key: cord-0872695-8je0vu8a authors: Favà, Alexandre; Donadeu, Laura; Sabé, Nuria; Pernin, Vincent; González‐Costello, José; Lladó, Laura; Meneghini, Maria; Charmetant, Xavier; García‐Romero, Elena; Cachero, Alba; Torija, Alba; Rodriguez‐Urquia, Ronny; Crespo, Elena; Teubel, Iris; Melilli, Edoardo; Montero, Nuria; Manonelles, Anna; Preyer, Rosemarie; Strecker, Kevin; Ovize, Anne; Lozano, Juan J.; Sidorova, Julia; Cruzado, Josep M.; Le Quintrec, Moglie; Thaunat, Olivier; Bestard, Oriol title: SARS‐CoV‐2‐specific serological and functional T cell immune responses during acute and early COVID‐19 convalescence in solid organ transplant patients date: 2021-04-12 journal: Am J Transplant DOI: 10.1111/ajt.16570 sha: c8e91871e247998c6e7b51adac2779ad3d9ee76e doc_id: 872695 cord_uid: 8je0vu8a The description of protective humoral and T cell immune responses specific against SARS‐CoV‐2 has been reported among immunocompetent (IC) individuals developing COVID‐19 infection. However, its characterization and determinants of poorer outcomes among the at‐risk solid organ transplant (SOT) patient population have not been thoroughly investigated. Cytokine‐producing T cell responses, such as IFN‐γ, IL‐2, IFN‐γ/IL‐2, IL‐6, IL‐21, and IL‐5, against main immunogenic SARS‐CoV‐2 antigens and IgM/IgG serological immunity were tracked in SOT (n = 28) during acute infection and at two consecutive time points over the following 40 days of convalescence and were compared to matched IC (n = 16) patients admitted with similar moderate/severe COVID‐19. We describe the development of a robust serological and functional T cell immune responses against SARS‐CoV‐2 among SOT patients, similar to IC patients during early convalescence. However, at the infection onset, SOT displayed lower IgG seroconversion rates (77% vs. 100%; p = .044), despite no differences on IgG titers, and a trend toward decreased SARS‐CoV‐2‐reactive T cell frequencies, especially against the membrane protein (7 [0–34] vs. 113 [15–245], p = .011, 2 [0–9] vs. 45 [5–74], p = .009, and 0 [0–2] vs. 13 [1–24], p = .020, IFN‐γ, IL‐2, and IFN‐γ/IL‐2 spots, respectively). In summary, our data suggest that despite a certain initial delay, SOT population achieve comparable functional immune responses than the general population after moderate/severe COVID‐19. While most people remain asymptomatic or develop only mild symptoms during COVID-19, 1,2 some specific group of patients seem to be at significantly higher risk of fatal outcomes, 3 and among them recipients of solid organ transplants (SOT) most likely because they receive chronic immunosuppressive therapy that predominantly targets T cell adaptive immunity. 4 Importantly, SOT patients represent an important prevalent high-risk population in whom the biology of the adaptive immunity specific to SARS-CoV-2 during COVID- 19 has not yet been thoroughly investigated. First studies evaluating immunocompetent (IC) convalescent individuals have shown the induction of neutralizing antibodies after primary infection [5] [6] [7] [8] which seem to be detectable essentially among patients with more severe forms of COVID-19. 9,10 Conversely, robust anti-viral T cell responses have been described after SARS-CoV-2 infection, which seem to correlate with the magnitude of SARS-CoV-2-specific IgG and IgA titers during the initial phase of convalescence 11 and with the severity of COVID-19 infection. 12 Interestingly, SARS-CoV-2-reactive T cell immunity seems to last for a longer period of time, even among seronegative convalescent patients 13 and can discriminate those patients with the poorest outcomes. 14 In this study, we aimed at investigating the IgM and IgG serological antibody responses as well as the SARS-CoV-2-reactive T cell responses against main four different structural viral proteins, Spike In this study, we evaluated 44 consecutive patients hospitalized be- Table 1 ). A total of 113 serially collected peripheral blood samples at three different time points of the disease were analyzed in this study-during the acute phase of infection (T1; median 16, IQR 12-19 days after symptom onset) and at two convalescence periods (T2; median 32, IQR 25-37 days, and T3; 49 days, IQR 43-53), which represented a median of 7 days, IQR 4-11 and 23 days, IQR 20-27 and 40 days, and IQR 37-44, after first positive PCR, respectively. Additionally, PBMC samples from 16 non-immunosuppressed patients on the waiting list for kidney transplantation that were obtained 2 years before the COVID-19 outbreak (November 2018) and were stored in our biobank facilities were used as healthy controls (HC). All clinical, demographic, and immunological patient characteristics as well as the main outcomes, such as mortality, or the need of invasive/non-invasive mechanical ventilation (MV) were recorded. COVID-19 disease severity was defined according to the level of oxygen support during hospitalization according to the World Health Organization interim guidance to define Acute Respiratory Distress Syndrome (bilateral opacities not explained by volume overload with an oxygen saturation/fraction of inspired oxygen ratio <315). 15 The study was approved by the Ethical Review Boards (PR115/20) at each center and patients were recruited in the study after providing a signed informed consent. IgG titers, and a trend toward decreased SARS-CoV-2-reactive T cell frequencies, especially against the membrane protein (7 [0-34] vs. 113 , p = .011, 2 [0 -9] vs. 45 , p = .009, and 0 [0-2] vs. 13 , p = .020, IFNγ, IL-2, and IFNγ/IL-2 spots, respectively). In summary, our data suggest that despite a certain initial delay, SOT population achieve comparable functional immune responses than the general population after moderate/severe COVID-19. adaptive immunity, basic (laboratory) research / science, clinical research / practice, COVID-19 infection, heart transplantation / cardiology, infection and infectious agents, kidney transplantation / nephrology, liver transplantation / hepatology, solid organ transplantation, T cell biology Detailed description is depicted in Data S1. IgM and IgG antibodies against SARS-CoV-2 were detected by a Overlapping peptide pools covering the whole Influenza virus antigen length (AID® Gmbh) were also tested. In each test, complete medium alone and Pokeweed (PWM) mitogen were used as negative and positive controls, respectively. Any antigen-specific ELISPOT test with less than 5 spots/2 × 10 5 PBMC was considered as negative when assessed in a qualitative manner. Precise information is provided in Data S1. Continuous variables were expressed as mean ±SD or median and IQR and categorical variables as number of total (n) and percentage (%). A comparison between groups was performed using Pearson's χ 2 test for categorical data. Continuous measurements were compared among groups using Kruskal-Wallis and Mann-Whitney U test for non-normally distributed data, while ANOVA and t tests were used when data were normally distributed. p-values <.05 were considered statistically significant. SARS-CoV-2-reactive cellular and humoral responses were centered and scaled and heatmap was built by means of the pheatmap R package 16 using Euclidean distance and complete method as agglomeration method. R package version 1.0.12 was used https://CRAN.R-proje ct.org/packa ge=pheatmap. All other analyses were performed using SPSS version 26 software, and graphs were generated using GraphPad Prism version 8.0 software (GraphPad Software). Our first analysis showed that while both SOT and IC patients displayed abnormally low total lymphocyte counts, this lymphopenia was more pronounced for SOT recipients (866 ± 427 vs. 1531 ± 490 in IC; p < .001). Total lymphocyte counts in HC were 1564 ±427 and were significantly higher than SOT at T1 (p < .001) ( Figure S2 ). As shown in Figure 2A and Figure Likewise, more predominant IgM responses were observed among SOT than IC, whereas IgG-specific antibodies were similarly detected. Conversely, non-SARS-CoV-2-reactive T cell immune responses against influenza and a polyclonal stimuli (PWM) were significantly weaker within both SOT and IC as compared to HC at baseline, which persisted during the convalescence period. No correlation was observed between absolute lymphocyte counts and SARS-CoV-2-reactive T cell frequencies for each antigenspecific cytokine-producing T cell (IFNγ, IL-2, IFNγ/IL-2, IL-6, IL-21, and IL-5) at any time point of the study (Table S1 ). A strong correlation was observed between all four SARS-CoV-2 antigen responses (Table S2) , showing a wide and different range of T cell frequencies. As illustrated in Figure 3A and described in While IC patients showed similarly high T cell immune responses against both antigens S and M, the highest immune response among SOT was only against antigen S. Of note, T cell responses against antigen E were barely detectable in all infected patients ( Figure S4A ). As illustrated in Figure S5A , a higher proportion of SARS-CoV-2 T cell non-responders was observed among SOT as compared to IC, and especially those IFNγ/IL-2-producing T cells. All infected patients showed detectable SARS-CoV-2-specific IgM titers at baseline ( Figure 5A) To investigate the degree of general immune impairment in patients In our study, 10 (22.7%) patients required MV or died during the follow-up, being nine SOT. As depicted in Table S8 , we did not find any Figure 7B) . Furthermore, the proportion of IgG seroconversion was numerically lower among those with worse outcomes (80% vs. 62.5%, p = .245). In terms of immunosuppression, while mycophenolate was broadly withdrawn in our cohort (Table S9 ), no differences were found between patients with or without CNI-based immunosuppressive regimens at T1. Also, no differences were observed at the successive time points for those patients who had the CNI withdrawn during the infection phase (data not shown). In this study, we investigated the magnitude and kinetics of adap- A widely reported viral-related effect is the severe peripheral lymphopenia observed during COVID-19 infection. [17] [18] [19] Indeed, it F I G U R E 2 Heatmaps generated by hierarchical clustering of SARS-CoV-2-specific and non-specific immune responses for SOT, IC patients, and HC, according to the COVID-19 disease severity (0 = no oxygen need; 1 = oxygen need; 2 = acute respiratory distress syndrome, 3 = death). Immune responses used for clustering were differentially expressed (fold change >2, false discovery rate p < .05). Gray Interestingly, a progressive emergence of both IL-5-and IL-21producing T cells was detected during the convalescent period in both groups. Although we did not phenotypically characterize these immune cells due to the lack of viable cell samples, these data suggest the fact that for an optimal B-cell activation, cognate T cell help, most likely through antigen-specific follicular helper T cells, is needed. 21 As similarly described in a recent published report, 28 Finally, we did not find SARS-CoV-2-reactive T cell responses against any of the four viral antigens in any HC thus, no evidence for T cell immune cross-reactivity was observed in out cohort, at least in vitro. Despite the presence of IL-6-producing T cell responses against SARS-CoV-2 in HC suggesting unspecific T cell stimulation, the assessment of SARS-CoV-2-reactive IL-6-producing T cell frequencies over time showed a similar pattern than that also observed in other T cell compartments. There are some limitations in this study such as the small sam- We thank CERCA Program / Generalitat de Catalunya for their institutional support. We want to particularly acknowledge the patients F I G U R E 6 T cell responses against non-specific SARS-CoV-2 antigens (influenza and PWM) at the different time points of study. Percentile 5-95 represented by whiskers; median and IQR inside the boxes. Intragroup paired analysis; *p < .05 evaluated with Friedman's test. Significant differences with healthy controls are shown by **p < .05 (analyzed by Mann-Whitney U test). 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