key: cord-1012845-64y43o0y authors: Andreano, Emanuele; Nicastri, Emanuele; Paciello, Ida; Pileri, Piero; Manganaro, Noemi; Piccini, Giulia; Manenti, Alessandro; Pantano, Elisa; Kabanova, Anna; Troisi, Marco; Vacca, Fabiola; Cardamone, Dario; De Santi, Concetta; Agrati, Chiara; Capobianchi, Maria Rosaria; Castilletti, Concetta; Emiliozzi, Arianna; Fabbiani, Massimiliano; Montagnani, Francesca; Montomoli, Emanuele; Sala, Claudia; Ippolito, Giuseppe; Rappuoli, Rino title: Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent patients date: 2020-05-09 journal: bioRxiv DOI: 10.1101/2020.05.05.078154 sha: c9095de51802ad81812c8bf6c6594ac6cb3d1ab2 doc_id: 1012845 cord_uid: 64y43o0y In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy and prevention of Covid-19. Human monoclonal antibodies have very good probability of being safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow natural production of antibodies. Supernatants from each cell were tested by ELISA for spike protein binding, and positive antibodies were further tested for neutralization of spike binding to receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro. Lead candidates to progress into the drug development pipeline will be selected from the panel of neutralizing antibodies identified with the procedure described in this study. One Sentence Summary Neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions. The impact of the SARS-CoV-2 pandemic, with more than 3.5 million cases, 250,000 deaths and 47 more than 25 million people unemployed in the United States alone, is unprecedented. This first 48 wave of infection is likely to be followed by additional waves in the next few years, until herd 49 immunity, acquired by vaccination or by infection, will constrain the circulation of the virus. It is 50 therefore imperative to develop therapeutic and preventive tools to face the next waves of SARS-51 CoV-2 infections as soon as possible. Among the many therapeutic options available, human 52 monoclonal antibodies (mAbs) are the ones that can be developed in the shortest period of time. In 53 fact, the extensive clinical experience with the safety of more than 50 commercial mAbs approved 54 to treat cancer, inflammatory and autoimmune disorders provides high confidence on their safety. 55 These advantages, combined with the urgency of the SARS-CoV-2 pandemic, support and justify 56 an accelerated regulatory pathway. In addition, the long industrial experience in developing and 57 manufacturing mAbs decreases the risks usually associated with the technical development of 58 investigational products. Finally, the incredible technical progress in the field allows to shorten the 59 conventional timelines and go from discovery to proof of concept trials in 5-6 months (1). Indeed, 60 in the case of Ebola, mAbs were the first therapeutic intervention recommended by the World 61 Health Organization (WHO) and they were developed faster than vaccines or other drugs (2). The SARS-CoV-2 spike glycoprotein (S-protein) has a pivotal role in viral pathogenesis and it is 63 considered the main target to elicit potent neutralizing antibodies and the focus for the development 64 of therapeutic and prophylactic tools against this virus (3, 4) . Indeed, SARS-CoV-2 entry into host 65 cells is mediated by the interaction between S-protein and the human angiotensin converting 66 enzyme 2 (ACE2) (3, 5) . The S-protein is a trimeric class I viral fusion protein which exists in a 67 metastable prefusion conformation and in a stable postfusion state. Each S-protein monomer is 68 composed of two distinct regions, the S1 and S2 subunits. Structural rearrangement occurs when the 69 receptor binding domain (RBD) present in the S1 subunit binds to the host cell membrane. This 70 4 interaction destabilizes the prefusion state of the S-protein triggering the transition into the 71 postfusion conformation which in turn results in the ingress of the virus particle into the host cell 72 (6). Single-cell RNA-seq analyses to evaluate the expression levels of ACE2 in different human 73 organs have shown that SARS-CoV-2, through the S-protein, can invade human cells in different 74 major physiological systems including the respiratory, cardiovascular, digestive and urinary 75 systems, thus enhancing the possibility of spreading and infection (7). To identify potent mAbs against SARS-CoV-2 we isolated over a 1,100 S-protein specific-memory 77 B cells derived from seven Covid-19 convalescent donors. As the S-protein RBD domain is mainly 78 exposed when this glycoprotein is in its prefusion state (6), we screened naturally produced mAbs 79 against either the S1/S2 subunits and the S-protein trimer stabilized in its prefusion conformation 80 (6). This strategy allows us to identify mAbs able to recognize linear epitopes as well as highly 81 neutralizing trimer specific regions on the S-protein surface. The potent neutralizing effect of trimer 82 specific mAbs has already been shown for other pathogens including the respiratory syncytial virus 83 (RSV) (8, 9) . 84 In this paper we report the identification of a panel of 318 mAbs from which we plan to select lead 85 candidates for clinical development. 89 Patients recovered from SARS-CoV-2 infection were enrolled in two ongoing clinical studies based 90 in Rome, Italy (National Institute for Infectious Diseases, IRCCS, Lazzaro Spallanzani) and Siena, 91 Italy (Azienda Ospedaliera Universitaria Senese). We firstly examined whether these patients had 92 anti-SARS-CoV-2 S-protein antibodies. Plasma samples were evaluated by enzyme linked 93 immunosorbent assay (ELISA) to assess the polyclonal response to the S-protein trimer, for their 94 ability to neutralize the binding of the spike protein to Vero E6 cells (neutralization of binding or 95 NOB assay) and for their potency in neutralizing the cytopathic effect caused by SARS-CoV-2 96 infection in vitro. Results shown in Table 1 and Figure 1 show that, among the seven donors 97 included in this study, six were able to produce high titers of SARS-CoV-2 S-protein specific 98 antibodies and in particular donors R-042, R-122 and R-188 showed the highest virus neutralizing 99 titers. Only one patient (R-276) mounted low anti-spike polyclonal response ( Fig. 1A-B ). Interestingly, despite no statistically significant correlation was observed when Pearson correlation 101 analysis was performed, it is possible to observe a trend of correlation between S-protein binding, 102 NOB titer and neutralization titer, suggesting that an abundant response against the S-protein trimer 103 in its prefusion conformation may be indicative of immunity against SARS-CoV-2 ( Fig. 1C-D) . A 104 bigger dataset may be needed to support this observation. MBCs binding to the SARS-CoV-2 S-protein trimer in its prefusion conformation. A total of 1,167 113 S-protein-binding MBCs were successfully retrieved with frequencies ranging from 0,17% to 114 1,41% (Table 2) . Following the sorting procedure, S-protein + MBCs were incubated over a layer of 115 3T3-CD40L feeder cells in the presence of IL-2 and IL-21 stimuli for two weeks to allow natural 116 production of immunoglobulins (10). Subsequently, MBC supernatants containing IgG or IgA were 117 tested for their ability to bind either the SARS-CoV-2 S-protein S1 + S2 subunits ( showing a broad array of neutralization potency ranging from 52% to over 90% (Fig. 4B ). In the 128 viral neutralization assay, supernatants containing naturally produced IgG or IgA were tested for 129 their ability to protect the layer of Vero E6 cells from the cytopathic effect triggered by SARS- (Table 2) . the first drugs to be tested in the Ebola outbreak and showed remarkable efficacy in preventing 151 mortality (2). Given the remarkable efficacy of this intervention, potent mAbs became the first, and 152 so far the only, drug to be recommended for Ebola by the WHO. In the case of SARS-CoV-2, where so far we do not have any effective therapeutic nor prophylactic 154 interventions, mAbs have the possibility to become one of the first drugs that can be used for 155 immediate therapy of any patient testing positive for the virus, and even to provide immediate 156 protection from infection in high risk populations. Preliminary evidences showed that plasma from 157 infected people improves the outcome of patients with severe disease, therefore it is highly possible 158 that a therapeutic and/or prophylactic mAb-based intervention can be highly effective (12). Furthermore, vaccination strategies inducing neutralizing antibodies have shown to protect non-160 8 human primates from infection (13). These results further stress the importance of mAbs as a 161 measure to counterattack SARS-CoV-2 infection and to constrain its circulation. In this work we addressed the question of whether mAbs recognizing SARS-CoV-2 can be 163 recovered from memory B cells of people who survived Covid-19 and whether some of them are 164 able to neutralize the virus. Our data show that SARS-CoV-2 specific mAbs can be successfully 165 isolated from most convalescent donors even if the frequency of S-protein specific memory B cells 166 is highly variable among them. In addition, approximately 28% of isolated mAbs were able to 167 inhibit the binding of the S-protein to the receptor(s) on Vero E6 cells. Finally a fraction of isolated 168 mAbs (N=17) were able to effectively neutralize SARS-CoV-2 with high potency when tested in 169 vitro. These data suggest that the method we implemented allows us to successfully retrieve mAbs 170 with potent neutralizing activity against SARS-CoV-2 and we plan to select the most promising 171 candidate(s) for drug development. Lead candidates will be further tested for the ability to generate 172 resistant viruses and for the ability to induce antibody-dependent disease enhancement in 173 appropriate models. ELISA assay with S1 and S2 subunits of SARS-CoV-2 S-protein 217 The presence of S1-and S2-binding antibodies in culture supernatants of monoclonal S-protein- To study the binding of the Covid-19 Spike protein to cell-surface receptor(s) we developed an 274 assay to assess recombinant Spike protein specific binding to target cells and neutralization thereof. To this aim the stabilized Spike protein was coupled to Streptavidin-PE (eBioscience # 12-4317-87, The infected cultures were incubated at 37°C, 5% CO 2 and monitored daily until approximately 80-298 90% of the cells exhibited cytopathic effect (CPE). Culture supernatants were then collected, 299 centrifuged at 4°C at 1,600 rpm for 8 minutes to allow removal of cell debris, aliquoted and stored 300 at -80°C as the harvested viral stock. Viral titers were determined in confluent monolayers of Vero 301 E6 cells seeded in 96-well plates using a 50% tissue culture infectious dose assay (TCID50). Cells 302 were infected with serial 1:10 dilutions (from 10-1 to 10-11) of the virus and incubated at 37°C, in a 303 humidified atmosphere with 5% CO 2 . Plates were monitored daily for the presence of SARS-CoV-2 304 induced CPE for 4 days using an inverted optical microscope. The virus titer was estimated 305 according to Spearman-Karber formula (14) and defined as the reciprocal of the highest viral 306 dilution leading to at least 50% CPE in inoculated wells. RR is an employee of GSK group of companies. We would like to thank the whole GSK Vaccines Pre-clinical Evidence Generation and Assay -Immunolgy function led by Dr Oretta Finco for their availability and support as well as Mrs We would also like to thank Dr. Mariagrazia Pizza and Dr. Simone Pecetta for initial insightful advice Jason McLellan and his team for generously providing the SARS-CoV-2 S-protein stabilized in its 368 prefusion conformation used in this study. Furthermore, we would like to thank Dr. Daniel Wrapp and Dr We gratefully acknowledge the Collaborators Members of INMI COVID-19 study group: Maria Alessandra Abbonizio We would like to thank all the other Members of the Covid-19 UNIT and of the Covid-19 TEAM Giacomo Zanelli, and the Covid-19 nursing staff Developing therapeutic monoclonal antibodies at pandemic pace Successful Ebola treatments promise to tame outbreak Structure, Function, and 543 Antigenicity of the SARS-CoV-2 Spike Glycoprotein The trinity of COVID-19: immunity, 545 inflammation and intervention Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2 Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Single-cell RNA-seq data analysis on the receptor 553 ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV 554 infection. Frontiers of medicine Structural, antigenic and immunogenic features of respiratory 556 syncytial virus glycoproteins relevant for vaccine development Immunological goals for respiratory syncytial virus vaccine development. Current 558 opinion in immunology 59 Isolation of human monoclonal antibodies from peripheral blood 561 B cells Human monoclonal antibodies for discovery, therapy, and 563 vaccine acceleration Effectiveness of convalescent plasma therapy in severe COVID-19 patients Rapid development of an inactivated 573 vaccine for SARS-CoV-2 One-hit models for virus inactivation studies This publication was supported by funds from the "Centro Regionale Medicina di Precisione" and by all the people who answered Table 2 . SARS-CoV-2 convalescent donors S-protein specific MBCs analyses. The Table 422 reports the number of S-protein-specific MBCs that were sorted and screened (for binding by 423 ELISA and for functionality by NOB and viral neutralization) for each subject enrolled in this 424 study; ND = Not Done.