An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

AbstractSARS-CoV-2 is the etiologic agent of COVID-19, currently causing a devastating pandemic for which pharmacological interventions are urgently needed. The virus enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. The nanobody binds with high affinity in the low nM range to the RBD, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations and that Ty1 sterically hinders RBD-ACE2 binding. This 12.8 kDa nanobody does not need an Fc domain to neutralize SARS-CoV-2, and can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.

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Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Science ◽

10.1126/science.abe3354 ◽

2020 ◽

Vol 370 (6519) ◽

pp. 950-957 ◽

Cited By ~ 21

Author(s):

M. Alejandra Tortorici ◽

Martina Beltramello ◽

Florian A. Lempp ◽

Dora Pinto ◽

Ha V. Dang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Viral Escape ◽

Angiotensin Converting Enzyme 2 ◽

Effector Functions ◽

Human Antibodies ◽

Binding Domains ◽

Isolation And Characterization ◽

Cryo Electron Microscopy ◽

Escape Mutants

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo–electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.

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Isolation and characterization of two new Staphylococcus aureus bacteriophages with potential to infect distinct bacteria from bovine mastitis

10.21203/rs.3.rs-229928/v1 ◽

2021 ◽

Author(s):

Bibiana Martins Barasuol ◽

Juliana Felipetto Cargnelutti ◽

Luis Antônio Sangioni ◽

Daniela Isabel Brayer Pereira ◽

Ana Paula Muterle Varela ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bovine Milk ◽

Bovine Mastitis ◽

Gc Content ◽

Etiologic Agent ◽

Cure Rate ◽

Long Tail ◽

Isolation And Characterization ◽

Double Stranded Dna

Abstract Bovine mastitis is an important disease of dairy cows, and Staphylococcus aureus is the etiologic agent most prevalent among the microorganisms. Mastitis caused by S. aureus present low cure rate with antimicrobials treatment and low vaccines efficacy. Bacteriophages or phages have been considered as an alternative for treating this disease. This study, we isolated and characterized two new S. aureus phages, namely B_UFSM4 and B_UFSM5, from bovine milk of cows with mastitis. The adsorptions rates were 10–20 min for B_UFSM4 and 20–30 min for B_UFSM5. Phages activities were relatively stable at pH 3–11; however, at temperatures of 50 °C-60ºC-70ºC/60 min, the phages were completely inactivated. These viruses presented infectivity in various bacteria isolated from bovine mastitis, where the lytic activity of phages B_UFSM4 and B_UFSM5 were 34.2%(13/38) and 42.1%(16/38), respectively, including isolates from S. aureus, Pseudomonas aeruginosa, Staphylococcus sciuri, and Rothia terrae. The complete genomes of B_UFSM4 and B_UFSM5 have 41.396 bp and 41.829 bp, with GC-content 33.97% and 33.98%, respectively. Both phages comprise 61 putative ORFs. The viruses have double stranded DNA and linear architecture. Phylogenic similarity was observed by proteome with Staphylococcus prophage phiPV83 (45,536 nt), Staphylococcus phage CN125 (44,492 nt) and Staphylococcus phage JS01 (43,458 nt). Based on the morphology, the phages belong to Siphoviridae family, presenting icosahedral head with a long tail, Caudovirales order and Biseptimavirus genus. Thus, two S. aureus phages (B_UFSM4 and B_UFSM5) were isolated and characterized, and these phages can be used as therapeutic or prophylactic candidates against S. aureus infections in cattle mastitis.

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First isolation and characterization of Brucella suis from yak

Genome ◽

10.1139/gen-2019-0101 ◽

2020 ◽

Vol 63 (8) ◽

pp. 397-405

Author(s):

Xiaowen Yang ◽

Ning Wang ◽

Xiaofang Cao ◽

Pengfei Bie ◽

Zhifeng Xing ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Health And Safety ◽

Host Cells ◽

Whole Genome ◽

Genetic Characteristics ◽

Qinghai Province ◽

Isolation And Characterization ◽

Smooth Strain

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

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Isolation and Characterization of a Thermally Stable Recombinant Anti-Caffeine Heavy-Chain Antibody Fragment

Analytical Chemistry ◽

10.1021/ac058044j ◽

2006 ◽

Vol 78 (13) ◽

pp. 4501-4508 ◽

Cited By ~ 81

Author(s):

Ruth C. Ladenson ◽

Dan L. Crimmins ◽

Yvonne Landt ◽

Jack H. Ladenson

Keyword(s):

Heavy Chain ◽

Antibody Fragment ◽

Thermally Stable ◽

Isolation And Characterization ◽

Heavy Chain Antibody

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Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-005-3401-3 ◽

2005 ◽

Vol 383 (2) ◽

pp. 218-223 ◽

Cited By ~ 4

Author(s):

Jiebo Mi ◽

Jin Yan ◽

Zhenquan Guo ◽

Meiping Zhao ◽

Wenbao Chang

Keyword(s):

Phage Display ◽

Antibody Fragment ◽

Recombinant Erythropoietin ◽

Antibody Library ◽

Single Chain ◽

Single Chain Antibody ◽

Isolation And Characterization ◽

Single Chain Antibody Fragment ◽

Phage Display Antibody

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Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

10.1101/2020.10.12.336016 ◽

2020 ◽

Author(s):

Dongchun Ni ◽

Kelvin Lau ◽

Frank Lehmann ◽

Andri Fränkl ◽

David Hacker ◽

...

Keyword(s):

Structural Rearrangement ◽

Host Cells ◽

Surface Glycoprotein ◽

Angiotensin Converting Enzyme 2 ◽

Cryo Electron Microscopy ◽

Monomeric Complex ◽

Electron Microscopy Analysis ◽

Microscopy Analysis ◽

Viral Surface

AbstractThe human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.

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Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

10.1101/2020.06.22.166033 ◽

2020 ◽

Cited By ~ 4

Author(s):

Tongqing Zhou ◽

I-Ting Teng ◽

Adam S. Olia ◽

Gabriele Cerutti ◽

Jason Gorman ◽

...

Keyword(s):

Region Of Interest ◽

Molecular Probes ◽

Terminal Sequence ◽

Protease Cleavage ◽

Isolation And Characterization ◽

Cryo Electron Microscopy ◽

Specific Protease ◽

A Site ◽

Biotinylated Probes

SummaryBiotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

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Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

10.1101/2021.02.02.428995 ◽

2021 ◽

Author(s):

Tal Noy-Porat ◽

Adva Mechaly ◽

Yinon Levy ◽

Efi Makdasi ◽

Ron Alcalay ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Phage Display Library ◽

Host Cells ◽

Terminal Domain ◽

Isolation And Characterization ◽

Recent Emergence ◽

Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

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