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1.
Cell Host Microbe ; 29(1): 44-57.e9, 2021 01 13.
Article in English | MEDLINE | ID: covidwho-1385265

ABSTRACT

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.


Subject(s)
SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Binding Sites , Epitopes/immunology , Gene Library , High-Throughput Nucleotide Sequencing , Humans , Protein Domains , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/chemistry
2.
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
3.
PLoS Pathog ; 17(4): e1009453, 2021 04.
Article in English | MEDLINE | ID: covidwho-1172889

ABSTRACT

There is intense interest in antibody immunity to coronaviruses. However, it is unknown if coronaviruses evolve to escape such immunity, and if so, how rapidly. Here we address this question by characterizing the historical evolution of human coronavirus 229E. We identify human sera from the 1980s and 1990s that have neutralizing titers against contemporaneous 229E that are comparable to the anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We test these sera against 229E strains isolated after sera collection, and find that neutralizing titers are lower against these "future" viruses. In some cases, sera that neutralize contemporaneous 229E viral strains with titers >1:100 do not detectably neutralize strains isolated 8-17 years later. The decreased neutralization of "future" viruses is due to antigenic evolution of the viral spike, especially in the receptor-binding domain. If these results extrapolate to other coronaviruses, then it may be advisable to periodically update SARS-CoV-2 vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus 229E, Human/genetics , Coronavirus 229E, Human/immunology , Immune Evasion , Humans , SARS-CoV-2/genetics , SARS-CoV-2/immunology
4.
J Infect Dis ; 223(2): 197-205, 2021 02 03.
Article in English | MEDLINE | ID: covidwho-1060937

ABSTRACT

Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop neutralizing antibodies that target the viral spike protein. In this study, we quantified how levels of these antibodies change in the months after SARS-CoV-2 infection by examining longitudinal samples collected approximately 30-152 days after symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about 4-fold from 1 to 4 months after symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike protein or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/blood , COVID-19/virology , Female , Humans , Male , Middle Aged , Prospective Studies , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Young Adult
5.
Nat Commun ; 11(1): 4378, 2020 09 01.
Article in English | MEDLINE | ID: covidwho-740036

ABSTRACT

Children are strikingly underrepresented in COVID-19 case counts. In the United States, children represent 22% of the population but only 1.7% of confirmed SARS-CoV-2 cases as of April 2, 2020. One possibility is that symptom-based viral testing is less likely to identify infected children, since they often experience milder disease than adults. Here, to better assess the frequency of pediatric SARS-CoV-2 infection, we serologically screen 1,775 residual samples from Seattle Children's Hospital collected from 1,076 children seeking medical care during March and April of 2020. Only one child was seropositive in March, but seven were seropositive in April for a period seroprevalence of ≈1%. Most seropositive children (6/8) were not suspected of having had COVID-19. The sera of seropositive children have neutralizing activity, including one that neutralized at a dilution > 1:18,000. Therefore, an increasing number of children seeking medical care were infected by SARS-CoV-2 during the early Seattle outbreak despite few positive viral tests.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Visitors to Patients , Adolescent , COVID-19 , COVID-19 Testing , Child , Child, Preschool , Clinical Laboratory Techniques/methods , Coronavirus Infections/blood , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Female , Hospitals , Humans , Infant , Infant, Newborn , Male , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Prospective Studies , SARS-CoV-2 , Seroepidemiologic Studies , Serologic Tests/methods , United States/epidemiology
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