An ultrapotent neutralizing bispecific antibody with broad spectrum against SARS-CoV-2 variants

In spite of the successful development of effective countermeasures against Covid-19, variants have and will continue to emerge that could compromise the efficacy of currently approved neutralizing antibodies and vaccines. Consequently, novel and more efficacious agents are urgently needed. We have developed a bispecific antibody, 2022, consisting of two antibodies, 2F8 and VHH18. 2F8 was isolated from our proprietary fully synthetic human IDEAL (Intelligently Designed and Engineered Antibody Library)-VH/VL library and VHH18 is a single domain antibody isolated from IDEAL-nanobody library. 2022 was constructed by attaching VHH18 to the C-terminal of Fc of 2F8. 2022 binds two non-overlapping epitopes simultaneously on the RBD of the SARS-CoV-2 spike protein and blocks the binding of RBD to human angiotensin-converting enzyme 2 (ACE2). 2022 potently neutralizes SARS-CoV-2 and all of the variants tested in both pseudovirus and live virus assays, including variants carrying mutations known to resist neutralizing antibodies approved under EUA and that reduce the protection efficiency of current effective vaccines. The half-maximum inhibitory concentration (IC50) of 2022 is 270 pM, 30 pM, 20 pM, and 1 pM, for wildtype, alpha, beta, and delta pseudovirus, respectively. In the live virus assay, 2022 has an IC50 of 30 pM, 13 pM, and 90 pM, for wildtype, beta, and delta live virus, respectively. In a mouse model of SARS-CoV-2, 2022 showed strong prophylactic and therapeutic effects. A single administration of 2022 intranasal (i.n.) or intraperitoneal (i.p.) 24 hours before virus challenge completely protected all mice from bodyweight loss, as compared with up to 20% loss of bodyweight in placebo treated mice. In addition, the lung viral titers were undetectable (FRNT assay) in all mice treated with 2022 either prophylactically or therapeutically, as compared with around 1X105 pfu/g lung tissue in placebo treated mice. In summary, bispecific antibody 2022 showed potent binding and neutralizing activity across a variety of SARS-CoV-2 variants and could be an attractive weapon to combat the ongoing waves of the COVID-19 pandemic propagated mainly by variants, especially, the much more contagious delta variant.

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An Ultrapotent Neutralizing Bispecific Antibody with Broad Spectrum Against SARS-CoV-2 Variants

10.21203/rs.3.rs-951257/v1 ◽

2021 ◽

Author(s):

Hui Zhang ◽

Haohui Huang ◽

Rong Li ◽

Lu Zhang ◽

Zhiwei Wang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Bispecific Antibody ◽

Herd Immunity ◽

Therapeutic Effects ◽

Bodyweight Loss ◽

Receptor Binding Domain ◽

Single Administration ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

High Affinity

Abstract Some variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are threatening our global efforts of herd immunity, novel and more efficacious agents are urgently needed. We have developed a bispecific antibody, 2022, which bonds with high affinity to two non-overlapping epitopes on the receptor-binding domain (RBD) simultaneously, blocks the binding of RBD to human angiotensin-converting enzyme 2 (ACE2), and potently neutralizes SARS-CoV-2 and all of the variants tested, including variants carrying mutations known to resist neutralizing antibodies approved under Emergency Use Authorization (EUA) and reduce the efficacy of existing vaccines. In a mouse model of SARS-CoV-2, 2022 showed strong prophylactic and therapeutic effects. A single administration of 2022 completely protected all mice from bodyweight loss, as compared with up to 20% loss of bodyweight in placebo treated mice, reduced the lung viral titers to undetectable in all mice treated with 2022 either prophylactically or therapeutically, as compared with around 1X105 pfu/g lung tissue in placebo treated mice. In summary, bispecific antibody 2022 showed potent binding and neutralizing activity across a variety of SARS-CoV-2 variants and could be an attractive weapon to combat the ongoing waves of the COVID-19 pandemic.

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Probing Morbillivirus Antisera Neutralization Using Functional Chimerism between Measles Virus and Canine Distemper Virus Envelope Glycoproteins

Viruses ◽

10.3390/v11080688 ◽

2019 ◽

Vol 11 (8) ◽

pp. 688 ◽

Cited By ~ 7

Author(s):

Miguel Angel Muñoz-Alía ◽

Stephen J. Russell

Keyword(s):

Neutralizing Antibodies ◽

Measles Virus ◽

Canine Distemper Virus ◽

Reverse Genetics ◽

Canine Distemper ◽

Virus Envelope ◽

Live Virus ◽

Virus Challenge ◽

Virus Attachment ◽

Globular Head

Measles virus (MeV) is monotypic. Live virus challenge provokes a broadly protective humoral immune response that neutralizes all known measles genotypes. The two surface glycoproteins, H and F, mediate virus attachment and entry, respectively, and neutralizing antibodies to H are considered the main correlate of protection. Herein, we made improvements to the MeV reverse genetics system and generated a panel of recombinant MeVs in which the globular head domain or stalk region of the H glycoprotein or the entire F protein, or both, were substituted with the corresponding protein domains from canine distemper virus (CDV), a closely related morbillivirus that resists neutralization by measles-immune sera. The viruses were tested for sensitivity to human or guinea pig neutralizing anti-MeV antisera and to ferret anti-CDV antisera. Virus neutralization was mediated by antibodies to both H and F proteins, with H being immunodominant in the case of MeV and F being so in the case of CDV. Additionally, the globular head domains of both MeV and CDV H proteins were immunodominant over their stalk regions. These data shed further light on the factors constraining the evolution of new morbillivirus serotypes.

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Profiling B cell immune responses to identify neutralizing antibodies from convalescent COVID-19 patients

10.21203/rs.3.rs-38173/v2 ◽

2020 ◽

Author(s):

Lisu Huang ◽

Bingqing Shen ◽

Yu Guo ◽

Shu Shen ◽

Heyu Huang ◽

...

Keyword(s):

B Cell ◽

Neutralizing Antibodies ◽

Therapeutic Option ◽

Antiviral Drug ◽

Cell Receptor ◽

High Serum ◽

Spike Protein ◽

Live Virus ◽

Angiotensin Converting Enzyme 2 ◽

Humoral Responses

Abstract The pandemic Coronavirus Disease 2019 (COVID-19) causes noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralizing antibodies for treatment purposes is a viable alternative. In this study, we aimed to profile the humoral responses and identify neutralizing antibodies against SARS-CoV-2 using high-throughput single-cell sequencing that tailored to B cell receptor sequencing. From two convalescent patients with high serum titer against SARS-COV-2, we identified seven antibodies specifically binding to SARS-CoV-2. Among these, the most potent antibody, P4A1 was demonstrated to block the binding of spike protein to its receptor angiotensin-converting enzyme 2 (ACE2), and prevent the viral infection in neutralization assays with pseudovirus as well as live virus at nM to sub-nM range. Moreover, antibody P4A1 can also bind strongly to spike protein with N354D/D364Y, R408I, W436R, V367F or D614G mutations respectively, suggesting that the antibody alone or in combination with other antibodies that recognize different variations of SARS-CoV-2, may provide a broad spectrum therapeutic option for COVID-19 patients. Authors Lisu Huang, Bingqing Shen, Yu Guo, and Shu Shen contributed equally to this work.

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A Novel, Killed-Virus Nasal Vaccinia Virus Vaccine

Clinical and Vaccine Immunology ◽

10.1128/cvi.00440-07 ◽

2007 ◽

Vol 15 (2) ◽

pp. 348-358 ◽

Cited By ~ 35

Author(s):

Anna U. Bielinska ◽

Alexander A. Chepurnov ◽

Jeffrey J. Landers ◽

Katarzyna W. Janczak ◽

Tatiana S. Chepurnova ◽

...

Keyword(s):

Vaccinia Virus ◽

Neutralizing Antibodies ◽

Clinical Symptoms ◽

Lethal Dose ◽

Minimal Risk ◽

Live Virus ◽

Virus Challenge ◽

Lethal Infection ◽

Nasal Vaccination ◽

Complete Disruption

ABSTRACT Live-virus vaccines for smallpox are effective but have risks that are no longer acceptable for routine use in populations at minimal risk of infection. We have developed a mucosal, killed-vaccinia virus (VV) vaccine based on antimicrobial nanoemulsion (NE) of soybean oil and detergent. Incubation of VV with 10% NE for at least 60 min causes the complete disruption and inactivation of VV. Simple mixtures of NE and VV (Western Reserve serotype) (VV/NE) applied to the nares of mice resulted in both systemic and mucosal anti-VV immunity, virus-neutralizing antibodies, and Th1-biased cellular responses. Nasal vaccination with VV/NE vaccine produced protection against lethal infection equal to vaccination by scarification, with 100% survival after challenge with 77 times the 50% lethal dose of live VV. However, animals protected with VV/NE immunization did after virus challenge have clinical symptoms more extensive than animals vaccinated by scarification. VV/NE-based vaccines are highly immunogenic and induce protective mucosal and systemic immunity without the need for an inflammatory adjuvant or infection with live virus.

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Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays

Journal of Clinical Microbiology ◽

10.1128/jcm.00527-21 ◽

2021 ◽

Author(s):

Anton M. Sholukh ◽

Andrew Fiore-Gartland ◽

Emily S. Ford ◽

Maurine D. Miner ◽

Yixuan J. Hou ◽

...

Keyword(s):

Geometric Mean ◽

Live Virus ◽

Angiotensin Converting Enzyme 2 ◽

Protein Receptor ◽

Virus Assay ◽

Viral Particles ◽

Antibody Assays ◽

High Concordance ◽

Highly Correlated ◽

Transmembrane Serine Protease

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81–0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD ( r = 0.63–0.89), but moderately correlated with nucleoprotein IgG ( r = 0.46–0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 249

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Bispecific antibody prevents SARS-CoV-2 escape and protects mice from disease

10.1101/2021.01.22.427567 ◽

2021 ◽

Author(s):

Raoul De Gasparo ◽

Mattia Pedotti ◽

Luca Simonelli ◽

Petr Nickl ◽

Frauke Muecksch ◽

...

Keyword(s):

Single Molecule ◽

Lung Inflammation ◽

Neutralizing Antibodies ◽

Bispecific Antibody ◽

Viral Escape ◽

Cellular Receptor ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Escape Mutants ◽

Novel Model

SummaryNeutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19)1,2. We developed a bispecific, IgG1-like molecule based on two antibodies derived from COVID-19 convalescent donors, C121 and C1353. CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, completely prevents S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 recognizes a broad panel of RBD variants and neutralizes SARS-CoV-2 and the escape mutants generated by the single monoclonals at sub-nanomolar concentrations. In a novel model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.

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Profiling B cell immune responses to identify neutralizing antibodies from convalescent COVID-19 patients

10.21203/rs.3.rs-38173/v1 ◽

2020 ◽

Author(s):

Lisu Huang ◽

Bingqing Shen ◽

Yu Guo ◽

Shu Shen ◽

Heyu Huang ◽

...

Keyword(s):

B Cell ◽

Neutralizing Antibodies ◽

Therapeutic Option ◽

Antiviral Drug ◽

Cell Receptor ◽

High Serum ◽

Spike Protein ◽

Live Virus ◽

Angiotensin Converting Enzyme 2 ◽

Humoral Responses

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A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection

Nature Communications ◽

10.1038/s41467-021-24905-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Tingting Li ◽

Hongmin Cai ◽

Hebang Yao ◽

Bingjie Zhou ◽

Ning Zhang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

In Vitro Selection ◽

Rapid Development ◽

Clinical Signs ◽

Live Virus ◽

Fusion Construct ◽

Virus Challenge ◽

Medical Interventions ◽

Single Domain Antibodies

AbstractSARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

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Evaluation of a Pseudovirus Neutralization Assay for SARS-CoV-2 and Correlation with Live Virus-Based Micro Neutralization Assay

Diagnostics ◽

10.3390/diagnostics11060994 ◽

2021 ◽

Vol 11 (6) ◽

pp. 994

Author(s):

Ahmed Majdi K. Tolah ◽

Sayed S. Sohrab ◽

Khaled Majdi K. Tolah ◽

Ahmed M. Hassan ◽

Sherif A. El-Kafrawy ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Epidemiological Studies ◽

Neutralization Assay ◽

Serum Samples ◽

Live Virus ◽

Spike Gene ◽

Microneutralization Assay ◽

Viral Particles ◽

Reliable Performance ◽

Prior Infection

The unusual cases of pneumonia outbreak were reported from Wuhan city in late December 2019. Serological testing provides a powerful tool for the identification of prior infection and for epidemiological studies. Pseudotype virus neutralization assays are widely used for many viruses and applications in the fields of serology. The accuracy of pseudotype neutralizing assay allows for its use in low biosafety lab and provides a safe and effective alternative to the use of wild-type viruses. In this study, we evaluated the performance of this assay compared to the standard microneutralization assay as a reference. The lentiviral pseudotype particles were generated harboring the Spike gene of SARS-CoV-2. The generated pseudotype particles assay was used to evaluate the activity of neutralizing antibodies in 300 human serum samples from a COVID-19 sero-epidemiological study. Testing of these samples resulted in 55 positive samples and 245 negative samples by pseudotype viral particles assay while microneutralization assay resulted in 64 positive and 236 negative by MN assay. Compared to the MN, the pseudotyped viral particles assay showed a sensitivity of 85.94% and a specificity of 100%. Based on the data generated from this study, the pseudotype-based neutralization assay showed a reliable performance for the detection of neutralizing antibodies against SARS-CoV-2 and can be used safely and efficiently as a diagnostic tool in a biosafety level 2 laboratory.

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A surrogate virus neutralization test to quantify antibody-mediated inhibition of SARS-CoV-2 in finger stick dried blood spot samples

Scientific Reports ◽

10.1038/s41598-021-94653-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Amelia E. Sancilio ◽

Richard T. D’Aquila ◽

Elizabeth M. McNally ◽

Matthew P. Velez ◽

Michael G. Ison ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Venous Blood ◽

Host Cells ◽

Dried Blood Spot ◽

Blood Collection ◽

Widespread Application ◽

Live Virus ◽

Dilution Series ◽

Wide Range ◽

Blood Spot

AbstractThe spike protein of SARS-CoV-2 engages the human angiotensin-converting enzyme 2 (ACE2) receptor to enter host cells, and neutralizing antibodies are effective at blocking this interaction to prevent infection. Widespread application of this important marker of protective immunity is limited by logistical and technical challenges associated with live virus methods and venous blood collection. To address this gap, we validated an immunoassay-based method for quantifying neutralization of the spike-ACE2 interaction in a single drop of capillary whole blood, collected on filter paper as a dried blood spot (DBS) sample. Samples are eluted overnight and incubated in the presence of spike antigen and ACE2 in a 96-well solid phase plate. Competitive immunoassay with electrochemiluminescent label is used to quantify neutralizing activity. The following measures of assay performance were evaluated: dilution series of confirmed positive and negative samples, agreement with results from matched DBS-serum samples, analysis of results from DBS samples with known COVID-19 status, and precision (intra-assay percent coefficient of variation; %CV) and reliability (inter-assay; %CV). Dilution series produced the expected pattern of dose–response. Agreement between results from serum and DBS samples was high, with concordance correlation = 0.991. Analysis of three control samples across the measurement range indicated acceptable levels of precision and reliability. Median % surrogate neutralization was 46.9 for PCR confirmed convalescent COVID-19 samples and 0.1 for negative samples. Large-scale testing is important for quantifying neutralizing antibodies that can provide protection against COVID-19 in order to estimate the level of immunity in the general population. DBS provides a minimally-invasive, low cost alternative to venous blood collection, and this scalable immunoassay-based method for quantifying inhibition of the spike-ACE2 interaction can be used as a surrogate for virus-based assays to expand testing across a wide range of settings and populations.

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