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1.
J Mol Biol ; 434(10): 167583, 2022 May 30.
Article in English | MEDLINE | ID: covidwho-1778319

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.


Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , COVID-19 , SARS-CoV-2 , Viral Proteins , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/metabolism , Cell Surface Display Techniques , Coronavirus RNA-Dependent RNA Polymerase/analysis , Enzyme-Linked Immunosorbent Assay , Humans , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/analysis , Viral Proteins/analysis
2.
Front Public Health ; 10: 712657, 2022.
Article in English | MEDLINE | ID: covidwho-1775961

ABSTRACT

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24-100.0) and specificity (CI 88.78-100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.


Subject(s)
Antibodies, Monoclonal , Bacterial Proteins , Listeria monocytogenes , Antibodies, Monoclonal/metabolism , Bacterial Proteins/immunology , Bacteriophages , Cell Surface Display Techniques , Humans , Listeria monocytogenes/isolation & purification
3.
Viruses ; 14(2)2022 02 14.
Article in English | MEDLINE | ID: covidwho-1687052

ABSTRACT

The evolution of the SARS-CoV-2 virus during the COVID-19 pandemic was accompanied by the emergence of new heavily mutated viral variants with increased infectivity and/or resistance to detection by the human immune system. To respond to the urgent need for advanced methods and materials to empower a better understanding of the mechanisms of virus's adaptation to human host cells and to the immuno-resistant human population, we suggested using recombinant filamentous bacteriophages, displaying on their surface foreign peptides termed "mimotopes", which mimic the structure of viral receptor-binding sites on the viral spike protein and can serve as molecular probes in the evaluation of molecular mechanisms of virus infectivity. In opposition to spike-binding antibodies that are commonly used in studying the interaction of the ACE2 receptor with SARS-CoV-2 variants in vitro, phage spike mimotopes targeted to other cellular receptors would allow discovery of their role in viral infection in vivo using cell culture, tissue, organs, or the whole organism. Phage mimotopes of the SARS-CoV-2 Spike S1 protein have been developed using a combination of phage display and molecular mimicry concepts, termed here "phage mimicry", supported by bioinformatics methods. The key elements of the phage mimicry concept include: (1) preparation of a collection of p8-type (landscape) phages, which interact with authentic active receptors of live human cells, presumably mimicking the binding interactions of human coronaviruses such as SARS-CoV-2 and its variants; (2) discovery of closely related amino acid clusters with similar 3D structural motifs on the surface of natural ligands (FGF1 and NRP1), of the model receptor of interest FGFR and the S1 spike protein; and (3) an ELISA analysis of the interaction between candidate phage mimotopes with FGFR3 (a potential alternative receptor) in comparison with ACE2 (the authentic receptor).


Subject(s)
Bacteriophages/genetics , Cell Surface Display Techniques/methods , Molecular Mimicry , Receptors, Cell Surface/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Bacteriophages/metabolism , Binding Sites , Humans , Protein Binding , Receptors, Cell Surface/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment
4.
Front Immunol ; 12: 771011, 2021.
Article in English | MEDLINE | ID: covidwho-1639016

ABSTRACT

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is an ongoing pandemic. Detection and vaccination are essential for disease control, but they are distinct and complex operations that require significant improvements. Here, we developed an integrated detection and vaccination system to greatly simplify these efforts. We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based system to accurately detect patient serum. Notably, although well-recognized by our newly developed detection system in which S-displaying insect cells acted as antigen, anti-S antibodies from many patients were barely detectable by Western blot, evidencing that COVID-19 patients primarily produce conformation-dependent anti-S antibodies. Furthermore, the same baculovirus constructs can display N (N-Bac) or S (S-Bac) on the baculovirus envelope and serve as vector vaccines. Animal experiments show that S-Bac or N-Bac immunization in mice elicited a strong and specific antibody response, and S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination.


Subject(s)
Baculoviridae/immunology , COVID-19 Vaccines/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Aged, 80 and over , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Baculoviridae/genetics , COVID-19/immunology , COVID-19/prevention & control , Cell Line , Cell Surface Display Techniques , Coronavirus Nucleocapsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Phosphoproteins/genetics , Phosphoproteins/immunology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spodoptera , Vaccination , Young Adult
5.
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
6.
Mol Immunol ; 141: 287-296, 2022 01.
Article in English | MEDLINE | ID: covidwho-1559780

ABSTRACT

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19 Serological Testing , COVID-19/diagnosis , Cell Surface Display Techniques , Epitopes/immunology , Receptors, Virus/metabolism , SARS-CoV-2/immunology , Single-Chain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Viral/blood , Antibody Specificity , Baculoviridae , COVID-19/prevention & control , Escherichia coli , Female , Genetic Vectors , Mice , Mice, Inbred BALB C , Models, Molecular , Peptide Library , Protein Conformation , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid , Single-Chain Antibodies/biosynthesis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
7.
Viruses ; 13(11)2021 11 02.
Article in English | MEDLINE | ID: covidwho-1502526

ABSTRACT

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the "pairing potential" of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the "inside" or "outside" of the infected cell. Their therapeutic potential will be discussed.


Subject(s)
Antibodies, Monoclonal/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Viroporin Proteins/immunology , Animals , Biomarkers , COS Cells , Cell Surface Display Techniques/methods , Chlorocebus aethiops , Epitopes/immunology , HEK293 Cells , Humans , Membrane Proteins/immunology , Protein Domains , Vero Cells
8.
Adv Sci (Weinh) ; 9(1): e2102181, 2022 01.
Article in English | MEDLINE | ID: covidwho-1487434

ABSTRACT

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID-19) pandemic is used to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS-CoV-2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , Binding Sites , Binding, Competitive , Cell Surface Display Techniques , Chlorocebus aethiops , HEK293 Cells , Humans , Peptide Library , SARS-CoV-2/drug effects , Somatic Hypermutation, Immunoglobulin , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
9.
J Microbiol Biotechnol ; 31(8): 1088-1097, 2021 Aug 28.
Article in English | MEDLINE | ID: covidwho-1399433

ABSTRACT

Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate-buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of Escherichia coli BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV removal efficacy of the bacteria and viral cytotoxicity after bacterial adsorption treatment. Although both constructs were successfully expressed, only BL21/lpp-Mx exhibited GNNV binding activity; BL21/lpp-Mx cells removed GNNV and protected GF-1 cells from GNNV infection more efficiently. Moreover, salinity affected the GNNV removal efficacy of BL21/lpp-Mx. Thus, our GNNV-binding bacterium is an efficient microparticle for removing GNNV from 10‰ brackish water and for preventing GNNV infection in groupers.


Subject(s)
Bacteria/metabolism , Fish Diseases/prevention & control , Fish Proteins/metabolism , Myxovirus Resistance Proteins/metabolism , RNA Virus Infections/veterinary , Animals , Antiviral Agents/metabolism , Bacteria/genetics , Bass , Cell Line , Cell Membrane/metabolism , Cell Surface Display Techniques , Cell Survival , Fish Diseases/virology , Fish Proteins/genetics , Myxovirus Resistance Proteins/genetics , Nodaviridae/isolation & purification , Nodaviridae/metabolism , RNA Virus Infections/prevention & control , RNA Virus Infections/virology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salinity , Virus Attachment
10.
STAR Protoc ; 2(3): 100617, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1386745

ABSTRACT

This protocol is a comprehensive guide to phage display-based selection of virus neutralizing VH antibody domains. It details three optimized parts including (1) construction of a large-sized (theoretically > 1011) naïve human antibody heavy chain domain library, (2) SARS-CoV-2 antigen expression and stable cell line construction, and (3) library panning for selection of SARS-CoV-2-specific antibody domains. Using this protocol, we identified a high-affinity neutralizing human VH antibody domain, VH ab8, which exhibits high prophylactic and therapeutic efficacy. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Variable Region/immunology , Peptide Library , SARS-CoV-2/immunology , Amino Acid Sequence , Base Sequence , COVID-19/virology , Cell Surface Display Techniques/methods , Humans , SARS-CoV-2/isolation & purification , Sequence Homology
11.
Front Immunol ; 12: 678570, 2021.
Article in English | MEDLINE | ID: covidwho-1295637

ABSTRACT

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , COVID-19/prevention & control , COVID-19/therapy , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cell Surface Display Techniques/methods , Data Mining/methods , Epitopes/immunology , Humans , Immunization, Passive/methods
12.
Viruses ; 13(6)2021 06 10.
Article in English | MEDLINE | ID: covidwho-1264532

ABSTRACT

The latest coronavirus disease outbreak, COVID-19, has brought attention to viral infections which have posed serious health threats to humankind throughout history. The rapid global spread of COVID-19 is attributed to the increased human mobility of today's world, yet the threat of viral infections to global public health is expected to increase continuously in part due to increasing human-animal interface. Development of antiviral agents is crucial to combat both existing and novel viral infections. Recently, there is a growing interest in peptide/protein-based drug molecules. Antibodies are becoming especially predominant in the drug market. Indeed, in a remarkably short period, four antibody therapeutics were authorized for emergency use in COVID-19 treatment in the US, Russia, and India as of November 2020. Phage display has been one of the most widely used screening methods for peptide/antibody drug discovery. Several phage display-derived biologics are already in the market, and the expiration of intellectual property rights of phage-display antibody discovery platforms suggests an increment in antibody drugs in the near future. This review summarizes the most common phage display libraries used in antiviral discovery, highlights the approaches employed to enhance the antiviral potency of selected peptides/antibody fragments, and finally provides a discussion about the present status of the developed antivirals in clinic.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Cell Surface Display Techniques/methods , Drug Discovery/methods , SARS-CoV-2/drug effects , Humans , Peptide Library
13.
Cell Rep ; 35(8): 109164, 2021 05 25.
Article in English | MEDLINE | ID: covidwho-1227990

ABSTRACT

A major goal of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine efforts is to elicit antibody responses that confer protection. Mapping the epitope targets of the SARS-CoV-2 antibody response is critical for vaccine design, diagnostics, and development of therapeutics. Here, we develop a pan-coronavirus phage display library to map antibody binding sites at high resolution within the complete viral proteomes of all known human-infecting coronaviruses in patients with mild or moderate/severe coronavirus disease 2019 (COVID-19). We find that the majority of immune responses to SARS-CoV-2 are targeted to the spike protein, nucleocapsid, and ORF1ab and include sites of mutation in current variants of concern. Some epitopes are identified in the majority of samples, while others are rare, and we find variation in the number of epitopes targeted between individuals. We find low levels of SARS-CoV-2 cross-reactivity in individuals with no exposure to the virus and significant cross-reactivity with endemic human coronaviruses (CoVs) in convalescent sera from patients with COVID-19.


Subject(s)
COVID-19/immunology , Epitopes/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Viral Proteins/immunology , Adult , Aged , Antibodies, Viral/immunology , Binding Sites, Antibody , COVID-19/virology , Cell Surface Display Techniques , Coronavirus/immunology , Cross Reactions , Female , HEK293 Cells , Humans , Immunity , Male , Middle Aged , Nucleocapsid Proteins/immunology , Polyproteins/immunology , Serology , Young Adult
14.
mSphere ; 6(2)2021 04 28.
Article in English | MEDLINE | ID: covidwho-1207481

ABSTRACT

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within 6 days post-symptom onset and sometimes within 1 day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient's comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention.IMPORTANCE The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Severity of Illness Index , COVID-19/pathology , Cell Surface Display Techniques , Enzyme-Linked Immunosorbent Assay , Epitopes/blood , Epitopes/immunology , Humans , Nucleocapsid/immunology , Phosphoproteins/immunology , Prognosis , Risk Factors
15.
Curr Microbiol ; 78(4): 1124-1134, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1122762

ABSTRACT

Phage display is one of the important and effective molecular biology techniques and has remained indispensable for research community since its discovery in the year 1985. As a large number of nucleotide fragments may be cloned into the phage genome, a phage library may harbour millions or sometimes billions of unique and distinctive displayed peptide ligands. The ligand-receptor interactions forming the basis of phage display have been well utilized in epitope mapping and antigen presentation on the surface of bacteriophages for screening novel vaccine candidates by using affinity selection-based strategy called biopanning. This versatile technique has been modified tremendously over last three decades, leading to generation of different platforms for combinatorial peptide display. The translation of new diagnostic tools thus developed has been used in situations arising due to pathogenic microbes, including bacteria and deadly viruses, such as Zika, Ebola, Hendra, Nipah, Hanta, MERS and SARS. In the current situation of pandemic of Coronavirus disease (COVID-19), a search for neutralizing antibodies is motivating the researchers to find therapeutic candidates against novel SARS-CoV-2. As phage display is an important technique for antibody selection, this review presents a concise summary of the very recent applications of phage display technique with a special reference to progress in diagnostics and therapeutics for coronavirus diseases. Hopefully, this technique can complement studies on host-pathogen interactions and assist novel strategies of drug discovery for coronaviruses.


Subject(s)
Antibodies, Viral/immunology , COVID-19/diagnosis , Cell Surface Display Techniques/methods , SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , Bacteriophage M13/genetics , Bacteriophage M13/metabolism , Bacteriophage T4/genetics , Bacteriophage T4/metabolism , Bacteriophage T7/genetics , Bacteriophage T7/metabolism , Escherichia coli/genetics , Escherichia coli/virology , Humans
16.
J Immunol Methods ; 490: 112952, 2021 03.
Article in English | MEDLINE | ID: covidwho-1065340

ABSTRACT

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Bacterial Proteins/metabolism , Biotin/analogs & derivatives , Biotin/metabolism , Cell Surface Display Techniques , Flow Cytometry , Fluorescent Dyes , HEK293 Cells , High-Throughput Screening Assays , Humans , Immunomagnetic Separation , Microspheres , Mutation/genetics , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
17.
Science ; 371(6531): 823-829, 2021 02 19.
Article in English | MEDLINE | ID: covidwho-1048643

ABSTRACT

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Broadly Neutralizing Antibodies/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Affinity , Binding Sites , Binding Sites, Antibody , Broadly Neutralizing Antibodies/genetics , Broadly Neutralizing Antibodies/metabolism , COVID-19/prevention & control , COVID-19/therapy , Cell Surface Display Techniques , Directed Molecular Evolution , Epitopes/immunology , Humans , Immunization, Passive , Immunoglobulin Fc Fragments/immunology , Mice, Inbred BALB C , Protein Domains , Protein Engineering , Receptors, Coronavirus/metabolism , SARS Virus/immunology , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/prevention & control , Severe Acute Respiratory Syndrome/therapy , Spike Glycoprotein, Coronavirus/metabolism
18.
FEMS Yeast Res ; 21(1)2021 03 04.
Article in English | MEDLINE | ID: covidwho-1038287

ABSTRACT

Viral infections pose intense burdens to healthcare systems and global economies. The correct diagnosis of viral diseases represents a crucial step towards effective treatments and control. Biosensors have been successfully implemented as accessible and accurate detection tests for some of the most important viruses. While most biosensors are based on physical or chemical interactions of cell-free components, the complexity of living microorganisms holds a poorly explored potential for viral detection in the face of the advances of synthetic biology. Indeed, cell-based biosensors have been praised for their versatility and economic attractiveness, however, yeast platforms for viral disease diagnostics are still limited to indirect antibody recognition. Here we propose a novel strategy for viral detection in Saccharomyces cerevisiae, which combines the transductive properties of G Protein-Coupled Receptors (GPCRs) with the Yeast Surface Display (YSD) of specific enzymes enrolled in the viral recognition process. The GPCR/YSD complex might allow for active virus detection through a modulated signal activated by a GPCR agonist, whose concentration correlates to the viral titer. Additionally, we explore this methodology in a case study for the detection of highly pathogenic coronaviruses that share the same cell receptor upon infection (i.e. the Angiotensin-Converting Enzyme 2, ACE2), as a conceptual example of the potential of the GPCR/YSD strategy for the diagnosis of COVID-19.


Subject(s)
COVID-19/diagnosis , COVID-19/metabolism , COVID-19/virology , Cell Surface Display Techniques , Host-Pathogen Interactions , Receptors, G-Protein-Coupled/metabolism , SARS-CoV-2/physiology , Two-Hybrid System Techniques , Animals , Biosensing Techniques , Cell Line , Humans , Molecular Diagnostic Techniques , Saccharomyces cerevisiae
19.
Curr Opin Biotechnol ; 68: 160-165, 2021 04.
Article in English | MEDLINE | ID: covidwho-970472

ABSTRACT

Bacteriophages have attracted extensive interest in vaccine design. This includes the use of phage display technology to select antigens, the use of engineered phages displaying target antigens in vaccine formulations, and phage DNA vaccines. However, the development of these approaches is limited in part by uncertainty regarding the underlying mechanisms by which phages elicit immunity. This has stymied the clinical development of this technology. Here we review the immunology of phage vaccines and highlight the gaps in our knowledge regarding the underlying mechanisms. First, we review the basic biology of phages and their use in vaccines. Next we discuss what is known about the mechanisms of immunity against engineered phages and phage DNA. Finally, we highlight the gaps in our understanding regarding the immunogenicity of these preparations. We argue that mechanistic insight into the immunology of phage vaccines is essential for the further development and clinical utility of these technologies.


Subject(s)
Bacteriophages , Vaccines , Bacteriophages/genetics , Cell Surface Display Techniques
20.
Expert Rev Anti Infect Ther ; 19(5): 557-558, 2021 05.
Article in English | MEDLINE | ID: covidwho-851600
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