Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

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SARS-CoV-2 IgG Antibodies Seroprevalence and Sera Neutralizing Activity in MEXICO: A National Cross-Sectional Study during 2020

Microorganisms ◽

10.3390/microorganisms9040850 ◽

2021 ◽

Vol 9 (4) ◽

pp. 850

Author(s):

José Esteban Muñoz-Medina ◽

Concepción Grajales-Muñiz ◽

Angel Gustavo Salas-Lais ◽

Larissa Fernandes-Matano ◽

Constantino López-Macías ◽

...

Keyword(s):

Immunosorbent Assay ◽

Neutralizing Antibodies ◽

Herd Immunity ◽

Cross Sectional Study ◽

Molecular Techniques ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Enzyme Linked Immunosorbent Assay ◽

Cross Sectional ◽

Neutralizing Activity

Until recently, the incidence of COVID-19 was primarily estimated using molecular diagnostic methods. However, the number of cases is vastly underreported using these methods. Seroprevalence studies estimate cumulative infection incidences and allow monitoring of transmission dynamics, and the presence of neutralizing antibodies in the population. In February 2020, the Mexican Social Security Institute began conducting anonymous unrelated sampling of residual sera from specimens across the country, excluding patients with fever within the previous two weeks and/or patients with an acute respiratory infection. Sampling was carried out weekly and began 17 days before Mexico’s first officially confirmed case. The 24,273 sera obtained were analyzed by chemiluminescent-linked immunosorbent assay (CLIA) IgG S1/S2 and, later, positive cases using this technique were also analyzed to determine the rate of neutralization using the enzyme-linked immunosorbent assay (ELISA). We identified 40 CLIA IgG positive cases before the first official report of SARS-CoV-2 infection in Mexico. The national seroprevalence was 3.5% in February and 33.5% in December. Neutralizing activity among IgG positives patients during overall study period was 86.1%. The extent of the SARS-CoV-2 infection in Mexico is 21 times higher than that reported by molecular techniques. Although the general population is still far from achieving herd immunity, epidemiological indicators should be re-estimated based on serological studies of this type.

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Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100104118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2100104118

Author(s):

Ryan J. Malonis ◽

James T. Earnest ◽

Arthur S. Kim ◽

Matthew Angeliadis ◽

Frederick W. Holtsberg ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Human Mabs ◽

Isolation And Characterization ◽

Neutralizing Capacity ◽

Rheumatological Disease ◽

Mayaro Virus ◽

Alphavirus Infection ◽

Globally Distributed

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

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Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants

10.1101/2022.01.12.22269023 ◽

2022 ◽

Author(s):

Zhaochun Chen ◽

Peng Zhang ◽

Yumiko Matsuoka ◽

Yaroslav Tsybovsky ◽

Kamille West ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Structural Basis ◽

Hamster Model ◽

Golden Syrian Hamster ◽

Phage Display Libraries ◽

Antibody Phage ◽

Antibody Phage Display ◽

Early Isolate

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity1. This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies1,2. Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe3. Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

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Previous Infection Combined with Vaccination Produces Neutralizing Antibodies With Potency Against SARS-CoV-2 Variants

10.1101/2021.08.27.21262744 ◽

2021 ◽

Author(s):

F Javier Ibarrondo ◽

Christian Hofmann ◽

Ayub Ali ◽

Paul Ayoub ◽

Donald B Kohn ◽

...

Keyword(s):

Antiviral Activity ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Original Sequence ◽

Neutralizing Activity ◽

Previous Infection ◽

Protein Mutations ◽

Antigenic Exposure ◽

Prior Infection ◽

Spike Sequence

SARS-CoV-2 continues to evolve in humans. Spike protein mutations increase transmission and potentially evade antibodies raised against the original sequence used in current vaccines. Our evaluation of serum neutralizing activity in both persons soon after SARS-CoV-2 infection (in April 2020 or earlier) or vaccination without prior infection confirmed that common spike mutations can reduce antibody antiviral activity. However, when the persons with prior infection were subsequently vaccinated, their antibodies attained an apparent biologic ceiling of neutralizing potency against all tested variants, equivalent to the original spike sequence. These findings indicate that additional antigenic exposure further improves antibody efficacy against variants.

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Development of specific immunity in laboratory animals after co-immunization against seasonal influenza and COVID-19

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-183 ◽

2022 ◽

Vol 98 (6) ◽

pp. 648-656

Author(s):

G. M. Ignatyev ◽

I. A. Leneva ◽

A. V. Atrasheuskaya ◽

L. I. Kozlovskaya ◽

N. P. Kartashova ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Seasonal Influenza ◽

Neutralization Test ◽

Elisa Test ◽

Control Measures ◽

Laboratory Animals ◽

Enzyme Linked Immunosorbent Assay ◽

Post Vaccination ◽

Specific Immunity ◽

Influenza Prevention

Introduction. In clinical practice, the differential diagnosis of COVID-19 can be challenging during the flu season, entailing serious consequences such as delays in appropriate control measures against the SARS-CoV-2 pandemic. Another problem is posed by co-infection of SARS-CoV-2 and influenza virus (IV), which significantly contributes to the severity of the COVID-19 disease. This study was aimed to explore the cross-impact of co-administration of Russian influenza and COVID-19 vaccines on development of specific immunity in laboratory animals.Materials and methods. The study was conducted on BALB/c mice. The animals were inoculated intramuscularly with the vaccine for COVID-19 prevention (CoviVac) and the vaccine for influenza prevention (Flu-M). The sera from the immunized animals were examined separately. Three IV strains were used in the hemagglutination inhibition assay. Antibodies (Abs) against SARS-CoV-2 were detected by an enzyme-linked immunosorbent assay (ELISA). The neutralization test was performed to detect virus neutralizing antibodies against SARS-CoV-2 and IV.Results. Relatively high titers of specific Abs were found in the groups of animals inoculated with one vaccine and with two vaccines concurrently. In the groups of animals inoculated with CoviVac and with two vaccines concurrently, both in the ELISA test and in the neutralization test, the average titers of specific Abs against SARSCoV- 2 did not demonstrate any statistical difference. The group of animals inoculated concurrently with two vaccines demonstrated statistically higher titers of Abs against IV after the second immunization compared to the group of animals inoculated with Flu-M.Discussion. The study has shown that post-vaccination immunity both to IV and to SARS-CoV-2 develops after co-vaccination with two vaccines. The observed enhanced post-vaccination immune response to IV in the coimmunized laboratory animals needs further research.Conclusion. The performed studies suggest the possibility of co-administration of two vaccines to prevent influenza and COVID-19.

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Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

10.1101/2021.02.03.429355 ◽

2021 ◽

Author(s):

Carl Graham ◽

Jeffrey Seow ◽

Isabella Huettner ◽

Hataf Khan ◽

Neophytos Kouphou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Infectious Virus ◽

Antigenic Drift ◽

Host Cells ◽

Receptor Binding Domain ◽

Neutralization Activity ◽

Neutralizing Activity ◽

Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

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Evaluation of a Novel Reporter Virus Neutralization Test for Serological Diagnosis of Zika and Dengue Virus Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.00975-17 ◽

2017 ◽

Vol 55 (10) ◽

pp. 3028-3036 ◽

Cited By ~ 21

Author(s):

Chao Shan ◽

Daniel A. Ortiz ◽

Yujiao Yang ◽

Susan J. Wong ◽

Laura D. Kramer ◽

...

Keyword(s):

Dengue Virus ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Reference Method ◽

Laboratory Diagnosis ◽

Virus Neutralization Test ◽

Virus Neutralization ◽

Enzyme Linked Immunosorbent Assay ◽

Reporter Virus ◽

Screening Assay

ABSTRACT Currently, the laboratory diagnosis of Zika virus (ZIKV) infection is primarily through the detection of ZIKV RNA or antibodies against ZIKV proteins. The detection of viral RNA is highly sensitive and specific, but periods of viremia and viruria are brief, limiting the utility of ZIKV RNA assays. Instead, most ZIKV infections are diagnosed serologically, using an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for screening, followed by a confirmatory plaque reduction neutralization test (PRNT). Typical turnaround times vary, due to assay incubation periods and a lack of clinical laboratories performing these tests. Recently, a novel luciferase-ZIKV- and -dengue virus (DENV)-based serological assay, which considerably improves the turnaround times and throughput for ZIKV diagnosis, was described. Using the traditional PRNT as a reference method, we evaluated the performance characteristics of the reporter virus neutralization test (RVNT) with 258 clinical serum specimens. The ZIKV RVNT produced primary ZIKV screening and secondary confirmation results in 4 days, with 100% reproducibility. As a screening assay, the ZIKV RVNT displayed excellent diagnostic accuracy, sensitivity, and specificity of 98.2%, 100%, and 98.1%, respectively. As a confirmatory assay, the ZIKV RVNT titers displayed 93.1% agreement with the traditional ZIKV PRNT titers. Overall, the RVNT accurately and reliably detects neutralizing antibodies in patient serum specimens, with improved turnaround times, and can be used for the serological detection of ZIKV infections. Due to the homogeneous 96-well format, the RVNT has also significantly improved the assay throughput to allow testing of a large number of specimens in a single run.

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Assessment of Mumps Virus-Specific Antibodies: Comparison of Plaque Reduction Neutralization Test and Enzyme-Linked Immunosorbent Assay Estimates

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz345 ◽

2019 ◽

Vol 220 (9) ◽

pp. 1462-1468 ◽

Cited By ~ 3

Author(s):

Stéphanie Ravault ◽

Damien Friel ◽

Emmanuel Di Paolo ◽

Adrian Caplanusi ◽

Paul Gillard ◽

...

Keyword(s):

Antibody Response ◽

Immunosorbent Assay ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Pearson Correlation ◽

Mumps Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Serological Response ◽

Moderate Correlation ◽

Plaque Reduction Neutralization Test

Abstract Background The plaque reduction neutralization test (PRNT), which measures a subset of immunoglobulin antibodies (functional neutralizing antibodies), and the enzyme-linked immunosorbent assay (ELISA), which measures total immunoglobulin (neutralizing and nonneutralizing antibodies), characterize different aspects of the anti–mumps virus antibody response after vaccination. Methods Data from a recent phase 3 clinical trial (NCT01681992) of 2 measles-mumps-rubella vaccines were used to compare anti-mumps antibody responses measured using an unenhanced PRNT (GSK; seropositivity cutoff and threshold, 2.5 and 4 times the 50% end-point dilution, respectively) with those estimated using an ELISA (thresholds, 5 and 10 ELISA units/mL, respectively). Results Of 3990 initially seronegative samples, 3284 (82.3%) were seropositive after vaccination for anti-mumps antibodies in both assays. The Pearson correlation coefficient for double-positive samples was 0.57, indicative of a moderate correlation. Receiver operating characteristic curve analysis showed that an ELISA threshold of 51.7 ELISA units/mL best corresponded to the PRNT seroresponse threshold. There was no obvious vaccine brand effect on the correlation between assays. Conclusions The moderate correlation between the anti-mumps antibody measurements obtained with PRNT and ELISA reflects different aspects of the serological response. In the absence of a well-defined protective serological threshold, PRNT provides complementary information on the antibody response, whereas ELISA remains a critically useful measurement of vaccine immunogenicity.

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Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes

Vaccines ◽

10.3390/vaccines8030386 ◽

2020 ◽

Vol 8 (3) ◽

pp. 386 ◽

Cited By ~ 8

Author(s):

Ferdinand Zettl ◽

Toni Luise Meister ◽

Tanja Vollmer ◽

Bastian Fischer ◽

Jörg Steinmann ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Pearson Correlation ◽

Spike Protein ◽

Virus Neutralization ◽

Pseudotype Virus ◽

Vesicular Stomatitis ◽

Level 1 ◽

Biosafety Level

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2, a new member of the genus Betacoronavirus, is a pandemic virus, which has caused numerous fatalities, particularly in the elderly and persons with underlying morbidities. At present, there are no approved vaccines nor antiviral therapies available. The detection and quantification of SARS-CoV-2-neutralizing antibodies plays a crucial role in the assessment of the immune status of convalescent COVID-19 patients, evaluation of recombinant therapeutic antibodies, and the evaluation of novel vaccines. To detect SARS-CoV-2-neutralizing antibodies, classically, a virus-neutralization test has to be performed at biosafety level 3, considerably limiting the general use of this test. In the present work, a biosafety level 1 pseudotype virus assay based on a propagation-incompetent vesicular stomatitis virus (VSV) has been used to determine the neutralizing antibody titers in convalescent COVID-19 patients. The neutralization titers in serum of two independently analyzed patient cohorts were available within 18 h and correlated well with those obtained with a classical SARS-CoV-2 neutralization test (Pearson correlation coefficients of r = 0.929 and r = 0.939, respectively). Most convalescent COVID-19 patients had only low titers of neutralizing antibodies (ND50 < 320). The sera of convalescent COVID-19 patients also neutralized pseudotype virus displaying the SARS-CoV-1 spike protein on their surface, which is homologous to the SARS-CoV-2 spike protein. In summary, we report a robust virus-neutralization assay, which can be used at low biosafety level 1 to rapidly quantify SARS-CoV-2-neutralizing antibodies in convalescent COVID-19 patients and vaccinated individuals.

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Identification of functionally distinct domains of human granulocyte- macrophage colony-stimulating factor using monoclonal antibodies

Blood ◽

10.1182/blood.v77.5.1033.1033 ◽

1991 ◽

Vol 77 (5) ◽

pp. 1033-1043 ◽

Cited By ~ 29

Author(s):

Y Kanakura ◽

SA Cannistra ◽

CB Brown ◽

M Nakamura ◽

GF Seelig ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Solid Phase ◽

Receptor Interaction ◽

Enzyme Linked Immunosorbent Assay ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40–77, 78–94, or 110–127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

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