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

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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A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

10.21203/rs.3.rs-75540/v1 ◽

2020 ◽

Author(s):

Dianfan Li ◽

Tingting Li ◽

Hongmin Cai ◽

Hebang Yao ◽

Bingjie Zhou ◽

...

Keyword(s):

Neutralizing Antibodies ◽

In Vitro Selection ◽

Binding Domain ◽

Host Cells ◽

Neutralization Activity ◽

Rapid Responses ◽

Therapeutic Development ◽

Single Domain Antibodies

Abstract SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

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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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A novel Schmallenberg virus subunit vaccine candidate protects IFNAR-/- mice against virulent SBV challenge

Scientific Reports ◽

10.1038/s41598-020-73424-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hani Boshra ◽

Gema Lorenzo ◽

Diego Charro ◽

Sandra Moreno ◽

Gabriel Soares Guerra ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Vaccine Candidate ◽

Clinical Signs ◽

Interferon Γ ◽

Schmallenberg Virus ◽

Immune Protection ◽

Large Ruminant ◽

Economical Alternative

AbstractSchmallenberg virus (SBV), an arthropod-transmitted pathogenic bunyavirus, continues to be a threat to the European livestock industry, causing morbidity and mortality among young ruminant livestock. Here, we describe a novel SBV subunit vaccine, based on bacterially expressed SBV nucleoprotein (SBV-N) administered with a veterinary-grade Saponin adjuvant. When assayed in an IFNAR-/- mouse model, SBV-N with Saponin induced strong non-neutralizing broadly virus-reactive antibodies, decreased clinical signs, as well as significantly reduced viremia. Vaccination assays also suggest that this level of immune protection is cell mediated, as evidenced by the lack of neutralizing antibodies, as well as interferon-γ secretion observed in vitro. Therefore, based on these results, bacterially expressed SBV-N, co-administered with veterinary-grade Saponin adjuvant may serve as a promising economical alternative to current SBV vaccines, and warrant further evaluation in large ruminant animal models. Moreover, we propose that this strategy may be applicable to other bunyaviruses.

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Alternative affinity tools: more attractive than antibodies?

Biochemical Journal ◽

10.1042/bj20101860 ◽

2011 ◽

Vol 436 (1) ◽

pp. 1-13 ◽

Cited By ~ 120

Author(s):

Vincent J. B. Ruigrok ◽

Mark Levisson ◽

Michel H. M. Eppink ◽

Hauke Smidt ◽

John van der Oost

Keyword(s):

Binding Proteins ◽

In Vitro Selection ◽

Rapid Development ◽

Physical Stability ◽

Production Costs ◽

Medical Setting ◽

Throughput Optimization ◽

Molecular Imprinted Polymers ◽

High Production

Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future developments.

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Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates

10.1101/2021.03.02.433390 ◽

2021 ◽

Cited By ~ 1

Author(s):

Joseph R. Francica ◽

Barbara J. Flynn ◽

Kathryn E. Foulds ◽

Amy T. Noe ◽

Anne P. Werner ◽

...

Keyword(s):

Nonhuman Primates ◽

Viral Infections ◽

Antibody Responses ◽

High Dose ◽

Post Challenge ◽

Igg Antibody ◽

Live Virus ◽

Virus Challenge ◽

Humoral Responses

AbstractAdjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FC receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3×106 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.

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A liposome-displayed hemagglutinin vaccine platform protects mice and ferrets from heterologous influenza virus challenge

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025759118 ◽

2021 ◽

Vol 118 (22) ◽

pp. e2025759118

Author(s):

Zachary R. Sia ◽

Xuedan He ◽

Ali Zhang ◽

Jann C. Ang ◽

Shuai Shao ◽

...

Keyword(s):

Influenza Virus ◽

Neutralizing Antibodies ◽

H3n2 Virus ◽

Antigen Delivery ◽

Vaccine Platform ◽

Virus Challenge ◽

H5n1 Influenza ◽

Adjuvant System ◽

Protein Particle

Recombinant influenza virus vaccines based on hemagglutinin (HA) hold the potential to accelerate production timelines and improve efficacy relative to traditional egg-based platforms. Here, we assess a vaccine adjuvant system comprised of immunogenic liposomes that spontaneously convert soluble antigens into a particle format, displayed on the bilayer surface. When trimeric H3 HA was presented on liposomes, antigen delivery to macrophages was improved in vitro, and strong functional antibody responses were induced following intramuscular immunization of mice. Protection was conferred against challenge with a heterologous strain of H3N2 virus, and naive mice were also protected following passive serum transfer. When admixed with the particle-forming liposomes, immunization reduced viral infection severity at vaccine doses as low as 2 ng HA, highlighting dose-sparing potential. In ferrets, immunization induced neutralizing antibodies that reduced the upper respiratory viral load upon challenge with a more modern, heterologous H3N2 viral strain. To demonstrate the flexibility and modular nature of the liposome system, 10 recombinant surface antigens representing distinct influenza virus strains were bound simultaneously to generate a highly multivalent protein particle that with 5 ng individual antigen dosing induced antibodies in mice that specifically recognized the constituent immunogens and conferred protection against heterologous H5N1 influenza virus challenge. Taken together, these results show that stable presentation of recombinant HA on immunogenic liposome surfaces in an arrayed fashion enhances functional immune responses and warrants further attention for the development of broadly protective influenza virus vaccines.

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Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naïve human antibody library

10.1101/2021.01.07.425806 ◽

2021 ◽

Author(s):

Benjamin Nikola Bell ◽

Abigail E. Powell ◽

Carlos Rodriguez ◽

Jennifer R Cochran ◽

Peter S. Kim

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

In Vitro Selection ◽

Surface Display ◽

Binding Domain ◽

Yeast Surface Display ◽

Surface Glycoprotein ◽

Human Antibody ◽

Receptor Binding Domain

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naive antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naive antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/mL in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naive antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.

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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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Immunoprotective Activity and Safety of a RespiratorySyncytial Virus Vaccine: Mucosal Delivery of Fusion Glycoprotein with aCpG OligodeoxynucleotideAdjuvant

Journal of Virology ◽

10.1128/jvi.77.24.13156-13160.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13156-13160 ◽

Cited By ~ 43

Author(s):

Gregory A. Prince ◽

James J. Mond ◽

David D. Porter ◽

Kevin C. Yim ◽

Steve J. Lan ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Neutralizing Antibody ◽

Live Virus ◽

Cotton Rat ◽

F Protein ◽

Virus Challenge ◽

Cpg Oligodeoxynucleotides ◽

Cotton Rats ◽

Syncytial Virus

ABSTRACT CpG oligodeoxynucleotides (ODN) were identified that stimulated immunoglobulin production and cell proliferation in cotton rat cells in vitro. Three of these ODN were used as a mucosal adjuvant in the noses of cotton rats immunized via this route with respiratory syncytial virus fusion (F) protein. The CpG ODN markedly increased the cotton rat humoral neutralizing-antibody response to respiratory syncytial virus. Such immunized animals had a marked reduction in the production of infectious virus after a live-virus challenge. Animals immunized with the combination of F protein and CpG developed enhanced pulmonary pathology consisting of alveolitis and interstitial pneumonitis after a live-virus challenge. Similar enhanced disease has been seen in cotton rats and children immunized with formalin-inactivated respiratory syncytial virus.

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An Adenovirus-Based Vaccine with a Double-Stranded RNA Adjuvant Protects Mice and Ferrets against H5N1 Avian Influenza in Oral Delivery Models

Clinical and Vaccine Immunology ◽

10.1128/cvi.00552-12 ◽

2012 ◽

Vol 20 (1) ◽

pp. 85-94 ◽

Cited By ~ 34

Author(s):

Ciaran D. Scallan ◽

Debora W. Tingley ◽

Jonathan D. Lindbloom ◽

James S. Toomey ◽

Sean N. Tucker

Keyword(s):

Avian Influenza ◽

Oral Delivery ◽

Neutralizing Antibodies ◽

Rapid Development ◽

Adenovirus Vector ◽

Oral Route ◽

Lethal Challenge ◽

Double Stranded Rna ◽

Virus Challenge ◽

H5n1 Avian Influenza

ABSTRACTAn oral gene-based avian influenza vaccine would allow rapid development and simplified distribution, but efficacy has previously been difficult to achieve by the oral route. This study assessed protection against avian influenza virus challenge using a chimeric adenovirus vector expressing hemagglutinin and a double-stranded RNA adjuvant. Immunized ferrets and mice were protected upon lethal challenge. Further, ferrets immunized by the peroral route induced cross-clade neutralizing antibodies, and the antibodies were selective against hemagglutinin, not the vector. Similarly, experiments in mice demonstrated selective immune responses against HA with peroral delivery and the ability to circumvent preexisting vector immunity.

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