A considerable fraction of B cells recognize SARS-CoV-2 with germline-encoded elements of their B cell receptor resulting in the production of neutralizing and non-neutralizing antibodies. We found that antibody sequences from different discovery cohorts shared biochemical properties and could be retrieved across validation cohorts confirming the stereotyped character of this naive response in COVID-19. While neutralizing antibody sequences were found independently of disease severity in line with serological data, individual non-neutralizing antibody sequences were associated with fatal clinical courses suggesting detrimental effects of these antibodies. We mined 200 immune repertoires of healthy individuals and 500 of patients with blood or solid cancers - all acquired prior to the pandemic - for SARS-CoV-2 antibody sequences. While the largely unmutated B cell rearrangements occurred in a substantial fraction of immune repertoires from young and healthy individuals, these sequences were less likely found in individuals over 60 years of age and in cancer. This reflects B cell repertoire restriction in aging and cancer and may to a certain extent explain the different clinical COVID-19 courses observed in these risk groups. Future studies will have to address if this stereotyped B cell response to SARS-CoV-2 emerging from unmutated antibody rearrangements will create long-lived memory.
Lisa Paschold, Donjete Simnica, Edith Willscher, Maria J.G.T. Vehreschild, Jochen Dutzmann, Daniel G. Sedding, Christoph Schultheiß, Mascha Binder
Four different endemic coronaviruses (eCoVs) are etiologic agents for the seasonal “common cold,” and these eCoVs share extensive sequence homology with human severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we show that individuals with as compared to without a relatively recent documented eCoV were tested at greater frequency for respiratory infections but had similar rate of SARS-CoV-2 acquisition. Importantly, the patients with a previously detected eCoV had less severe coronavirus disease-2019 (COVID-19) illness. Our observations suggest that pre-existing immune responses against endemic human coronaviruses can mitigate disease manifestations from SARS-CoV-2 infection.
Manish Sagar, Katherine Reifler, Michael Rossi, Nancy S. Miller, Pranay Sinha, Laura White, Joseph P. Mizgerd
BACKGROUND. Serological assays are of critical importance to investigate correlates of response and protection in COVID-19, to define previous exposure to SARS-CoV-2 in populations and to verify the development of an adaptive immune response in infected individuals. METHODS. We studied 509 confirmed COVID-19 patients from the San Raffaele Hospital of Milan and 480 pre-pandemic organ donor sera collected in 2010-2012. Using fluid-phase luciferase immune precipitation (LIPS) assays, we characterized IgG, IgM, IgA antibodies to the spike Receptor Binding Domain (RBD), S1+S2, nucleocapsid, and ORF6 to 10 of SARS-CoV-2, to the HCoV-OC43 and HCoV-HKU1 betacoronaviruses spike S2, and the H1N1Ca2009 flu virus hemagglutinin. Sequential samples at 1 and 3 months post-hospital discharge were also tested in 95 patients for SARS-CoV-2 RBD antibodies. RESULTS. Antibodies developed rapidly against multiple SARS-CoV-2 antigens in 95% of patients by 4 weeks post-symptoms onset and IgG to the RBD increased until the 3rd month of follow-up. We observed a major synchronous expansion of antibodies to the HCoV-OC43 and HCoV-HKU1 spike S2. A likely co-infection with influenza was neither linked to a more severe presentation of the disease nor to a worse outcome. Of the measured antibody responses positivity for IgG against the SARS-CoV-2 spike RBD was predictive of survival. CONCLUSIONS. The measurement of antibodies to selected epitopes of SARS-CoV-2 antigens can offer a more accurate assessment of the humoral response in patients and its impact on survival. The presence of partially cross-reactive antibodies with other betacoronoviruses is likely to impact on serological assay specificity and interpretation.
Massimiliano Secchi, Elena Bazzigaluppi, Cristina Brigatti, Ilaria Marzinotto, Cristina Tresoldi, Patrizia Rovere-Querini, Andrea Poli, Antonella Castagna, Gabriella Scarlatti, Alberto Zangrillo, Fabio Ciceri, Lorenzo Piemonti, Vito Lampasona
BACKGROUND. Understanding outcomes and immunologic characteristics of cellular therapy recipients with SARS-CoV-2 is critical to performing these potentially life-saving therapies in the COVID-19 era. In this study of recipients of allogeneic (Allo) and autologous (Auto) hematopoietic cell transplant and CD19-directed chimeric antigen receptor T cell therapy (CAR-T) at Memorial Sloan Kettering Cancer Center, we aimed to identify clinical variables associated with COVID-19 severity and assess lymphocyte populations. METHODS. We retrospectively investigated patients diagnosed between March 15th and May 7th, 2020. In a subset of patients, lymphocyte immunophenotyping, quantitative real-time PCR from nasopharyngeal swabs, and SARS-CoV-2 antibody status were available. RESULTS. We identified 77 SARS-CoV-2 + cellular therapy recipients (Allo = 35, Auto = 37, CAR-T = 5; median time from cellular therapy 782 days (IQR 354,1611). Overall survival at 30 days was 78%. Clinical variables significantly associated with the composite endpoint of non-rebreather or higher oxygen requirement and death (n events = 25/77) included number of co-morbidities (HR 5.41, P = 0.004), infiltrates (HR 3.08, P = 0.032), and neutropenia (HR 1.15, P = 0.04). Worsening graft-versus-host-disease was not identified among Allo subjects. Immune profiling revealed reductions and rapid recovery in lymphocyte populations across lymphocyte subsets. Antibody responses were seen in a subset of patients. CONCLUSION. In this series of Allo, Auto, and CAR-T recipients, we report overall favorable clinical outcomes for COVID-19 patients without active malignancy and provide preliminary insights into the lymphocyte populations that are key for the anti-viral response and immune reconstitution. FUNDING. NIH P01 CA23766, NIH/NCI P30 CA008748.
Gunjan L. Shah, Susan DeWolf, Yeon Joo Lee, Roni Tamari, Parastoo B. Dahi, Jessica A. Lavery, Josel D. Ruiz, Sean M. Devlin, Christina Cho, Jonathan U. Peled, Ioannis Politikos, Michael Scordo, N. Esther Babady, Tania Jain, Santosha Vardhana, Anthony F. Daniyan, Craig S. Sauter, Juliet N. Barker, Sergio A. Giralt, Cheryl Goss, Peter Maslak, Tobias M. Hohl, Mini Kamboj, Lakshmi Ramanathan, Marcel R.M. van den Brink, Esperanza B. Papadopoulos, Genovefa A. Papanicolaou, Miguel-Angel Perales
Background: Marked progress is achieved in understanding the physiopathology of COVID-19 that caused global pandemics. However, CD4+ T cell population that is critical for antibody response in COVID-19 is poorly understood. Methods: In this study, we provided a comprehensive analysis of peripheral CD4+ T cells of 13 COVID-19 convalescent patients, as defined as confirmed free of SARS-CoV-2 for 2-4 weeks, using flow cytometry, magnetic chemiluminescence enzyme antibody immunoassay and correlated the data with clinical characteristics. Results: We observed that relative to healthy individuals, convalescent patients displayed an altered peripheral CD4+ T cell spectrum. Specifically, consistent with other viral infections, cTFH1 cell associated with SARS-CoV-2 targeting antibodies, which was found to skew with disease severity as more severe individuals showed higher frequency of TEM and TFH-EM cells but a lower frequency of TCM, TFH-CM and TNaive cells, relative to mild and moderate patients. Interestingly, higher frequency of cTFH-EM cells correlated with lower number of recorded admission blood oxygen level in convalescent patients. These observations might constitute residual effects by which COVID-19 can impact the homeostasis of CD4+ T cells in the long-term and explain the highest ratio of class-switched virus-specific antibody producing individuals found in our severe COVID-19 cohort. Conclusion: Together, our study demonstrated close connection between CD4+ T cells and antibody production in COVID-19 convalescents.Funding: This study was supported by Six Talent Peaks Project in Jiangsu Province and the National Natural Science Foundation of China (NSFC) grants 81970759.
Fang Gong, Yaping Dai, Ting Zheng, Liang Cheng, Dan Zhao, Hao Wang, Min Liu, Hao Pei, Tengchuan Jin, Di Yu, Pengcheng Zhou
COVID-19 has emerged as a global pandemic caused by SARS-CoV-2. So far, viral targets of cellular immunity and factors determining successful mounting of T-cell responses are poorly defined. We therefore analyzed cellular responses to membrane, nucleocapsid and spike protein in individuals suffering from moderate or severe infection and after recovery from mild disease. We demonstrate that the CoV-2 specific CD4+ T-helper cell response is directed against all three proteins with comparable magnitude, ex vivo proliferation and portions of responding patients. However, deceased individuals were more frequently amongst non-responders. Higher patient age and comorbidity index correlated with increased frequencies of CoV-2 specific CD4+ T-cells, harboring higher portions of IL-2-, but lower portions of IFNγ secreting cells. Diminished frequencies of membrane protein reactive IFNγ+ T cells were particularly associated with higher Acute Physiology And Chronic Health Evaluation II scores in patients admitted to intensive care. CoV-2 specific T cells exhibited elevated PD-1 expression in active patients as compared to recovered individuals with previous mild disease. In summary, our data suggest a link between individual patient predisposition with respect to age and comorbidity and impairment of CoV-2 specific Th1-type cellular immunity, thereby supporting a concept of altered T-cell function in patients at risk.
Arne Sattler, Stefan Angermair, Helena Stockmann, Katrin Moira Heim, Dmytro Khadzhynov, Sascha Treskatsch, Fabian Halleck, Martin E. Kreis, Katja Kotsch
BACKGROUND. COVID-19 patients develop pneumonia generally associated to lymphopenia and severe inflammatory response due to uncontrolled cytokine release. These mediators are transcriptionally regulated by the JAK-STAT signaling pathways, which can be disabled by small molecules. METHODS. A group of subjects (n = 20) was treated with baricitinib according to an off-label use of the drug. The study was designed as an observational longitudinal trial and approved by the local ethical committee. The patients were treated with baricitinib 4 mg twice daily for 2 days, followed by 4 mg per day for the remaining 7 days. Changes in the immune phenotype and expression of pSTAT3 in blood cells were evaluated and correlated with serum-derived cytokine levels and antibodies anti-SARS-CoV-2. In a single treated patient, we evaluated also the alteration of myeloid cell functional activity. RESULTS. We provided evidences that baricitinib-treated patients have a marked reduction in serum levels of interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α, a rapid recovery in circulating T and B cell frequencies, and increased antibody production against SARS-CoV-2 spike protein, which were clinically associated with a reduction in oxygen flow need and progressive increase in the P/F. CONCLUSION. Baricitinib prevented the progression towards a severe/extreme form of the viral disease by modulating the patients’ immune landscape and these changes were associated with a safer and favorable clinical outcome of patients with COVID-19 pneumonia. TRIAL REGISTRATION. The ClinicalTrials.gov identifier of this project is protocol NCT04438629. FUNDING. This work was supported by Fondazione Cariverona (ENACT Project) and Fondazione TIM.
Vincenzo Bronte, Stefano Ugel, Elisa Tinazzi, Antonio Vella, Francesco De Sanctis, Stefania Canè, Veronica Batani, Rosalinda Trovato, Alessandra Fiore, Varvara Petrova, Francesca Hofer, Roza Maria Barouni, Chiara Musiu, Simone Caligola, Laura Pinton, Lorena Torroni, Enrico Polati, Katia Donadello, Simonetta Friso, Francesca Pizzolo, Manuela Iezzi, Federica Facciotti, Pier Giuseppe Pelicci, Daniela Righetti, Paolo Bazzoni, Mariaelisa Rampudda, Andrea C. Comel, Walter Mosaner, Claudio Lunardi, Oliviero Olivieri
SARS-CoV-2 is responsible for development of COVID-19 in infected individuals, who can either exhibit mild symptoms or progress towards a life-threatening acute respiratory distress syndrome (ARDS). Exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8+ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. Here, we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood and lung of COVID-19 patients with different clinical severity in comparison with healthy individuals. Furthermore, these subpopulations and their association with antiviral effector CD8+ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Our results indicate that inflammatory transitional and non-classical monocytes and CD1c+ conventional dendritic cells preferentially migrate from blood to lungs in patients with severe COVID-19. Thus, this study increases the knowledge on specific myeloid subsets involved in the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies to fight SARS-CoV-2 infection.
Ildefonso Sánchez-Cerrillo, Pedro Landete, Beatriz Aldave, Santiago Sánchez-Alonso, Ana Sánchez-Azofra, Ana Marcos-Jiménez, Elena Ávalos, Ana Alcaraz-Serna, Ignacio de los Santos, Tamara Mateu-Albero, Laura Esparcia, Celia López-Sanz, Pedro Martínez-Fleta, Ligia Gabrie, Luciana del Campo Guerola, Hortensia de la Fuente, María J Calzada, Isidoro González-Álvaro, Arantzazu Alfranca, Francisco Sánchez-Madrid, Cecilia Muñoz-Calleja, Joan B. Soriano, Julio Ancochea, Enrique Martín-Gayo
Convalescent plasma is a leading treatment for COVID-19, but there is a paucity of data identifying therapeutic efficacy. Among 126 potential convalescent plasma donors, the humoral immune response was evaluated by a SARS-CoV-2 virus neutralization assay using Vero-E6-TMPRSS2 cells, commercial IgG and IgA ELISA to spike(S) protein S1 domain (Euroimmun), IgA, IgG and IgM indirect ELISAs to the full-length S or S-receptor binding domain(S-RBD), and an IgG avidity assay. Multiple linear regression and predictive models were utilized to assess the correlations between antibody responses with demographic and clinical characteristics. IgG titers were greater than either IgM or IgA for S1, full length S, and S-RBD in the overall population. Of the 126 plasma samples, 101(80%) had detectable neutralizing antibody(nAb) titers. Using nAb titers as the reference, the IgG ELISAs confirmed between 95-98% of the nAb positive, but only 20-32% of the nAb negative samples. Male sex, older age, and hospitalization with COVID-19 were associated with increased antibody responses across the serological assays. There was substantial heterogeneity in the antibody response among potential convalescent plasma donors, but sex, age, and hospitalization emerged as factors that can be used to identify individuals with a high likelihood of having strong antiviral antibody responses.
Sabra L. Klein, Andrew Pekosz, Han-Sol Park, Rebecca L. Ursin, Janna R. Shapiro, Sarah E. Benner, Kirsten Littlefield, Swetha Kumar, Harnish Mukesh Naik, Michael Betenbaugh, Ruchee Shrestha, Annie A. Wu, Robert M. Hughes, Imani Burgess, Patrizio Caturegli, Oliver Laeyendecker, Thomas C. Quinn, David J. Sullivan, Shmuel Shoham, Andrew D. Redd, Evan M. Bloch, Arturo Casadevall, Aaron A. R. Tobian
Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyper-inflammation and thrombotic microangiopathy, thereby increasing COVID-19 mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies and NETs/human aortic endothelial cell (HAEC) co-cultures. Increased plasma levels of NETs, tissue factor (TF) activity and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAEC. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against SARS-CoV-2 that exploit complement or NETosis inhibition.
Panagiotis Skendros, Alexandros Mitsios, Akrivi Chrysanthopoulou, Dimitrios C. Mastellos, Simeon Metallidis, Petros Rafailidis, Maria Ntinopoulou, Eleni Sertaridou, Victoria Tsironidou, Christina Tsigalou, Maria G. Tektonidou, Theocharis Konstantinidis, Charalampos Papagoras, Ioannis Mitroulis, Georgios Germanidis, John D. Lambris, Konstantinos Ritis
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