key: cord-1008547-4ig0mopw authors: Hueso, Thomas; Pouderoux, Cécile; Péré, Hélène; Beaumont, Anne-Lise; Raillon, Laure-Anne; Ader, Florence; Chatenoud, Lucienne; Eshagh, Déborah; Szwebel, Tali-Anne; Martinot, Martin; Camou, Fabrice; Crickx, Etienne; Michel, Marc; Mahevas, Matthieu; Boutboul, David; Azoulay, Elie; Joseph, Adrien; Hermine, Olivier; Rouzaud, Claire; Faguer, Stanislas; Petua, Philippe; Pommeret, Fanny; Clerc, Sébastien; Planquette, Benjamin; Merabet, Fatiha; London, Jonathan; Zeller, Valérie; Ghez, David; Veyer, David; Ouedrani, Amani; Gallian, Pierre; Pacanowski, Jérôme; Mékinian, Arsène; Garnier, Marc; Pirenne, France; Tiberghien, Pierre; Lacombe, Karine title: Convalescent plasma therapy for B-cell–depleted patients with protracted COVID-19 date: 2020-11-12 journal: Blood DOI: 10.1182/blood.2020008423 sha: d47fa7f9daff9cf67380207ff555c28970bf73f9 doc_id: 1008547 cord_uid: 4ig0mopw Abstract Anti-CD20 monoclonal antibodies are widely used for the treatment of hematological malignancies or autoimmune disease but may be responsible for a secondary humoral deficiency. In the context of COVID-19 infection, this may prevent the elicitation of a specific SARS-CoV-2 antibody response. We report a series of 17 consecutive patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms, negative immunoglobulin G (IgG)-IgM SARS-CoV-2 serology, and positive RNAemia measured by digital polymerase chain reaction who were treated with 4 units of COVID-19 convalescent plasma. Within 48 hours of transfusion, all but 1 patient experienced an improvement of clinical symptoms. The inflammatory syndrome abated within a week. Only 1 patient who needed mechanical ventilation for severe COVID-19 disease died of bacterial pneumonia. SARS-CoV-2 RNAemia decreased to below the sensitivity threshold in all 9 evaluated patients. In 3 patients, virus-specific T-cell responses were analyzed using T-cell enzyme-linked immunospot assay before convalescent plasma transfusion. All showed a maintained SARS-CoV-2 T-cell response and poor cross-response to other coronaviruses. No adverse event was reported. Convalescent plasma with anti–SARS-CoV-2 antibodies appears to be a very promising approach in the context of protracted COVID-19 symptoms in patients unable to mount a specific humoral response to SARS-CoV-2. Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, Imagine Institute, INSERM, Paris, France; 16 Intensive Care Unit, Hôpital Saint-Louis, AP-HP, Paris Diderot Sorbonne University, Paris, France; 17 Laboratory of Molecular Mechanisms of Hematologic Disorders and Therapeutic Implications, Imagine Institute, Paris, France; 18 Department of Clinical Hematology, Hôpital Necker-Enfants-Malades, Paris, France; 19 Infectious Diseases Department, Necker-Enfants Malades Hospital, AP-HP, Paris, France; 20 Anti-CD20 monoclonal antibodies are widely used for the treatment of hematological malignancies or autoimmune disease but may be responsible for a secondary humoral deficiency. In the context of COVID-19 infection, this may prevent the elicitation of a specific SARS-CoV-2 antibody response. We report a series of 17 consecutive patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms, negative immunoglobulin G (IgG)-IgM SARS-CoV-2 serology, and positive RNAemia measured by digital polymerase chain reaction who were treated with 4 units of COVID-19 convalescent plasma. Within 48 hours of transfusion, all but 1 patient experienced an improvement of clinical symptoms. The inflammatory syndrome abated within a week. Only 1 patient who needed mechanical ventilation for severe COVID-19 disease died of bacterial pneumonia. SARS-CoV- Anti-CD20 monoclonal antibodies (MoAbs), such as rituximab, represent the cornerstone of treatment for most patients with B-cell malignancies and, to a lesser extent, patients with autoimmune disease. 1,2 Repeated administrations of rituximab may lead to prolonged B-cell depletion, which impairs the adaptive immune response and the ability to produce neutralizing antibodies. 3, 4 Patients with hematological malignancies or autoimmune diseases may be at higher risk for severe forms of COVID-19. [5] [6] [7] Those patients are often excluded from clinical trials testing COVID-19 drugs and urgently need therapeutic options. In the past, convalescent plasma transfusion (CPT) has been used for numerous viral epidemics, such as severe acute respiratory syndrome, Middle East respiratory syndrome, or influenza. 8 This therapeutic strategy appears to be promising for severe COVID-19, as well. [9] [10] [11] [12] As a proof of concept, this approach should be of particular interest in patients who are unable to produce neutralizing antibodies. In this article, we report the safety and efficacy of CPT in 17 patients with profound B-cell lymphopenia and protracted COVID-19 disease. This nationwide, observational, and multicenter study was conducted in 13 French hospitals from 1 May 2020 to 30 June 2020. All patients presenting with a B-cell immunodeficiency and prolonged COVID-19 symptoms, confirmed by SARS-CoV-2-specific reverse transcription polymerase chain reaction (RT-PCR) in respiratory samples and without seroconversion, were eligible for CPT. The severity of COVID-19 disease was evaluated using the World Health Organization classification. 13 Patients gave their written informed consent for the retrospective data collection, and ethical clearance was obtained from the French Infectious Diseases Society. Convalescent donors were eligible for plasma donation 15 days after resolution of COVID-19 disease. Collected apheresis plasma underwent pathogen reduction (Intercept blood system; Cerus, Concord, CA) and standard testing, as per current regulations in France. Additionally, anti-SARS-CoV-2 antibody content was assessed in each donation, with a requirement for a SARS-CoV-2 seroneutralization titer $ 40 and/or an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (EUROIMMUN, Bussy-Saint-Martin, France) ratio . 5.6 as further described in the supplemental Data (available on the Blood Web site). 14 Convalescent plasma was delivered through the National Early Access Program 15 . SARS-CoV-2 serology was performed using the IgG enzymelinked immunosorbent assays in use in the different hospitals. 16 SARS-CoV-2 RNAemia was quantified using droplet-based digital RT-PCR (ddPCR) technology (Stilla Technologies, Villejuif, France), based on a COVID-19 Multiplex Crystal Digital PCR detection kit (ApexBio, Houston, TX). 17, 18 Virus-specific T-cell responses were analyzed before CPT in peripheral blood mononuclear cells using an interferon-g (IFN-g) enzyme-linked immunospot assay after the addition of individual 15-mers 11-aa overlapping peptide pools of different SARS-CoV-2 proteins or of common coronavirus proteins. 19 Each patient received 2 consecutive transfusions of 2 ABOcompatible convalescent plasma units (200-220 mL each) at days 0 and 11. The Elisa ratio and neutralization titers of transfused convalescent plasma units are detailed in supplemental Table 1 . Clinical parameters (temperature and oxygen need) were collected daily from day 15 before to day 17 after the last plasma transfusion. Biological parameters, including inflammatory markers (C-reactive protein [CRP], ferritin) and circulating lymphocyte subpopulations, were also assessed. When available, plasma interleukin-6 (IL-6) was quantified in a subset of patients who did not receive tocilizumab. Seventeen consecutive patients treated with CPT were included (Table 1 ). Fifteen patients were treated for hematological malignancies, 1 patient was treated for multiple sclerosis, and 1 patient was diagnosed with common variable immune deficiency during COVID-19 disease. Fifteen patients had received anti-CD20 MoAbs within the last 2 years (median number of cycles, 7; range, [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , with an interval between the last rituximab injection and symptom onset of 4 months (range, 3-6). Patients had protracted COVID-19 symptoms for a median of 56 days (range, 7-83). The patient with symptomatic COVID-19 for only 7 days (following chemotherapy) experienced a prior asymptomatic phase over the previous 8 weeks (as evidenced by positive nasopharyngeal swab), suggesting that he was, in fact, experiencing a protracted form of COVID-19. Ten patients required oxygen by nasal prongs or noninvasive ventilation, and 2 required mechanical ventilation. Specific treatments had been administered before CPT in 11 patients. Three had shown a temporary clinical improvement following remdesivir (n 5 2) or tocilizumab (n 5 1) but had relapsed within a few days after treatment completion. A severe hypogammaglobulinemia (median, 3.5 g/L; range, 1.8-14) was noted in 15 patients, whereas the remaining 2 patients received gamma globulin supplementation. No patient previously treated with anti-CD20 MoAbs had detectable circulating B cells. Two patients with pancytopenia had a positive SARS-CoV-2 RT-PCR in the bone marrow aspirate (Table 1) . No serious adverse effect was observed during or after CPT. Almost all patients experienced a very fast clinical improvement. Fever abated within the first 48 hours, and all 10 oxygendependent patients could be weaned from the oxygen mask or noninvasive ventilation within a median of 5 days (range, 1-45) after CPT. Among the 2 patients requiring mechanical ventilation, 1 died 7 days after CPT from ventilation-associated pneumonia, and 1 could be weaned from mechanical ventilation, although he still required oxygen. Biological parameters improved, in particular, CRP, ferritin, and IL-6 levels. All 16 living patients were asymptomatic for COVID-19 2 weeks after CPT. Although RT-PCR on nasopharyngeal swab remained positive in 5 patients, monitoring of circulating SARS-CoV-2 using ddPCR technology performed in 9 patients showed a decrease in RNAemia within 7 to 14 days, which correlated with clinical improvement. Quantification of SARS-CoV-2-specific T cells secreting INF-g before the first plasma administration. As shown in Figure 1 , a strong positive response was detected, in particular, toward peptides of the Spike glycoprotein (CoV-S1). In 2 patients, a high response was also detected to the N nucleoprotein and M membrane protein. The response to peptides from other common b coronaviruses (OC43-S1 and OC43-S2) and a coronaviruses (229E-S1 and 229E-S2) was negative in 2 patients, whereas the third patient exhibited a positive response to peptides of a common a coronavirus (229E-S2). This case series reports the clinical benefit of CPT in 17 consecutive patients with profound B-cell lymphopenia and protracted COVID-19 disease. While COVID-19-specific treatments induced a transient decrease of fever or CRP, all failed to improve sustainably the course of the disease. Conversely, CPT was associated with a striking improvement of clinical symptoms and biological parameters in 16 out of 17 patients and a decrease of SARS-CoV-2 RNAemia within 7 to 14 days. The possibility that some patients were recovering before CPT cannot be totally excluded. However, the deleterious clinical course in all 17 patients before CPT, the close temporal association between plasma administration and clinical improvement, as well as the extent of the clinical responses make this possibility unlikely. 20 In our series, almost all patients had a profound hypogammaglobulinemia associated with an absence of circulating B cells, and none had mounted a neutralizing antibody response after several weeks of symptoms. This suggests that prior treatment with anti-CD20 MoAbs or innate immunodeficiency resulted in a severely impaired adaptive humoral response that was responsible for persistent SARS-CoV-2 shedding and a protracted SARS-CoV-2 infection. 21 The only patient who had detectable circulating B lymphocytes was receiving ibrutinib for chronic lymphocytic leukemia. It is likely that these remaining B cells actually represent residual chronic lymphocytic leukemia B-cell clones that are unable to produce specific SARS-CoV-2 antibodies. 22 Interestingly, the rapid clinical improvement observed after CPT correlated strongly with virological clearance, as demonstrated by the decrease of SARS-CoV-2 RNAemia using an innovative ddPCR technology that allows precise quantification of SARS-CoV-2 RNAemia. 23, 24 These results support the concept that passive immunotherapy in such patients provides the neutralizing SARS-CoV-2 antibodies that are mandatory for viral clearance. By contrast, virus-specific T-cell responses tested in 3 patients prior to plasma injection revealed a very high number of circulating SARS-CoV-2-specific IFN-g-producing T cells. One must note the prominent response to the structural spike (S) glycoprotein that expresses the specific immunodominant epitopes of SARS-CoV-2, which is the target of the neutralizing SARS-CoV-2 antibodies. Conversely, a poor cross-reacting response to other coronaviruses was detected. 25 These ancillary results suggest that specific T-cell responses to SARS-CoV-2 are not sufficient to control viral infection in the absence of neutralizing antibodies. It remains possible that these T cells might work synergistically with the antibodies brought by CPT. Cycles of anti-CD20 therapy, median (range) 7 (4-18) Gammaglobulinemia, median (range), g/L ‡ 3.5 (1.8-14) Time between COVID-19 symptoms onset and last anti-CD20 therapy, median (range), mo COVID-19 severity (WHO score) Data sharing requests should be sent to Thomas Hueso (hueso.th@ gmail.com). The online version of this article contains a data supplement. There is a Blood Commentary on this article in this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734. Rituximab in B-cell hematologic malignancies: a review of 20 years of clinical experience Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebocontrolled multicenter trial B-cell depletion with rituximab in relapsing-remitting multiple sclerosis Consequences of B-cell-depleting therapy: hypogammaglobulinemia and impaired B-cell reconstitution Severe COVID-19-associated pneumonia in 3 patients with systemic sclerosis treated with rituximab Determinants of COVID-19 disease severity in patients with cancer Persisting SARS-CoV-2 viraemia after rituximab therapy: two cases with fatal outcome and a review of the literature Convalescent Plasma Study Group. The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis Collecting and evaluating convalescent plasma for COVID-19 treatment: why and how? Treatment of 5 critically ill patients with COVID-19 with convalescent plasma Effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening COVID-19: a randomized clinical trial Plasma from donors recovered from the new Coronavirus 2019 as therapy for critical patients with COVID-19 (COVID-19 plasma study): a multicentre study protocol WHO Working Group on the Clinical Characterisation and Management of COVID-19 Infection. A minimal common outcome measure set for COVID-19 clinical research Lower prevalence of antibodies neutralizing SARS-CoV-2 in group O French blood donors ANSM. COVID-19: L'ANSM encadre le recours possibleà l'utilisation de plasma de personnes convalescentes pour des patients ne pouvantêtre inclus dans les essais cliniques-Point d'information Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease COVID-19-related collapsing glomerulopathy in a kidney transplant recipient Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients Humoral and cellular immune responses after influenza vaccination in kidney transplant recipients Convalescent plasma for persisting COVID-19 following therapeutic lymphocyte depletion: a report of rapid recovery Effect of convalescent plasma therapy on viral shedding and survival in patients with Coronavirus Disease Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy Detecting biomarkers with microdroplet technology ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals The authors thank the following colleagues who helped with data collection: Stephanie Guillet, Thomas Perpoint, Célia Azoulay, Florence Runyo, Lucie Oberic, and Jean-Benoit Arlet. They also acknowledge Veronique Saada, Emmanuelle Gallois, Franck Griscelli, and Agathe Dubuisson (Gustave Roussy Cancer Centre) for technical support and