Received: 5 February 2021 | Revised: 29 March 2021 | Accepted: 29 March 2021 DOI: 10.1111/ajt.16599 B R I E F C O M M U N I C AT I O N Changes in humoral immune response after SARS-­CoV-­2 infection in liver transplant recipients compared to immunocompetent patients Aránzazu Caballero-­Marcos1,2 | Magdalena Salcedo1,2 | Roberto Alonso-­Fernández3 | Manuel Rodríguez-­Perálvarez2,4 | María Olmedo3 | Javier Graus Morales5 | 6,7 8 9 Valentín Cuervas-­Mons | Alba Cachero | Carmelo Loinaz-­Segurola | 10 2,11 12 Mercedes Iñarrairaegui | Lluís Castells | Sonia Pascual | 2,13 14 Carmen Vinaixa-­Aunés | Rocío González-­Grande | Alejandra Otero15 | 16 17 18 Santiago Tomé | Javier Tejedor-­Tejada | José María Álamo-­Martínez | 19 20 Luisa González-­Diéguez | Flor Nogueras-­Lopez | Gerardo Blanco-­Fernández21 | 22 23 2,24 Gema Muñoz-­Bartolo | Francisco Javier Bustamante | Emilio Fábrega | 1,2 5 Mario Romero-­Cristóbal | Rosa Martin-­Mateos | 2 6 Julia Del Rio-­Izquierdo | Ana Arias-­Milla | Laura Calatayud25 | 9 1 Alberto A. Marcacuzco-­Quinto | Víctor Fernández-­Alonso | 11 2,26 Concepción Gómez-­Gavara | Jordi Colmenero | Patricia Muñoz3 | 27 José A. Pons | the Spanish Society of Liver Transplantation (SETH) 1 Hepatology and Liver Transplantation Unit, Hospital General Universitario Gregorio Marañón, Universidad Complutense, Madrid, Spain 2 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain 3 Deparment of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain 4 Department of Hepatology and Liver Transplantation, Hospital Universitario Reina Sofía, IMIBIC, Cordoba, Spain 5 Department of Digestive Diseases, Hospital Ramón y Cajal, IRYCIS, Madrid, Spain 6 Hepatology and Liver Transplant Unit, Hospital Puerta de Hierro, IDIPHIMSA, Universidad Autónoma de Madrid, Madrid, Spain 7 Instituto de Investigación Puerta de Hierro Segovia de Aran (IDIPHISA), Madrid, Spain 8 Liver Transplant Unit, Hospital Universitari de Bellvitge, IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain 9 Department of Hepatology/HPB-­surgery/Transplantation, Hospital Universitario 12 de Octubre, Madrid, Spain 10 Liver Unit, Clinica Universidad de Navarra, Pamplona, Spain 11 Department of Internal Medicine, Liver Unit, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain 12 Liver Unit, Hospital General Universitario de Alicante, Alicante, Spain 13 Department of Hepatology and Liver Transplantation, Hospital Universitario y Politécnico La Fe, Valencia, Spain 14 Department of Liver Transplantation, Hospital Regional Universitario de Málaga, Malaga, Spain 15 Liver Transplant Unit, Hospital de A Coruña, A Coruña, Spain 16 Department of Liver Transplantation, Hospital Universitario de Santiago, Santiago de Compostela, Spain 17 Department of Gastroenterology, Hepatology and Liver Transplantation Unit, Hospital Universitario Rio Hortega, Valladolid, Spain 18 Liver Transplant Unit, Hospital Virgen del Rocío, Seville, Spain Abbreviations: ACE, angiotensin-­converting enzyme; ACE2, angiotensin-­converting enzyme 2; ARB, angiotensin II receptor blockers; CI, confidence interval; COVID-­19, coronavirus disease 2019; LT, liver transplant; OR, odds ratio; RT-­P CR, real-­time reverse transcription-­polymerase chain reaction; SARS-­CoV-­2 , severe acute respiratory syndrome coronavirus 2; SD, standard deviation. Aránzazu Caballero-­Marcos and Magdalena Salcedo contributed equally to the present work and may be considered co-­f irst authors. © 2021 The American Society of Transplantation and the American Society of Transplant Surgeons Am J Transplant. 2021;00:1–9.  amjtransplant.com | 1 2 | CABALLERO-­MARCOS et al. 19 Liver Unit and Division of Gastroenterology and Hepatology, Hospital Universitario Central de Asturias, Oviedo, Spain 20 Department of Hepatology and Liver Transplantation, Hospital Virgen de las Nieves, Granada, Spain 21 Department of HPB surgery and Liver Transplantation, Complejo Hospitalario Universitario de Badajoz, Badajoz, Spain 22 Paediatric Liver Service, University Hospital La Paz, Madrid, Spain 23 Liver Transplant and Hepatology Unit, Cruces University Hospital, Barakaldo, Spain 24 Department of Digestive Diseases, IDIVAL, Hospital Universitario Marqués de Valdecilla, Santander, Spain 25 Deparment of Clinical Microbiology and Infectious Diseases, Hospital Universitari de Bellvitge, IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain 26 Liver Transplant Unit, Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain 27 Liver Transplantation Unit, Liver Unit, Department of Surgery, IMIB, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain Correspondence Magdalena Salcedo, Hepatology and The protective capacity and duration of humoral immunity after SARS-­CoV-­2 infection Liver Transplantation Unit, Hospital are not yet understood in solid organ transplant recipients. A prospective multicenter General Universitario Gregorio Marañón, Universidad Complutense, Madrid, Spain. study was performed to evaluate the persistence of anti-­nucleocapsid IgG antibodies Email: magdalena.salcedo@icloud.com in liver transplant recipients 6 months after coronavirus disease 2019 (COVID-­19) res- olution. A total of 71 liver transplant recipients were matched with 71 immunocompe- tent controls by a propensity score including variables with a well-­known prognostic impact in COVID-­19. Paired case–­control serological data were also available in 62 liver transplant patients and 62 controls at month 3 after COVID-­19. Liver transplant recipients showed a lower incidence of anti-­nucleocapsid IgG antibodies at 3 months (77.4% vs. 100%, p < .001) and at 6 months (63.4% vs. 90.1%, p < .001). Lower levels of antibodies were also observed in liver transplant patients at 3 (p = .001) and 6 months (p < .001) after COVID-­19. In transplant patients, female gender (OR = 13.49, 95% CI: 2.17–­83.8), a longer interval since transplantation (OR = 1.19, 95% CI: 1.03–­1.36), and therapy with renin–­angiotensin–­aldosterone system inhibitors (OR = 7.11, 95% CI: 1.47–­34.50) were independently associated with persistence of antibodies beyond 6 months after COVID-­19. Therefore, as compared with immunocompetent patients, liver transplant recipients show a lower prevalence of anti-­SARS-­CoV-­2 antibodies and more pronounced antibody levels decline. KEYWORDS clinical research/practice, immune regulation, immunosuppressant, immunosuppression/ immune modulation, infection and infectious agents-­viral, infectious disease, liver transplantation/hepatology 1 | I NTRO D U C TI O N disease have been associated with a more rapid and intense ap- pearance of antibodies. 3,4 Coronavirus disease 2019 (COVID-­19) continues to raise uncer- However, no studies have evaluated the medium-­term humoral re- tainties about the medium-­ and long-­term clinical course after sponse and its protective role in liver transplant (LT) recipients. As im- disease resolution. Severe acute respiratory syndrome coronavi- munosuppressed patients may show weakened immune response to rus 2 (SARS-­C oV-­2) infection produces early detectable humoral infections, it is paramount to understand the extent and duration of hu- immune responses in most cases reported to date; however, the moral immunity after COVID-­19 resolution to delineate surveillance and duration and protective capacity of the humoral immune re- vaccination protocols. In this prospective nationwide study, we aimed to sponse are still unknown. Several studies have shown the appear- analyze the incidence, evolution, and conditioning factors of SARS-­CoV-­2 ance of neutralizing and protective anti-­S ARS-­C oV-­2 antibodies humoral response within the first 12 months post-­SARS-­CoV-­2 infection after infection, which confer protection against reinfection in the in LT recipients as compared to immunocompetent individuals. We herein following 6 months.1,2 Older age and a more severe course of the present preliminary results at 6 months post-­SARS-­CoV-­2 infection. CABALLERO-­MARCOS et al. | 3 2 | PATI E NT S A N D M E TH O DS tested by ARCHITECT were considered reactive at a cutoff index of 1.4 or greater based on previous head-­to-­head evaluations of sev- 2.1 | Study design eral immunoassays; the previously reported sensitivity and speci- ficity of the anti-­nucleocapsid assay was of 92.7% (90.20–­94.8) and This was a prospective nationwide study endorsed by the Spanish 99.9% (99.4–­100), respectively. 8 Each laboratory from the different Society of Liver Transplantation (SETH). The study was approved by participating centers processed and transported specimens accord- the research ethics committee of the Hospital Gregorio Marañón ing to standard procedures. Serum levels of immunosuppressive (HGUGM 24 August 2020, 19/2020) and the research protocol was drugs were determined in each participant center. registered at Clini​calTr​ials.gov (NCT04410471). The study was per- formed according to the principles of the Declaration of Helsinki and European Union regulation 2016/679. 2.2.2 | Clinical evaluation LT patients with COVID-­19 were prospectively enrolled as part of a nationwide study conducted from February 28 to April 7, 2020 in All clinical information was extracted from reliable electronic medi- 5 Spain. A total of 101 LT recipients infected with SARS-­CoV-­2 from 23 cal data sources and recorded in a Red-­C ap database. Demographic centers were initially included. Serological data were available in 71 of data, comorbidities, clinical features, laboratory parameters, and 101 LT recipients at 6 months, and they were compared with an iden- transplant-­related information were documented. Severe COVID-­19 tical number of immunocompetent individuals who were diagnosed was defined as admission to the intensive care unit, requirement with COVID-­19 at the Hospital Gregorio Marañón within the same of mechanical ventilation, or death, whichever occurred first, ac- timeframe (control group). Study exclusion criteria were as follows: cording to a previous study describing the clinical characteristics of death within the first 3 months after SARS-­CoV-­2 infection, active COVID-­19 in China.9 Regarding immunosuppression, management chemotherapy, previous therapy with immunoglobulins or convales- protocols for COVID-­19 in LT patients were broadly similar among cent plasma transfusions, and lack of willingness or ability to provide the different centers following the recommendations of the Spanish informed consent. In the LT group, clinical operational tolerance was Society of Liver Transplantation and the Ministry of Health through- an additional exclusion criterion, as LT recipients not receiving immu- out the study period. All patients were managed in accordance with nosuppression could be considered as immunocompetent. Cases and COVID-­19 protocols, which encouraged clinicians to reduce, but not 6 controls were matched by a propensity score analysis in a 1/1 ratio. to withdraw, immunosuppression in liver transplant recipients. The propensity score was calculated by multiple logistic regression in- cluding variables with a well-­known prognostic impact in COVID-­19: age, gender, comorbidities (diabetes, arterial hypertension, and car- 2.3 | Statistical analysis diovascular disease), hospital admission, requirement of mechanical ventilation, and admission to the intensive care unit. The nearest Continuous variables are reported as mean and standard devia- neighbor approach was used to match LT patients and immunocom- tions (SD) or as median and interquartile range, as appropriate. petent controls to ensure that both groups were comparable in terms Categorical variables are described as absolute numbers and per- of clinical characteristics and severity of COVID-­19. centages. To assess factors associated with persistence of antibod- ies at 6 months post-­infection, the X 2 test was used, with the Fisher correction, whenever appropriate. Differences between antibod- 2.2 | Data collection ies levels in both groups were compared by the U Mann–­Whitney test. Univariate and multivariate logistic regression analyses were 2.2.1 | Laboratory assays performed to identify the independent predictors of persistence of antibodies beyond 6 months in the transplant group. Enter method COVID-­19 RNA testing of nasopharyngeal/oropharyngeal swab was followed, including variables showing a p ≤ .10 in the univariate specimens was performed by real-­time reverse transcriptase-­ analysis in the multivariable model. The statistical analyses were polymerase chain reaction (RT-­PCR) assay7 at 3 and 6 months after performed using the SPSS version 22.0 (IBM Corp). Every hypoth- SARS-­CoV-­2 infection. The main outcome was the presence of esis tested was two-­t ailed and considered significant at p < .05. anti-­SARS-­Cov-­2 binding antibodies at 12 months after infection. Determination of anti-­SARS-­Cov-­2 antibodies was additionally per- formed at 3 and 6 months. We herein present preliminary results 3 | R E S U LT S at 6 months. Detection of SARS-­CoV-­2 IgG targeting nucleocap- sid protein in serum samples was performed at the Microbiology 3.1 | Study population and baseline characteristics Laboratory in the Hospital Gregorio Marañón using the Abbott ARCHITECT i2000 chemiluminescent microparticle immunoassay The evaluation of SARS-­Cov-­2 humoral response at 6 months after (Abbott). The resulting chemiluminescent reaction was measured as COVID-­19 included a total of 142 patients, with 71 in each study a relative light unit and used to calculate an index value. Specimens group. No patient was excluded from the study, however, due to 4 | CABALLERO-­MARCOS et al. logistical difficulties, no serum sample was available in 30 of the (5.6%) were receiving prednisone as a part of the maintenance im- 101 LT recipients. LT patients with and without available serum munosuppressive regime when they were diagnosed with COVID-­19 samples were comparable in terms of age distribution, sex, diabe- (Table S3). tes, hypertension, COVID-­19 severity, and hospital admission (Table The median time from LT to COVID-­19 was 8.11 years (IQR: 2.87–­ S1). COVID-­19 was confirmed in all patients by a RT-­PCR assay of 13.26). Most of the patients required hospital admission (85.92%), nasopharyngeal swab specimens, between March 6, 2020 and April but the rate of severe COVID-­19 was 9.15%. COVID-­19 therapy 7, 2020. In 116 cases (58 case–­control pairs), serological data were differed between LT recipients and controls. LT patients more fre- available at both months 3 and 6 post-­infection (Figure 1). According quently received azithromycin compared with controls (60.6% vs. to propensity score matching, both groups were comparable in 15.5%, p < .001), whereas lopinavir therapy was much less frequent terms of age distribution, sex, comorbidities, COVID-­19 severity, in LT recipients (28.2% vs. 95.8%, p < .001) (Table 1). and hospital admission (Table S2). All patients presented sympto- matic COVID-­19. The majority of patients were from the Madrid area (n = 43; 42.6%), which was the autonomous region with the 3.2 | Incidence and quantitative assessment of highest absolute number of COVID-­19 confirmed cases during the antibodies against SARS-­CoV-­2 . first wave in Spain.5 The mean of participants per center was 4.39 (range: 1–­13). LT recipients showed reduced humoral immune response to SARS-­ Among the 142 patients, males predominated (74.6%), with a CoV-­2 infection as compared with immunocompetent controls at median age of 65.5 years old (range: 25–­93) (Table 1). Arterial hyper- 3 months (77.4% vs. 100%, p < .001) and at 6 months (63.4% vs. tension was the most common comorbidity (64.8%), followed by di- 90.1%, p < .001) after COVID-­19 diagnosis (Table 2). The quan- abetes mellitus (44.4%), cardiovascular disease (12.7%), and chronic titative analysis also showed significantly lower levels of anti-­ obstructive pulmonary disease (7%) (Table 1). Almost half of the nucleocapsid IgG antibodies in LT recipients at month 3 (4.28 vs. patients (43.7%) received angiotensin-­converting enzyme inhibitors [IQR: 1.64–­5.83] vs. 5.41 [IQR: 4.15–­6 .95], p = .002) and at month (ACE) or angiotensin II receptor blockers (ARB), without differences 6 (1.94 [IQR: 0.51–­4.26] vs. 4.33 [IQR: 1.98–­6 .15], p < .001) post-­ between LT recipients and controls (p = .398). All LT patients were infection (Figure 2). Although a decline of anti-­nucleocapsid IgG receiving chronic immunosuppression. Tacrolimus was the predomi- levels was observed in both study groups, it was more pronounced nant immunosuppressant (n = 44; 62%), followed by mycophenolate in LT recipients as analyzed by the ratio between the index val- mofetil (n = 35; 49.3%) and everolimus (n = 15; 21.1%). Eight patients ues at months 6 and 3 (0.627 vs. 0.784, p = .001) (Figure 3). F I G U R E 1 Study protocol and follow-­up. No patients were excluded from the study. Serum samples were not available in all patients at 3 and 6 months after COVID-­19 due to logistical difficulties CABALLERO-­MARCOS et al. | 5 TA B L E 1 Clinical characteristics of Immunocompetent 142 patients with paired case–­control Liver transplant patients patients serological determinations at month 6 (n = 71) (n = 71) p according to the study group Age (years) 65 (60–­71) 66 (57–­73) .931 Sex (male) 54 (76.1) 52 (73.2) .847 Previous medical history Diabetes Mellitus 30 (42.3) 33 (46.5) .736 Hypertension 44 (62.0) 48 (67.6) .598 ACE inhibitors or ARB 28 (39.4) 34 (47.9) .398 Cardiovascular disease 8 (11.3) 10 (14.1) .802 Chronic obstructive 5 (7) 5 (7) 1.000 pulmonary disease Asthma 7 (9.9) 5 (7) .764 Clinical characteristics Non-­severe COVID−19 63 (88.7) 64 (90.1) 1.000 Hospital admission 60 (84.5) 62 (87.3) .810 Interval since 8.11 (2.87–­13.26) NA NA NA transplantation (years) COVID−19-­specific therapy Lopinavir 20 (28.2) 68 (95.8) <.001 Interferon beta 1 (1.4) 28 (39.4) <.001 Hydroxychloroquine 64 (91.1) 67 (94.4) .532 Azithromycin 43 (60.6) 11 (15.5) <.001 Remdesivir 0 (0) 1 (1.4) 1.000 Tocilizumab 5 (7) 10 (14.1) .275 Corticosteroids (boluses) 4 (5.6) 5 (7) 1.000 Immunosuppression at baseline Tacrolimus 44 (62) NA NA NA Cyclosporine 4 (5.6) NA NA NA Mycophenolate 35 (49.3) NA NA NA Corticosteroids 8 (11.4) NA NA NA (maintenance) Everolimus 15 (21.1) NA NA NA Data are expressed as median (IQR) or n (%). Severe COVID-­19 was defined as a requirement for respiratory support, admission to the intensive care unit, and/or death. TA B L E 2 Observed incidence of anti-­nucleocapsid IgG antibodies and levels according to the study group Liver transplant patients Immunocompetent patients Month 3 n = 62 n = 62 p Anti-­nucleocapsid IgG detected; n (%) 48 (77.4) 62 (100) <.001 Anti-­nucleocapsid IgG levels; median (IQR) 4.28 (1.64–­5.83) 5.41 (4.15–­6.95) .002 Month 6 n = 71 n = 71 p Anti-­nucleocapsid IgG 45 (63.4) 64 (90.1) <.001 detected; n (%) Anti-­nucleocapsid IgG 1.94 (0.51–­4.26) 4.33 (1.98–­6.15) <.001 levels; median (IQR) 6 | CABALLERO-­MARCOS et al. Furthermore, a higher frequency of loss of antibodies at 6 months 3.3 | Predictors of persistence of antibodies was observed in LT recipients compared to immunocompetent against SARS-­CoV-­2 in LT patients beyond 6 months controls, although without reaching statistical significance (17.8% vs. 5.2%, p = .055) (Table S4). This analysis was restricted to pa- Baseline predictors of anti-­SARS-­CoV-­2 IgG antibodies in LT pa- tients with available serum samples at 3 and 6 months and detec- tients (n = 71) were screened using binary logistic regression analysis tion of antibodies at 3 months. (Table 3). Multivariate analysis identified the following independ- We also evaluated incidence and levels of anti-­SARS-­CoV-­2 ent predictors of persistence of anti-­SARS-­CoV-­2 IgG antibodies antibodies at 6 months excluding patients treated with interferon at 6 months post-­infection: female gender (odds ratio [OR] = 13.49, beta since its immunomodulatory activity could affect the immune 95% confidence interval [CI]: 2.17–­83.8, p = .005), interval since response. However, LT recipients continued to show a reduced LT (OR = 1.19, 95% CI: 1.03–­1.36, p = .018), and treatment with humoral immune response to SARS-­CoV-­2 infection as compared ACE inhibitors or ARB (OR = 7.11, 95% CI: 1.47–­34.50, p = .015). with immunocompetent controls (62.9% vs. 95.3%, p < .001). The Interestingly, we failed to link the type of immunosuppressive quantitative analysis also showed significantly lower levels of anti-­ therapy with the persistence of anti-­SARS-­CoV-­2 IgG antibodies at nucleocapsid IgG antibodies in LT recipients (1.89 [IQR: 0.49–­4.24] 6 months post-­infection. vs. 4.36 [IQR: 1.98–­6.15], p < .001; Table S5). The two study groups remained comparable in terms of age, sex, disease severity, and co- morbidities (Table S6). 4 | DISCUSSION We report the first longitudinal study analyzing the specific hu- moral immune response (anti-­nucleocapsid IgG antibodies) against SARS-­CoV-­2 in LT recipients after symptomatic SARS-­CoV-­2 in- fection compared with immunocompetent controls. Evidence of post-­infection immunity was observed in LT patients regardless of chronic exposure to immunosuppressive agents; however, despite a similar epidemiological pattern and disease severity, LT patients showed an earlier and more pronounced decline of serum levels of anti-­nucleocapsid IgG antibodies as compared with immunocompe- tent controls. Seroconversion rates after SARS-­CoV-­2 symptomatic infection range from 91% to 99%. 2,10 Moreover, SARS-­CoV-­2 infection pro- duces variable antibody durability2,11 and may induce strong mem- ory B-­cell responses, despite low plasma neutralizing activity.11,12 F I G U R E 2 Observed levels of anti-­nucleocapsid IgG antibodies Chronic immunosuppression may influence immunological response at 3 and 6 months post-­infection in liver transplant patients (dark against pathogens. Therapy with calcineurin inhibitors preferen- gray bars) and immunocompetent controls (light gray bars). Bars represent mean levels of antibodies. Error bars indicate the 95% tially inhibits the primary immune response to new antigens,13 and confidence interval therefore a weaker response to SARS-­CoV-­2 would be expected as F I G U R E 3 Anti-­SARS-­CoV-­2 IgG levels kinetics. The kinetic is presented for each liver transplant patient (n = 58) and immunocompetent control (n = 58) CABALLERO-­MARCOS et al. | 7 TA B L E 3 Clinical predictors of detectable anti-­SARS-­CoV-­2 IgG antibodies in liver transplant patients 6 months after COVID-­19 (n = 71) Univariate analysis Multivariate analysis Variables OR (95% CI) p OR (95% CI) p Age 1.06 (1.00–­1.10) .026 1.01 (0.94–­1.08) .873 Sex (female) 3.46 (0.89–­13.47) .073 13.49 (2.17–­83.80) .005 Interval since liver transplantation 1.17 (1.06–­1.28) .002 1.19 (1.03–­1.37) .018 Diabetes Mellitus 0.78 (0.29–­2.06) .613 Hypertension 2.21 (0.82–­5.99) .117 ACE inhibitors or ARB 9.58 (2.51–­36.57) .001 7.11 (1.47–­34.51) .015 Cardiovascular disease 0.54 (0.12–­2.36) .410 Severe COVID−19 1.85 (0.34–­9.90) .474 Hospital admission 0.60 (0.15–­2.51) .487 Lopinavir 3.03 (0.89–­10.36) .076 2.33 (0.46–­11.76) .365 Hydroxychloroquine 0.67 (0.12–­3.71) .643 Azithromycin 1.55 (0.58–­4.15) .380 Tocilizumab 0.86 (0.13–­5.50) .871 Corticosteroids (boluses) 1.79 (0.18–­18.11) .624 Tacrolimus a 0.25 (0.08–­0.78) .017 0.37 (0.08–­1.67) .193 Cyclosporinea 1.79 (0.18–­18.11) .624 Mycophenolatea 0.96 (0.36–­2.51) .928 Corticosteroids (maintenance)a 0.30 (0.07–­1.38) .122 Everolimusa 2.79 (0.71–­11.00) .143 Month 6 tacrolimus 1.02 (0.53–­1.99) .947 Month 6 trough concentrations (tacrolimus) 0.79 (0.59–­1.07) .131 Month 6 mycophenolate 1.18 (0.67–­2.10) .568 Month 6 corticosteroids 0.96 (0.57–­1.64) .888 Month 6 everolimus 1.27 (0.72–­2.24) .415 Bold values indicate the variables independently associated with antibody persistence at 6 months in the multivariate analysis. a These variables pertain to active immunosuppression therapy at COVID-­19 diagnosis. compared with immunocompetent individuals. Interestingly, a more factor of severe COVID-­19 in a dose-­dependent manner5 while tac- pronounced IgG decrease after SARS-­CoV-­2 infection has been rolimus can decrease mortality rates. 21 Regarding humoral immune described in kidney transplant patients treated with calcineurin response, we failed to demonstrate an association between the type inhibitors.14 Mycophenolate mofetil has also been associated with of immunosuppression and the persistence of protective antibodies. 15 a reduction in post-­infection antibody production, as well as de- This could be explained by the heterogeneity of immunosuppression creased humoral immune response to the influenza vaccine.16-­18 protocols in our cohort and by the limited sample size. Conversely, more intense humoral immune response has been de- Another relevant finding of our investigation is that humoral scribed in older patients, particularly in those with a more severe response against COVID-­19 was more intense in patients receiving COVID-­19.10,19,20 In our study, LT recipients and immunocompetent ACE inhibitors or ARB. The angiotensin-­converting enzyme 2 (ACE2) patients were matched according to these factors, being their im- is a cellular receptor which is required for SARS-­CoV-­2 entry and munocompromised status a possible conditioning factor of lower propagation in host cells. Experimental evidence have shown that humoral immune response. Chronic immunosuppression is con- treatment with ACE inhibitors or ARB can increase ACE2 expres- sidered a double-­edge sword in COVID-­19. While it may facilitate sion, 22 thus theoretically promoting SARS-­CoV-­2 susceptibility and viral replication in the early phase of the infection, it could also replication, that could ultimately result in stronger humoral immune ameliorate the aberrant immune response which the most severe response. Female gender was another independent predictor of an- forms of the disease produce. This may explain why LT patients have tibody development and persistence after COVID-­19. This interest- twofold greater incidence rates of COVID-­19 but slightly reduced ing finding could be explained by the stronger innate and adaptive mortality as compared with age-­ and gender-­matched general pop- immunity, and estrogen upregulated ACE2 expression in women. 23 5 ulations. However, not all immunosuppressive drugs may have the Finally, we also found that the interval since LT was another inde- same effect in COVID-­19. Mycophenolate mofetil therapy is a risk pendent predictor of antibody development. As a greater interval 8 | CABALLERO-­MARCOS et al. since LT is generally associated with lower immunosuppression load Moreover, none of these agents were identified as predictors of and older age, these results were expected. antibody persistence in the multivariate analysis. Although cau- While SARS-­CoV-­2 antibodies seem to protect against reinfection, tion must be exercised when attempting to generalize our results, antibody levels following infection generally decline. The kinetics of this is the first study demonstrating a key aspect of the immune waning antibodies appear to differ according to the severity of infec- response against SARS-­C oV-­2 infection that may have implica- tion. A greater reduction of antibodies has been shown in less severe tions for the vaccination of LT population. COVID-­19 cases,24 although in patients with greater severity of the In conclusion, LT patients exhibit a lower persistence of anti-­ disease and higher antibody levels, these levels will also eventually de- nucleocapsid IgG antibodies within the first 6 months post-­infection cline.4 We observed persistence of anti–­nucleocapsid IgG antibodies and more pronounced antibody levels decline. These results call for at 6 months after the onset of symptoms in most patients. Similar find- the need for specific studies regarding vaccination in solid organ ings have recently been described in kidney transplant recipients.14 transplant recipients receiving chronic immunosuppression to adapt Persistence of anti-­spike IgG antibodies 6 months after COVID-­19 has dosing and surveillance protocols. also been described in other studies,11 although accompanied by a de- cline in SARS-­CoV-­2-­specific CD4+T cells and CD8+T cells. D I S C LO S U R E Other elements of innate or cellular immunity may confer pro- The authors of this manuscript have no conflicts of interest to dis- tection to SARS-­CoV-­2 reinfection despite the absence of measur- close as described by the American Journal of Transplantation. able antibodies. Interestingly, the incidence of reinfection has been inversely associated with baseline antibody levels, including those DATA AVA I L A B I L I T Y S TAT E M E N T below the positive threshold.1 Ongoing follow-­up studies and fu- The data that support the findings of this study are available from ture research are needed after infection and vaccination, including the corresponding author upon reasonable request. the evaluation of waning antibodies and persistence of B-­cell and T-­cell memory to SARS-­CoV-­2. Furthermore, given the lower hu- ORCID moral immune response we have demonstrated in LT recipients Aránzazu Caballero-­Marcos https://orcid. after COVID-­19, it will be crucial to specifically assess the magnitude org/0000-0001-8116-213X and duration of protection against reinfection by vaccination in this Magdalena Salcedo https://orcid.org/0000-0002-1239-5746 population. Additionally, as LT recipients present a higher risk of de- Manuel Rodríguez-­Perálvarez https://orcid. veloping COVID-­195 and can act as disease vectors, the effect of org/0000-0002-1795-2919 protection of vaccination on transmission should also be assessed María Olmedo https://orcid.org/0000-0003-2398-9619 in future studies. Javier Graus Morales https://orcid.org/0000-0002-0328-8041 Our study is not without limitations. The high incidence of Valentín Cuervas-­Mons https://orcid.org/0000-0003-3086-9463 post-­infection antibodies in both study groups may be conditioned Alba Cachero https://orcid.org/0000-0002-0010-4534 by the high proportion of patients with pneumonia that required Carmelo Loinaz-­Segurola https://orcid.org/0000-0002-1873-0568 hospitalization, which represents a more severe disease to that Mercedes Iñarrairaegui https://orcid.org/0000-0001-9180-4693 observed in the general population. This may be explained by the Lluís Castells https://orcid.org/0000-0002-6672-5931 difficulties in accessing PCR diagnosis in the mildest or asymptom- Sonia Pascual https://orcid.org/0000-0002-4265-5019 atic COVID-­19 cases that existed during the first wave in Spain. Carmen Vinaixa-­Aunés https://orcid.org/0000-0001-5060-4556 On the other hand, we are aware that the test we used for the Rocío González-­Grande https://orcid.org/0000-0002-7691-5755 detection of antibodies, which is one of the most widely used, is Alejandra Otero https://orcid.org/0000-0002-9016-3705 not factually quantitative, and although it correlates well with the Javier Tejedor-­Tejada https://orcid.org/0000-0002-3585-5733 amount of antibodies present in the sample, it is not a titration José María Álamo-­Martínez https://orcid. technique in the strictest sense. Furthermore, the shorter half-­life org/0000-0002-8503-7552 of antibodies targeting nucleocapsid compared with antibodies Luisa González-­Diéguez https://orcid.org/0000-0003-1098-3891 targeting spike protein25 could have underestimated the propor- Flor Nogueras-­Lopez https://orcid.org/0000-0002-2420-1005 tion of seropositive patients in our study. Finally, COVID-­19 ther- Gerardo Blanco-­Fernández https://orcid. apies were heterogeneous in our cohort and a potential impact org/0000-0003-4845-5306 on subsequent humoral response cannot be ruled out. This het- Gema Muñoz-­Bartolo https://orcid.org/0000-0002-6207-7463 erogeneity could be explained by the potential pharmacological Francisco Javier Bustamante https://orcid. interactions of antiviral therapy with immunosuppression; con- org/0000-0002-5280-3038 sequently, this type of therapy was generally avoided in LT pa- Emilio Fábrega https://orcid.org/0000-0003-1876-3973 tients. Furthermore, as interferon beta therapy may lead to acute Mario Romero-­Cristóbal https://orcid.org/0000-0003-1633-2862 rejection and immune-­m ediated complications it was rarely used. Rosa Martin-­Mateos https://orcid.org/0000-0001-5874-211X However, when we analyzed humoral immune response excluding Ana Arias-­Milla https://orcid.org/0000-0002-1040-3140 patients treated with interferon beta, we obtained similar results. Laura Calatayud https://orcid.org/0000-0001-6182-4538 CABALLERO-­MARCOS et al. | 9 16. Duchini A, Hendry RM, Nyberg LM, et al. Immune response to in- Víctor Fernández-­Alonso https://orcid.org/0000-0002-4018-9931 fluenza vaccine in adult liver transplant recipients. Liver Transplant. Jordi Colmenero https://orcid.org/0000-0001-6024-8479 2001;7(4):311-­313. Patricia Muñoz https://orcid.org/0000-0001-5706-5583 17. Soesman N, Rimmelzwaan GF, Nieuwkoop NJ, et al. Efficacy of in- fluenza vaccination in adult liver transplant recipients. J Med Virol. José A. Pons https://orcid.org/0000-0003-4606-1557 2000;61(1):85-­93. 18. Khedmat H, Karbasi-­Afshar R, Izadi M, et al. 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