scholarly journals Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody

2019 ◽  
Vol 20 (20) ◽  
pp. 5073 ◽  
Author(s):  
Kim ◽  
Kim ◽  
Kim ◽  
Chang ◽  
Shim ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Therapeutic Antibody ◽  
Pulmonary Diseases ◽  
Human Antibody ◽  
Receptor Binding Domain ◽  
Hydrophobic Residues ◽  
Charged Amino Acids ◽  
Original Clone ◽  
Complementarity Determining Regions ◽  
Positively Charged

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe aggravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process, however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. We first isolated a MERS-CoV neutralizing antibody that is reactive to the receptor-binding domain (RBD) of spike (S) glycoprotein. To increase stability, we introduced mutations into the complementarity-determining regions (CDRs) of the antibody. In the HCDRs (excluding HCDR3) in this clone, two hydrophobic residues were replaced with Glu, two residues were replaced with Asp, and four residues were replaced with positively charged amino acids. In LCDRs, only two Leu residues were replaced with Val. These modifications successfully generated a clone with significantly greater stability and equivalent reactivity and neutralizing activity following nebulization compared to the original clone. In summary, we generated a MERS-CoV neutralizing human antibody that is reactive to recombinant MERS-CoV S RBD protein for delivery via a pulmonary route by introducing stabilizing mutations into five CDRs.

scholarly journals A Potent and Protective Human Neutralizing Antibody Against SARS-CoV-2 Variants

2021 ◽  
Vol 12 ◽  
Author(s):  
Sisi Shan ◽  
Chee Keng Mok ◽  
Shuyuan Zhang ◽  
Jun Lan ◽  
Jizhou Li ◽  
...  
Keyword(s):  
Prophylactic Treatment ◽  
Infectious Virus ◽  
Neutralizing Antibody ◽  
Human Antibody ◽  
Lung Pathology ◽  
Receptor Binding Domain ◽  
Isolation And Characterization ◽  
Normal Body Weight ◽  
Conserved Epitope

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge and spread around the world, antibodies and vaccines to confer broad and potent neutralizing activity are urgently needed. Through the isolation and characterization of monoclonal antibodies (mAbs) from individuals infected with SARS-CoV-2, we identified one antibody, P36-5D2, capable of neutralizing the major SARS-CoV-2 variants of concern. Crystal and electron cryo-microscopy (cryo-EM) structure analyses revealed that P36-5D2 targeted to a conserved epitope on the receptor-binding domain of the spike protein, withstanding the three key mutations—K417N, E484K, and N501Y—found in the variants that are responsible for escape from many potent neutralizing mAbs, including some already approved for emergency use authorization (EUA). A single intraperitoneal (IP) injection of P36-5D2 as a prophylactic treatment completely protected animals from challenge of infectious SARS-CoV-2 Alpha and Beta. Treated animals manifested normal body weight and were devoid of infection-associated death up to 14 days. A substantial decrease of the infectious virus in the lungs and brain, as well as reduced lung pathology, was found in these animals compared to the controls. Thus, P36-5D2 represents a new and desirable human antibody against the current and emerging SARS-CoV-2 variants.

scholarly journals Epitope–Paratope Interaction of a Neutralizing Human Anti-Hepatitis B Virus PreS1 Antibody That Recognizes the Receptor-Binding Motif

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 754
Author(s):  
Jisu Hong ◽  
Youngjin Choi ◽  
Yoonjoo Choi ◽  
Jiwoo Lee ◽  
Hyo Jeong Hong
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Hbv Infection ◽  
Binding Motif ◽  
Alanine Scanning Mutagenesis ◽  
B Virus ◽  
Complementarity Determining Regions

Hepatitis B virus (HBV) is a global health burden that causes acute and chronic hepatitis. To develop an HBV-neutralizing antibody that effectively prevents HBV infection, we previously generated a human anti-preS1 monoclonal antibody (1A8) that binds to genotypes A–D and validated its HBV-neutralizing activity in vitro. In the present study, we aimed to determine the fine epitope and paratope of 1A8 to understand the mechanism of HBV neutralization. We performed alanine-scanning mutagenesis on the preS1 (aa 19–34, genotype C) and the heavy (HCDR) and light (LCDR) chain complementarity-determining regions. The 1A8 recognized the three residues (Leu22, Gly23, and Phe25) within the highly conserved receptor-binding motif (NPLGFFP) of the preS1, while four CDR residues of 1A8 were critical in antigen binding. Structural analysis of the epitope–paratope interaction by molecular modeling revealed that Leu100 in the HCDR3, Ala50 in the HCDR2, and Tyr96 in the LCDR3 closely interacted with Leu22, Gly23, and Phe25 of the preS1. Additionally, we found that 1A8 also binds to the receptor-binding motif (NPLGFLP) of infrequently occurring HBV. The results suggest that 1A8 may broadly and effectively block HBV entry and thus have potential as a promising candidate for the prevention and treatment of HBV infection.

scholarly journals The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

2009 ◽  
Vol 284 (24) ◽  
pp. 16317-16324 ◽  
Author(s):  
Sandra Mueller ◽  
Gunnar Kleinau ◽  
Mariusz W. Szkudlinski ◽  
Holger Jaeschke ◽  
Gerd Krause ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thyroid Stimulating Hormone ◽  
Hinge Region ◽  
Camp Production ◽  
Glycoprotein Hormone ◽  
Charged Amino Acids ◽  
Bovine Thyroid ◽  
Positively Charged ◽  
Bovine Tsh ◽  
Signaling Activity

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu297, Glu303, and Asp382) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu297, Glu303, or Asp382 and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp382 seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.

scholarly journals Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19

2021 ◽  
Vol 22 (5) ◽  
pp. 2723
Author(s):  
Linhua Tian ◽  
Elzafir B. Elsheikh ◽  
Paul N. Patrone ◽  
Anthony J. Kearsley ◽  
Adolfas K. Gaigalas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Epidemiologic Studies ◽  
Cross Reactivity ◽  
Receptor Binding Domain ◽  
Proof Of Concept ◽  
Reference Standard ◽  
Improve Measurement ◽  
Antibody Titers

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

scholarly journals The C Terminus of Foamy Retrovirus Gag Contains Determinants for Encapsidation of Pol Protein into Virions

2008 ◽  
Vol 82 (21) ◽  
pp. 10803-10810 ◽  
Author(s):  
Eun-Gyung Lee ◽  
Maxine L. Linial
Keyword(s):  
Site Directed Mutagenesis ◽  
Rna Packaging ◽  
Interaction Domain ◽  
Virus Particles ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Foamy Viruses ◽  
Substitution Mutations ◽  
Gag Assembly ◽  
Positively Charged

ABSTRACT Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.

scholarly journals Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

2017 ◽  
Vol 114 (35) ◽  
pp. E7348-E7357 ◽  
Author(s):  
Jesper Pallesen ◽  
Nianshuang Wang ◽  
Kizzmekia S. Corbett ◽  
Daniel Wrapp ◽  
Robert N. Kirchdoerfer ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Case Fatality ◽  
Neutralizing Antibody ◽  
Human Populations ◽  
Receptor Binding Domain ◽  
Potential Mechanism ◽  
Primary Target ◽  
Humoral Immune ◽  
Receptor Recognition ◽  
Antibody Titers

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.

scholarly journals ANALYSIS OF IMMUNE ESCAPE VARIANTS FROM ANTIBODY-BASED THERAPEUTICS AGAINST COVID-19.

2021 ◽  
Author(s):  
Daniele Focosi ◽  
Fabrizio Maggi ◽  
Massimo Franchini ◽  
Scott McConnell ◽  
Arturo Casadevall
Keyword(s):  
Public Health ◽  
Monoclonal Antibodies ◽  
Immune Evasion ◽  
Immune Escape ◽  
Selective Pressure ◽  
Neutralizing Antibody ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Receptor Binding Domain ◽  
Amino Acid Mutations

Accelerated SARS-CoV-2 evolution under selective pressure by massive deployment of neutralizing antibody-based therapeutics is a concern with potentially severe implications for public health. We review here reports of documented immune escape after treatment with monoclonal antibodies and COVID19 convalescent plasma (CCP). While the former is mainly associated with specific single amino acid mutations at residues within the receptor-binding domain (e.g., E484K/Q, Q493R, and S494P), the few cases of immune evasion after CCP were associated with recurrent deletions within the N-terminal domain of Spike protein (e.g, delHV69-70, delLGVY141-144 and delAL243-244). Continuous genomic monitoring of non-responders is needed to better understand immune escape frequencies and fitness of emerging variants.

scholarly journals AbDiver - A tool to explore the natural antibody landscape to aid therapeutic design

2021 ◽  
Author(s):  
Jakub Mlokosiewicz ◽  
Piotr Deszynski ◽  
Wiktoria Wilman ◽  
Igor Jaszczyszyn ◽  
Rajkumar Ganesan ◽  
...  
Keyword(s):  
Rational Design ◽  
Therapeutic Antibody ◽  
Human Antibody ◽  
Use Case ◽  
Therapeutic Antibodies ◽  
Road Map ◽  
Antibody Sequence ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

Motivation: Rational design of therapeutic antibodies can be improved by harnessing the natural sequence diversity of these molecules. Our understanding of the diversity of antibodies has recently been greatly facilitated through the deposition of hundreds of millions of human antibody sequences in next-generation sequencing (NGS) repositories. Contrasting a query therapeutic antibody sequence to naturally observed diversity in similar antibody sequences from NGS can provide a mutational road-map for antibody engineers designing biotherapeutics. Because of the sheer scale of the antibody NGS datasets, performing queries across them is computationally challenging. Results: To facilitate harnessing antibody NGS data, we developed AbDiver (http://naturalantibody.com/abdiver), a free portal allowing users to compare their query sequences to those observed in the natural repertoires. AbDiver offers three antibody-specific use-cases: 1) compare a query antibody to positional variability statistics precomputed from multiple independent studies 2) retrieve close full variable sequence matches to a query antibody and 3) retrieve CDR3 or clonotype matches to a query antibody. We applied our system to a set of 742 therapeutic antibodies, demonstrating that for each use-case our system can retrieve relevant results for most sequences. AbDiver facilitates the navigation of vast antibody mutation space for the purpose of rational therapeutic antibody de-sign and engineering. Availability: AbDiver is freely accessible at http://naturalantibody.com/abdiver

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