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1.
Viruses ; 12(5)2020 05 06.
Article in English | MEDLINE | ID: covidwho-1389513

ABSTRACT

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.


Subject(s)
Antibodies, Viral/immunology , Neutralization Tests/methods , Spike Glycoprotein, Coronavirus/analysis , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Containment of Biohazards , HEK293 Cells , Humans , Lentivirus , Peptidyl-Dipeptidase A/metabolism , Plasma/immunology
2.
Front Immunol ; 12: 710263, 2021.
Article in English | MEDLINE | ID: covidwho-1315952

ABSTRACT

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunization Schedule , Immunogenicity, Vaccine , Mutation , Protein Domains/genetics , Protein Domains/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/virology , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Linoleic Acids , Mice , Mice, Inbred BALB C , Nanoparticles/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Treatment Outcome , Vero Cells
3.
Sci Transl Med ; 13(600)2021 06 30.
Article in English | MEDLINE | ID: covidwho-1262380

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with mutations in key antibody epitopes has raised concerns that antigenic evolution could erode adaptive immunity elicited by prior infection or vaccination. The susceptibility of immunity to viral evolution is shaped in part by the breadth of epitopes targeted by antibodies elicited by vaccination or natural infection. To investigate how human antibody responses to vaccines are influenced by viral mutations, we used deep mutational scanning to compare the specificity of polyclonal antibodies elicited by either two doses of the mRNA-1273 COVID-19 vaccine or natural infection with SARS-CoV-2. The neutralizing activity of vaccine-elicited antibodies was more targeted to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein compared to antibodies elicited by natural infection. However, within the RBD, binding of vaccine-elicited antibodies was more broadly distributed across epitopes compared to infection-elicited antibodies. This greater binding breadth means that single RBD mutations have less impact on neutralization by vaccine sera compared to convalescent sera. Therefore, antibody immunity acquired by natural infection or different modes of vaccination may have a differing susceptibility to erosion by SARS-CoV-2 evolution.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/therapy , COVID-19 Vaccines , Humans , Immunization, Passive , RNA, Messenger , Spike Glycoprotein, Coronavirus , Vaccination
4.
Cell Host Microbe ; 29(3): 463-476.e6, 2021 03 10.
Article in English | MEDLINE | ID: covidwho-1071171

ABSTRACT

The evolution of SARS-CoV-2 could impair recognition of the virus by human antibody-mediated immunity. To facilitate prospective surveillance for such evolution, we map how convalescent plasma antibodies are impacted by all mutations to the spike's receptor-binding domain (RBD), the main target of plasma neutralizing activity. Binding by polyclonal plasma antibodies is affected by mutations in three main epitopes in the RBD, but longitudinal samples reveal that the impact of these mutations on antibody binding varies substantially both among individuals and within the same individual over time. Despite this inter- and intra-person heterogeneity, the mutations that most reduce antibody binding usually occur at just a few sites in the RBD's receptor-binding motif. The most important site is E484, where neutralization by some plasma is reduced >10-fold by several mutations, including one in the emerging 20H/501Y.V2 and 20J/501Y.V3 SARS-CoV-2 lineages. Going forward, these plasma escape maps can inform surveillance of SARS-CoV-2 evolution.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibodies, Viral/chemistry , Binding Sites , Cell Line , Female , Humans , Male , Middle Aged , Mutation , Prospective Studies , Protein Binding , Protein Domains , Receptors, Virus/genetics , Receptors, Virus/immunology , Young Adult
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