Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent patients

ABSTRACTIn 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 SummaryNeutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions.

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Identification of Human SARS-CoV-2 Monoclonal Antibodies from Convalescent Patients Using EBV Immortalization

Antibodies ◽

10.3390/antib10030026 ◽

2021 ◽

Vol 10 (3) ◽

pp. 26

Author(s):

Rut Valgardsdottir ◽

Irene Cattaneo ◽

Gavino Napolitano ◽

Annibale Raglio ◽

Orietta Spinelli ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Recombinant Proteins ◽

Memory B Cells ◽

Spike Protein ◽

Lombardy Region ◽

Functional Assays ◽

Ebv Infection ◽

Cell Clones ◽

In Vitro Expansion

We report the isolation of two human IgG1k monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike protein. These mAbs were isolated from two donors who had recovered from COVID-19 infection during the first pandemic peak in the Lombardy region of Italy, the first European and initially most affected region in March 2020. We used the method of EBV immortalization of purified memory B cells and supernatant screening with a spike S1/2 assay for mAb isolation. This method allowed rapid isolation of clones, with one donor showing about 7% of clones positive against spike protein, whereas the other donor did not produce positive clones out of 91 tested. RNA was extracted from positive clones 39–47 days post-EBV infection, allowing VH and VL sequencing. The same clones were sequenced again after a further 100 days in culture, showing that no mutation had taken place during in vitro expansion. The B cell clones could be expanded in culture for more than 4 months after EBV immortalization and secreted the antibodies stably during that time, allowing to purify mg quantities of each mAb for functional assays without generating recombinant proteins. Unfortunately, neither mAb had significant neutralizing activity in a virus infection assay with several different SARS-CoV-2 isolates. The antibody sequences are made freely available.

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A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo

Journal of Virology ◽

10.1128/jvi.01126-21 ◽

2021 ◽

Author(s):

Kevin Chiem ◽

Desarey Morales Vasquez ◽

Jesus A. Silvas ◽

Jun-Gyu Park ◽

Michael S. Piepenbrink ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vaccine Efficacy ◽

Neutralizing Antibodies ◽

Viral Infections ◽

Rapid Assessment ◽

The United States ◽

Viral Fitness ◽

Human Monoclonal Antibodies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, β) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro , as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of human monoclonal antibodies (hMAbs) with neutralizing activity against SARS-CoV-2, including variants of concern (VoC) in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

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A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

10.1101/2021.04.16.440101 ◽

2021 ◽

Author(s):

Antonia Sophia Peter ◽

Edith Roth ◽

Sebastian R. Schulz ◽

Kirsten Fraedrich ◽

Tobit Steinmetz ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Human Immunoglobulin ◽

Spike Protein ◽

Infection Model ◽

Immunoglobulin Gene ◽

Binding Studies ◽

Human Monoclonal Antibodies ◽

Region Gene ◽

Viral Spread

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly generate human monoclonal antibodies. After immunizing these mice against the spike protein of SARS-CoV-2, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralized SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of neutralizing antibodies binds to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2 induced weight loss. Thus, we report two clusters of potent non-competing SARS-CoV-2 neutralizing antibodies providing potential candidates for therapy and prophylaxis of COVID-19. The study further supports the use of transgenic animals with human immunoglobulin gene repertoires in pandemic preparedness initiatives.

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An Exposed Domain in the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Induces Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.78.13.7217-7226.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 7217-7226 ◽

Cited By ~ 59

Author(s):

Tong Zhou ◽

Hong Wang ◽

Danlin Luo ◽

Thomas Rowe ◽

Zheng Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Vero Cells ◽

Spike Protein ◽

Protein Fragment

ABSTRACT Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.

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In vitro primary immunization of B lymphocytes for producing human monoclonal antibodies against tumor-associated antigens

Human Antibodies ◽

10.3233/hab-1993-4105 ◽

1993 ◽

Vol 4 (1) ◽

pp. 26-30 ◽

Cited By ~ 1

Author(s):

Yun Li ◽

Julian M. Ashby ◽

Oleg Eremin

Keyword(s):

Monoclonal Antibodies ◽

B Lymphocytes ◽

Primary Immunization ◽

Human Monoclonal Antibodies ◽

Tumor Associated Antigens

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Fc-Independent Protection from SARS-CoV-2 Infection by Recombinant Human Monoclonal Antibodies

Antibodies ◽

10.3390/antib10040045 ◽

2021 ◽

Vol 10 (4) ◽

pp. 45

Author(s):

Tal Noy-Porat ◽

Avishay Edri ◽

Ron Alcalay ◽

Efi Makdasi ◽

David Gur ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Complement System ◽

Neutralizing Antibodies ◽

Human Monoclonal Antibodies ◽

Antibody Treatment ◽

Future Design ◽

The Complement System

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.

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Faculty Opinions recommendation of An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019537.231468 ◽

2004 ◽

Author(s):

Frederick Hayden

Keyword(s):

Monoclonal Antibodies ◽

B Cells ◽

Efficient Method ◽

Memory B Cells ◽

Sars Coronavirus ◽

Human Monoclonal Antibodies ◽

Potent Neutralization

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Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

Science Translational Medicine ◽

10.1126/scitranslmed.abd6990 ◽

2021 ◽

pp. eabd6990

Author(s):

Sang Il Kim ◽

Jinsung Noh ◽

Sujeong Kim ◽

Younggeun Choi ◽

Duck Kyun Yoo ◽

...

Keyword(s):

B Cells ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

De Novo ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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