of vital Barcelona, Spain reagents/tools/patients Samuel Servei de Neurologia- Major role in the acquisition López-Maza, Neuroimmunologia, Centre of data Mónica Immunology Division, Drafting/revision of the MD d’Esclerosi Múltiple de Martı́nez Hospital Universitari Vall manuscript for content, Catalunya (Cemcat), Vall Gallo, MD, d’Hebron and Diagnostic including medical writing for d’Hebron Institut de Recerca, PhD Immunology Research content; study concept or Hospital Universitari Vall Group, Vall d’Hebron design; analysis or d’Hebron, Departament de Research Institute (VHIR), interpretation of data; and Medicina, Universitat Barcelona, Spain additional contributions: Autònoma de Barcelona, contribution of vital Spain reagents/tools/patients Pedro José Department of Major role in the acquisition Mireia Servei de Neurologia- Major role in the acquisition Melgarejo Neurology, Hospital of data Castillo Neuroimmunologia, Centre of data Otálora, MD General Universitario d’Esclerosi Múltiple de Gregorio Marañón, Madrid, Catalunya (Cemcat), Vall Spain d’Hebron Institut de Recerca, Hospital Universitari Vall Mariano Department of Neurology, Major role in the acquisition d’Hebron, Departament de Ruiz-Ortiz, Hospital Universitario 12 de of data Medicina, Universitat MD Octubre, Madrid, Spain Autònoma de Barcelona, Spain Continued Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 11 Appendix (continued) Appendix (continued) Name Location Contribution Name Location Contribution Agustin Servei de Neurologia- Major role in the acquisition Joaquı́n Servei de Neurologia- Major role in the acquisition Pappolla, Neuroimmunologia, Centre of data Castilló, MD, Neuroimmunologia, Centre of data MD d’Esclerosi Múltiple de PhD d’Esclerosi Múltiple de Catalunya (Cemcat), Vall Catalunya (Cemcat), Vall d’Hebron Institut de Recerca, d’Hebron Institut de Recerca, Hospital Universitari Vall Hospital Universitari Vall d’Hebron, Departament de d’Hebron, Departament de Medicina, Universitat Medicina, Universitat Autònoma de Barcelona, Autònoma de Barcelona, Spain Spain Breogán Servei de Neurologia- Major role in the acquisition Jordi Rı́o, Servei de Neurologia- Major role in the acquisition Rodrı́guez Neuroimmunologia, Centre of data MD, PhD Neuroimmunologia, Centre of data Acevedo, MD d’Esclerosi Múltiple de d’Esclerosi Múltiple de Catalunya (Cemcat), Vall Catalunya (Cemcat), Vall d’Hebron Institut de Recerca, d’Hebron Institut de Recerca, Hospital Universitari Vall Hospital Universitari Vall d’Hebron, Departament de d’Hebron, Departament de Medicina, Universitat Medicina, Universitat Autònoma de Barcelona, Autònoma de Barcelona, Spain Spain Luciana Servei de Neurologia- Major role in the acquisition Carmen Servei de Neurologia- Drafting/revision of the Midaglia, MD Neuroimmunologia, Centre of data Espejo, MD, Neuroimmunologia, Centre manuscript for content, d’Esclerosi Múltiple de PhD d’Esclerosi Múltiple de including medical writing for Catalunya (Cemcat), Vall Catalunya (Cemcat), Vall content; study concept or d’Hebron Institut de Recerca, d’Hebron Institut de Recerca, design; and analysis or Hospital Universitari Vall Hospital Universitari Vall interpretation of data d’Hebron, Departament de d’Hebron, Departament de Medicina, Universitat Medicina, Universitat Autònoma de Barcelona, Autònoma de Barcelona, Spain Spain Angela Vidal- Servei de Neurologia- Major role in the acquisition Manuel Servei de Neurologia- Drafting/revision of the Jordana, MD Neuroimmunologia, Centre of data Comabella, Neuroimmunologia, Centre manuscript for content, d’Esclerosi Múltiple de MD, PhD d’Esclerosi Múltiple de including medical writing for Catalunya (Cemcat), Vall Catalunya (Cemcat), Vall content, and analysis or d’Hebron Institut de Recerca, d’Hebron Institut de Recerca, interpretation of data Hospital Universitari Vall Hospital Universitari Vall d’Hebron, Departament de d’Hebron, Departament de Medicina, Universitat Medicina, Universitat Autònoma de Barcelona, Autònoma de Barcelona, Spain Spain Alvaro Cobo- Servei de Neurologia- Major role in the acquisition Carlos Nos, Servei de Neurologia- Major role in the acquisition Calvo, MD, Neuroimmunologia, Centre of data MD Neuroimmunologia, Centre of data PhD d’Esclerosi Múltiple de d’Esclerosi Múltiple de Catalunya (Cemcat), Vall Catalunya (Cemcat), Vall d’Hebron Institut de Recerca, d’Hebron Institut de Recerca, Hospital Universitari Vall Hospital Universitari Vall d’Hebron, Departament de d’Hebron, Departament de Medicina, Universitat Medicina, Universitat Autònoma de Barcelona, Autònoma de Barcelona, Spain Spain Carmen Tur, Servei de Neurologia- Major role in the acquisition Jaume Servei de Neurologia- Drafting/revision of the MD, PhD Neuroimmunologia, Centre of data Sastre- Neuroimmunologia, Centre manuscript for content, d’Esclerosi Múltiple de Garriga, MD, d’Esclerosi Múltiple de including medical writing for Catalunya (Cemcat), Vall PhD Catalunya (Cemcat), Vall content; major role in the d’Hebron Institut de Recerca, d’Hebron Institut de Recerca, acquisition of data; study Hospital Universitari Vall Hospital Universitari Vall concept or design; and d’Hebron, Departament de d’Hebron, Departament de analysis or interpretation of Medicina, Universitat Medicina, Universitat data Autònoma de Barcelona, Autònoma de Barcelona, Spain Spain Ingrid Galán, Servei de Neurologia- Major role in the acquisition Mar Tintore, Servei de Neurologia- Drafting/revision of the MD Neuroimmunologia, Centre of data MD, PhD Neuroimmunologia, Centre manuscript for content, d’Esclerosi Múltiple de d’Esclerosi Múltiple de including medical writing for Catalunya (Cemcat), Vall Catalunya (Cemcat), Vall content; major role in the d’Hebron Institut de Recerca, d’Hebron Institut de Recerca, acquisition of data; study Hospital Universitari Vall Hospital Universitari Vall concept or design; analysis d’Hebron, Departament de d’Hebron, Departament de or interpretation of data; Medicina, Universitat Medicina, Universitat and additional Autònoma de Barcelona, Autònoma de Barcelona, contributions: study Spain Spain supervision 12 Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 Neurology.org/NN 15. Radbruch A, Chang HD. A long-term perspective on immunity to COVID. Nature. 2021;595(7867):359-360. doi:10.1038/d41586-021-01557-z. Appendix (continued) 16. Post N, Eddy D, Huntley C, et al. Antibody response to SARS-CoV-2 infection in humans: a systematic review. PLoS One. 2020;15(12):e0244126. doi:10.1371/ Name Location Contribution journal.pone.0244126. 17. Bonelli F, Sarasini A, Zierold C, et al. Clinical and analytical performance of an Xavier Servei de Neurologia- Drafting/revision of the automated serological test that identifies S1/S2-neutralizing IgG in COVID-19 pa- Montalban, Neuroimmunologia, Centre manuscript for content, tients semiquantitatively. J Clin Microbiol. 2020;58(9):e01224-20. doi:10.1128/ MD, PhD d’Esclerosi Múltiple de including medical writing for JCM.01224-20. Catalunya (Cemcat), Vall content; major role in the 18. Grant OC, Montgomery D, Ito K, Woods RJ. Analysis of the SARS-CoV-2 spike d’Hebron Institut de Recerca, acquisition of data; study protein glycan shield reveals implications for immune recognition. Sci Rep. 2020; Hospital Universitari Vall concept or design; analysis 10(1):14991. doi:10.1038/s41598-020-71748-7. d’Hebron, Departament de or interpretation of data; 19. Wu J, Liang B, Chen C, et al. SARS-CoV-2 infection induces sustained humoral Medicina, Universitat and additional immune responses in convalescent patients following symptomatic COVID-19. Nat Autònoma de Barcelona, contributions: study Commun. 2021;12(1):1813. doi:10.1038/s41467-021-22034-1. Spain supervision 20. Baker D, Roberts CAK, Pryce G, et al. COVID-19 vaccine-readiness for anti-CD20- depleting therapy in autoimmune diseases. Clin Exp Immunol. 2020;202(2):149-161. doi:10.1111/cei.13495. 21. Bigaut K, Kremer L, Fabacher T, et al. Impact of disease-modifying treatments of References multiple sclerosis on anti-SARS-CoV-2 antibodies: an observational study. 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Lancet Microbe. 2021;2(6): Accessed July 4, 2021. ecdc.europa.eu/en/covid-19/surveillance/case-definition. e240-e249. doi:10.1016/S2666-5247(21)00025-2. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 9, Number 2 | March 2022 13