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1.
Cell Rep ; 39(7): 110812, 2022 May 17.
Article in English | MEDLINE | ID: covidwho-1803708

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral , COVID-19/drug therapy , Epitopes , Humans
2.
Int J Biol Macromol ; 209(Pt A): 1379-1388, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1796724

ABSTRACT

SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 µg mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.


Subject(s)
SARS-CoV-2 , Single-Domain Antibodies , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Protein Binding , Protein Engineering , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus/chemistry
3.
Front Public Health ; 10: 847384, 2022.
Article in English | MEDLINE | ID: covidwho-1792872

ABSTRACT

Background: Immunity and clinical protection induced by mRNA vaccines against SARS-CoV-2 have been shown to decline overtime. To gather information on the immunity profile deemed sufficient in protecting against hospitalization, we tested IgG levels, interferon-gamma (IFN-γ) secretion, and neutralizing antibodies 180 days (d180) after the second shot of BNT162b vaccine, in HW. Methods: A total of 392 subjects were enrolled. All received BioNTech/Pfizer from February 2020 to April 2021. The vaccine-specific humoral response was quantitatively determined by testing for IgG anti-S1 domain of SARS-CoV-spike protein. Live virus microneutralization (MN) was evaluated by an assay performing incubation of serial 2-fold dilution of human serum samples, starting from 1:10 to 1:5120, with an equal volume of Wuhan strain and Delta VOC viral solution and assessing the presence/absence of a cytopathic effect. SARS-CoV-2-spike protein-specific T-cell response was determined by a commercial IFN-γ release assay. Results: In 352 individuals, at d180, IgG levels decreased substantially but no results below the assay's positivity threshold were observed. Overall, 22 naive (8.1%) had values above the highest threshold. Among COVID-naive, the impact of age, which was observed at earlier stages, disappeared at d180, while it remained significant for 81 who had experienced a previous infection. Following the predictive model of protection by Khoury, we transformed the neutralizing titers in IU/ml and used a 54 IU/ml threshold to identify subjects with 50% protective immunity. Overall, live virus MN showed almost all subjects with previous exposure to SARS-CoV-2 neutralized the virus as compared to 33% of naive double-dosed subjects (p < 0.0001). All previously exposed subjects had strong IFN-γ secretion (>200 mIU/ml); among 271 naive, 7 (2.58%) and 17 (6.27%) subjects did not show borderline or strong secretion, respectively. Conclusions: In naive subjects, low IgG titers are relatively long-lasting. Only a third of naive subjects maintain neutralizing responses. After specific stimulation, a very limited number of naive were unable to produce IFN-γ. The results attained in the small group of subjects with breakthrough infection suggest that simultaneous neutralizing antibody titers <20, binding antibody levels/ml <200, and IFN-γ <1,000 mIU/ml in subjects older than 58 may identify at-risk groups.


Subject(s)
COVID-19 Vaccines , COVID-19 , Antibodies, Neutralizing/pharmacology , Antibodies, Viral , COVID-19/prevention & control , Health Personnel , Humans , Immunity, Humoral , Immunoglobulin G , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/pharmacology
5.
Nat Commun ; 13(1): 1152, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730284

ABSTRACT

In spring 2021, an increasing number of infections was observed caused by the hitherto rarely described SARS-CoV-2 variant A.27 in south-west Germany. From December 2020 to June 2021 this lineage has been detected in 31 countries. Phylogeographic analyses of A.27 sequences obtained from national and international databases reveal a global spread of this lineage through multiple introductions from its inferred origin in Western Africa. Variant A.27 is characterized by a mutational pattern in the spike gene that includes the L18F, L452R and N501Y spike amino acid substitutions found in various variants of concern but lacks the globally dominant D614G. Neutralization assays demonstrate an escape of A.27 from convalescent and vaccine-elicited antibody-mediated immunity. Moreover, the therapeutic monoclonal antibody Bamlanivimab and partially the REGN-COV2 cocktail fail to block infection by A.27. Our data emphasize the need for continued global monitoring of novel lineages because of the independent evolution of new escape mutations.


Subject(s)
COVID-19/immunology , COVID-19/virology , Pandemics , SARS-CoV-2/immunology , Africa, Western/epidemiology , Amino Acid Substitution , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , Antiviral Agents/pharmacology , COVID-19/transmission , Drug Combinations , Germany/epidemiology , Global Health , Humans , Immune Evasion/genetics , Mutation , Phylogeography , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
6.
Cell ; 185(3): 447-456.e11, 2022 02 03.
Article in English | MEDLINE | ID: covidwho-1712497

ABSTRACT

The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally dominant. Further, the high number of mutations in the viral spike protein raised concerns that the virus might evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant against most therapeutic antibodies but remained susceptible to inhibition by sotrovimab. Similarly, the Omicron spike evaded neutralization by antibodies from convalescent patients or individuals vaccinated with the BioNTech-Pfizer vaccine (BNT162b2) with 12- to 44-fold higher efficiency than the spike of the Delta variant. Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1 (Astra Zeneca-Oxford)/BNT162b2 vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omicron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization with BNT162b2 might not adequately protect against severe disease induced by this variant.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adaptive Immunity , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , Cell Line , Chlorocebus aethiops , Female , Humans , Male , Protein Binding , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vaccination , Vero Cells
8.
Viruses ; 14(2)2022 02 14.
Article in English | MEDLINE | ID: covidwho-1687056

ABSTRACT

Omicron was designated by the WHO as a VOC on 26 November 2021, only 4 days after its sequence was first submitted. However, the impact of Omicron on current antibodies and vaccines remains unknown and evaluations are still a few weeks away. We analysed the mutations in the Omicron variant against epitopes. In our database, 132 epitopes of the 120 antibodies are classified into five groups, namely NTD, RBD-1, RBD-2, RBD-3, and RBD-4. The Omicron mutations impact all epitopes in NTD, RBD-1, RBD-2, and RBD-3, with no antibody epitopes spared by these mutations. Only four out of 120 antibodies may confer full resistance to mutations in the Omicron spike, since all antibodies in these three groups contain one or more epitopes that are affected by these mutations. Of all antibodies under EUA, the neutralisation potential of Etesevimab, Bamlanivimab, Casirivimab, Imdevima, Cilgavimab, Tixagevimab, Sotrovimab, and Regdanvimab might be dampened to varying degrees. Our analysis suggests the impact of Omicron on current therapeutic antibodies by the Omicron spike mutations may also apply to current COVID-19 vaccines.


Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Viral/pharmacology , Computer Simulation , Mutation/immunology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Monoclonal/classification , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neutralizing/pharmacology , Databases, Factual , Epitopes/immunology , Humans , Immunoglobulin G/pharmacology , Neutralization Tests , Spike Glycoprotein, Coronavirus/immunology
10.
Signal Transduct Target Ther ; 7(1): 44, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1683982

ABSTRACT

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.


Subject(s)
Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Immunoglobulin Fc Fragments/pharmacology , Recombinant Fusion Proteins/pharmacology , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/genetics , Antibodies, Viral/biosynthesis , Antibodies, Viral/genetics , Antibody Affinity , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/genetics , Models, Molecular , Neutralization Tests , Protein Binding/drug effects , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/immunology , Single-Domain Antibodies/biosynthesis , Single-Domain Antibodies/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
12.
Emerg Microbes Infect ; 11(1): 548-551, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1642258

ABSTRACT

The neutralizing antibody is a potential therapeutic for the ongoing COVID-19 pandemic. As an antiviral agent, numerous mAbs recognize the epitopes that overlap with ACE2-binding sites in the SARS-CoV-2-RBD. Some studies have shown that residual changes on the spike protein can significantly decrease the efficiency of neutralizing antibodies. To address this issue, a therapeutic cocktail could be an effective countermeasure. In the present study, we isolated a fully human neutralizing antibody, JS026, from a convalescent patient. The comparative analysis revealed that JS026 binding to SARS-CoV-2-RBD mainly located between epitopes for class 2 and class 3 mAbs as opposed to that of class 1 (etesevimab) antibodies. A cocktail of etesevimab and JS026 increased neutralizing efficacy against both wild-type SARS-CoV-2 and the recent emergence of Alpha, Beta, Gamma, and Delta variants. JS026 and the cocktail reduced virus titers in the infected lungs of hACE2 transgenic mice and relieved pathological changes. These findings would benefit antibody-based therapeutic countermeasures in the treatment of COVID-19.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neutralizing/pharmacology , SARS-CoV-2 , Animals , Antibodies, Viral , COVID-19 , Humans , Mice , Mice, Transgenic , Pandemics , SARS-CoV-2/drug effects
13.
MAbs ; 14(1): 2002236, 2022.
Article in English | MEDLINE | ID: covidwho-1585298

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cell Surface Display Techniques , Immunoglobulin G/immunology , Peptide Library , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Specificity , Binding Sites, Antibody , COVID-19/metabolism , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Epitopes , Female , Host-Pathogen Interactions , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , Mesocricetus , SARS-CoV-2/pathogenicity , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Single-Domain Antibodies/pharmacology , Vero Cells
14.
Front Immunol ; 12: 766821, 2021.
Article in English | MEDLINE | ID: covidwho-1581335

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , HEK293 Cells , Humans , Immunization, Passive , Mice
15.
Nat Commun ; 12(1): 6977, 2021 11 30.
Article in English | MEDLINE | ID: covidwho-1545609

ABSTRACT

Despite an unprecedented global gain in knowledge since the emergence of SARS-CoV-2, almost all mechanistic knowledge related to the molecular and cellular details of viral replication, pathology and virulence has been generated using early prototypic isolates of SARS-CoV-2. Here, using atomic force microscopy and molecular dynamics, we investigated how these mutations quantitatively affected the kinetic, thermodynamic and structural properties of RBD-ACE2 complex formation. We observed for several variants of concern a significant increase in the RBD-ACE2 complex stability. While the N501Y and E484Q mutations are particularly important for the greater stability, the N501Y mutation is unlikely to significantly affect antibody neutralization. This work provides unprecedented atomistic detail on the binding of SARS-CoV-2 variants and provides insight into the impact of viral mutations on infection-induced immunity.


Subject(s)
Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/pharmacology , COVID-19/therapy , COVID-19/virology , Humans , Kinetics , Microscopy, Atomic Force , Molecular Dynamics Simulation , Mutation , Protein Binding/drug effects , Protein Interaction Domains and Motifs , Protein Stability , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Thermodynamics
16.
Sci Rep ; 11(1): 22848, 2021 11 24.
Article in English | MEDLINE | ID: covidwho-1532101

ABSTRACT

While mRNA vaccines against SARS-CoV-2 are exceedingly effective in preventing symptomatic infection, their immune response features remain to be clarified. In the present prospective study, 225 healthy individuals in Japan, who received two BNT162b2 doses, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT50; assessed using infectious virions) with various determinants were examined and the potency of sera against variants of concerns was determined. Significant rise in NT50s was seen in sera on day 28 post-1st dose. A moderate inverse correlation was seen between NT50s and ages, but no correlation seen between NT50s and adverse effects. NT50s and SARS-CoV-2-S1-binding-IgG levels on day 28 post-1st dose and pain scores following the 2nd dose were greater in women than in men. The average half-life of NT50s was ~ 68 days, and 23.6% (49 out of 208 individuals) failed to show detectable neutralizing activity on day 150. While sera from elite-responders (NT50s > 1,500: the top 4% among the participants) potently to moderately blocked all variants of concerns examined, some sera with low NT50s failed to block the B.1.351-beta strain. Since BNT162b2-elicited immunity against SARS-CoV-2 is short, an additional vaccine or other protective measures are needed.


Subject(s)
/adverse effects , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/pharmacokinetics , Drug-Related Side Effects and Adverse Reactions , Female , Humans , Immunogenicity, Vaccine/immunology , Immunologic Tests , Japan , Kinetics , Male , Middle Aged , Prospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
17.
Clin Pharmacol Ther ; 111(3): 572-578, 2022 03.
Article in English | MEDLINE | ID: covidwho-1527428

ABSTRACT

Leveraging limited clinical and nonclinical data through modeling approaches facilitates new drug development and regulatory decision making amid the coronavirus disease 2019 (COVID-19) pandemic. Model-informed drug development (MIDD) is an essential tool to integrate those data and generate evidence to (i) provide support for effectiveness in repurposed or new compounds to combat COVID-19 and dose selection when clinical data are lacking; (ii) assess efficacy under practical situations such as dose reduction to overcome supply issues or emergence of resistant variant strains; (iii) demonstrate applicability of MIDD for full extrapolation to adolescents and sometimes to young pediatric patients; and (iv) evaluate the appropriateness for prolonging a dosing interval to reduce the frequency of hospital visits during the pandemic. Ongoing research activities of MIDD reflect our continuous effort and commitment in bridging knowledge gaps that leads to the availability of effective treatments through innovation. Case examples are presented to illustrate how MIDD has been used in various stages of drug development and has the potential to inform regulatory decision making.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 , Drug Development/methods , Models, Biological , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , COVID-19/epidemiology , Drug Approval , Drug Repositioning , Humans , Pharmacology, Clinical/methods , SARS-CoV-2/immunology
18.
Front Immunol ; 12: 750386, 2021.
Article in English | MEDLINE | ID: covidwho-1515534

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Epitopes/immunology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Animals , Binding Sites, Antibody/immunology , COVID-19/drug therapy , Cell Line, Tumor , Chlorocebus aethiops , Female , Glycosylation , HEK293 Cells , Humans , Mice, Inbred BALB C , Neutralization Tests , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
19.
Theranostics ; 12(1): 1-17, 2022.
Article in English | MEDLINE | ID: covidwho-1512993

ABSTRACT

Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro. Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , COVID-19/drug therapy , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Binding Sites , Binding, Competitive , COVID-19/virology , Cricetinae , Cryoelectron Microscopy , Crystallography, X-Ray , Dogs , Epitopes , Female , Humans , Madin Darby Canine Kidney Cells , Neutralization Tests , Protein Domains , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
20.
J Appl Microbiol ; 132(3): 2421-2430, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1488216

ABSTRACT

AIMS: COVID-19 pandemic caused by SARS-CoV-2 has become a public health crisis worldwide. In this study, we aimed at demonstrating the neutralizing potential of the IgY produced after immunizing chicken with a recombinant SARS-CoV-2 spike protein S1 subunit. METHODS AND RESULTS: E. coli BL21 carrying plasmid pET28a-S1 was induced with IPTG for the expression of SARS-CoV-2 S1 protein. The recombinant His-tagged S1 was purified and verified by SDS-PAGE, Western blot and biolayer interferometry (BLI) assay. Then S1 protein emulsified with Freund's adjuvant was used to immunize layer chickens. Specific IgY against S1 (S1-IgY) produced from egg yolks of these chickens exhibited a high titer (1:25,600) and a strong binding affinity to S1 (KD  = 318 nmol L-1 ). The neutralizing ability of S1-IgY was quantified by a SARS-CoV-2 pseudotyped virus-based neutralization assay with an IC50  value of 0.99 mg ml-1 . In addition, S1-IgY exhibited a strong ability in blocking the binding of SARS-CoV-2 S1 to hACE2, and it could partially compete with hACE2 for the binding sites on S1 by BLI assays. CONCLUSIONS: We demonstrated here that after immunization of chickens with our recombinant S1 protein, IgY neutralizing antibodies were generated against the SARS-CoV-2 spike protein S1 subunit; therefore, showing the potential use of IgY to block the entry of this virus. SIGNIFICANCE AND IMPACT OF THE STUDY: IgY targeting S1 subunit of SARS-CoV-2 could be a promising candidate for pre- and post-exposure prophylaxis or treatment of COVID-19. Administration of IgY-based oral preparation, oral or nasal spray may have profound implications for blocking SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/pharmacology , Immunoglobulins/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Virus Internalization/drug effects , Animals , COVID-19 , Chickens , Egg Yolk/immunology , Humans , Pandemics
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