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1.
J Virol ; 95(19): e0068521, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1486511

ABSTRACT

The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here, we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7, B.1.351, and P.1 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralizing antibody approaches. Furthermore, we report a preclinical characterization package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralization capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralized four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections. IMPORTANCE Mutational drift of SARS-CoV-2 risks rendering both therapeutics and vaccines less effective. Receptor decoy strategies utilizing soluble human ACE2 may overcome the risk of viral mutational escape since mutations disrupting viral interaction with the ACE2 decoy will by necessity decrease virulence, thereby preventing meaningful escape. The solution described here of a soluble ACE2 receptor decoy is significant for the following reasons: while previous ACE2-based therapeutics have been described, ours has novel features, including (i) mutations within ACE2 to remove catalytical activity and systemic interference with the renin/angiotensin system, (ii) abrogated FcγR engagement, reduced risk of antibody-dependent enhancement of infection, and reduced risk of hyperinflammation, and (iii) streamlined antibody-like purification process and scale-up manufacturability indicating that this receptor decoy could be produced quickly and easily at scale. Finally, we demonstrate that ACE2-based therapeutics confer a broad-spectrum neutralization potency for ACE2-tropic viruses, including SARS-CoV-2 variants of concern in contrast to therapeutic MAb.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Antibodies, Neutralizing/immunology , Antibody-Dependent Enhancement , COVID-19/immunology , HEK293 Cells , Humans , Kinetics , Mutation , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism
2.
J Virol ; 95(19): e0068521, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1410201

ABSTRACT

The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here, we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7, B.1.351, and P.1 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralizing antibody approaches. Furthermore, we report a preclinical characterization package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralization capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralized four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections. IMPORTANCE Mutational drift of SARS-CoV-2 risks rendering both therapeutics and vaccines less effective. Receptor decoy strategies utilizing soluble human ACE2 may overcome the risk of viral mutational escape since mutations disrupting viral interaction with the ACE2 decoy will by necessity decrease virulence, thereby preventing meaningful escape. The solution described here of a soluble ACE2 receptor decoy is significant for the following reasons: while previous ACE2-based therapeutics have been described, ours has novel features, including (i) mutations within ACE2 to remove catalytical activity and systemic interference with the renin/angiotensin system, (ii) abrogated FcγR engagement, reduced risk of antibody-dependent enhancement of infection, and reduced risk of hyperinflammation, and (iii) streamlined antibody-like purification process and scale-up manufacturability indicating that this receptor decoy could be produced quickly and easily at scale. Finally, we demonstrate that ACE2-based therapeutics confer a broad-spectrum neutralization potency for ACE2-tropic viruses, including SARS-CoV-2 variants of concern in contrast to therapeutic MAb.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Antibodies, Neutralizing/immunology , Antibody-Dependent Enhancement , COVID-19/immunology , HEK293 Cells , Humans , Kinetics , Mutation , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism
3.
Transfusion ; 61(10): 2837-2843, 2021 10.
Article in English | MEDLINE | ID: covidwho-1360538

ABSTRACT

BACKGROUND: Convalescent plasma (CP) therapy for coronavirus disease (COVID-19) provides virus-neutralizing antibodies that may ameliorate the outcome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. The effectiveness of CP likely depends on its antiviral neutralizing potency and is determined using in vitro neutralizing antibody assays. STUDY DESIGN AND METHODS: We evaluated abilities of three immunoassays for anti-spike antibodies (EUROimmun, Ortho, Roche), a pseudotype-based neutralization assay, and two assays that quantify ACE2 binding of spike protein (GenScript and hemagglutination test [HAT]-based assay) to predict neutralizing antibody titers in 113 CP donations. Assay outputs were analyzed through linear regression and calculation of sensitivities and specificities by receiver operator characteristic (ROC) analysis. RESULTS: Median values of plasma samples containing neutralizing antibodies produced conversion factors for assay unitage of ×6.5 (pseudotype), ×19 (GenScript), ×3.4 (HAT assay), ×0.08 (EUROimmun), ×1.64 (Roche), and ×0.10 (Ortho). All selected assays were sufficient in identifying the high titer donations based on ROC analysis; area over curve ranged from 91.7% for HAT and GenScript assay to 95.6% for pseudotype assay. However, their ability to predict the actual neutralizing antibody levels varied substantially as shown by linear regression correlation values (from 0.27 for Ortho to 0.61 for pseudotype assay). DISCUSSION: Overall, the study data demonstrate that all selected assays were effective in identifying donations with high neutralizing antibody levels and are potentially suitable as surrogate assays for donation selection for CP therapy.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing/methods , Immunoassay/methods , SARS-CoV-2/immunology , COVID-19/therapy , Humans , Immunization, Passive , Neutralization Tests
4.
Antiviral Res ; 194: 105147, 2021 10.
Article in English | MEDLINE | ID: covidwho-1347484

ABSTRACT

The SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) was previously engineered into a high affinity tetravalent format (ACE2-Fc-TD) that is a potential decoy protein in SARS-CoV-2 infection.We report that this protein shows greatly enhanced binding to SARS-CoV-2 spike proteins of the SARS-CoV-2 variants of concern B.1.1.7 (alpha variant, originally isolated in the United Kingdom) and B.1.351 (beta variant, originally isolated in South Africa) with picomolar compared with nanomolar Kd values. In addition, ACE2-Fc-TD displays greater neutralization of SARS-CoV-2 pseudotype viruses compared to a dimeric ACE2-Fc, with enhanced activity on variant B.1.351. This tetrameric decoy protein would be a valuable addition to SARS-CoV-2 therapeutic approaches, especially where vaccination cannot be used but also should there be any future coronavirus pandemics.


Subject(s)
Angiotensin-Converting Enzyme 2/pharmacology , Antiviral Agents/metabolism , COVID-19/prevention & control , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/enzymology , COVID-19/virology , Cell Line , Humans , Kinetics , Mutation , Protein Binding , Protein Domains , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
5.
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
6.
Viruses ; 13(2)2021 01 31.
Article in English | MEDLINE | ID: covidwho-1069882

ABSTRACT

Serological assays detecting neutralising antibodies are important for determining the immune responses following infection or vaccination and are also often considered a correlate of protection. The target of neutralising antibodies is usually located in the Envelope protein on the viral surface, which mediates cell entry. As such, presentation of the Envelope protein on a lentiviral particle represents a convenient alternative to handling of a potentially high containment virus or for those viruses with no established cell culture system. The flexibility, relative safety and, in most cases, ease of production of lentiviral pseudotypes, have led to their use in serological assays for many applications such as the evaluation of candidate vaccines, screening and characterization of anti-viral therapeutics, and sero-surveillance. Above all, the speed of production of the lentiviral pseudotypes, once the envelope sequence is published, makes them important tools in the response to viral outbreaks, as shown during the COVID-19 pandemic in 2020. In this review, we provide an overview of the landscape of the serological applications of pseudotyped lentiviral vectors, with a brief discussion on their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , COVID-19/therapy , Genetic Therapy/methods , Genetic Vectors/genetics , Animals , Antiviral Agents , COVID-19/blood , COVID-19 Vaccines/administration & dosage , Humans , Lentivirus/genetics , SARS-CoV-2/isolation & purification
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