SARS-CoV-2 spike receptor-binding domain (RBD)-specific binding antibodies in serum were assessed by ELISA as described previously. SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction (NT50) in relative light units was observed relative to the average of the virus control wells.Įnzyme-linked immunosorbent assay (ELISA) ![]() After 48 h, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer’s instructions. The mixture was incubated at 37 ☌ for 1 h before adding to HEK293T-hACE2 cells. Three-fold serial dilutions of heat-inactivated serum samples were prepared and mixed with 50 μl of pseudovirus. To determine the neutralization activity of human serum, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 × 10 4 cells per well overnight. The supernatants containing the pseudotype viruses were collected 48h after transfection pseudotype viruses were purified by filtration with 0.45-μm filter. Pseudoviruses of SARS-CoV-2 variants were generated by using WA1/2020 strain (Wuhan/WIV04/2019, GISAID accession ID: EPI_ISL_402124), B.1.1.7 variant (Alpha, GISAID accession ID: EPI_ISL_601443), B.1.351 variant (Beta, GISAID accession ID: EPI_ISL_712096), or B.1.617.2 (Delta, GISAID accession ID: EPI_ISL_2020950). 5, 6 In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene) and spike protein expressing pcDNA3.1-SARS-CoV-2 SΔCT were co-transfected into HEK293T cells (ATCC CRL_3216) with lipofectamine 2000 (ThermoFisher Scientific). The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were used to measure pseudovirus neutralizing antibodies as described previously. ![]() Participants were asked to provide blood samples and nasal swabs. Breakthrough SARS-CoV-2 infection was defined as a positive nasopharyngeal swab PCR test after being fully vaccinated (at least 2 weeks following two doses of the BNT162b2 or mRNA-1273 vaccines or at least 2 weeks after a single dose of the vaccine). All participants provided informed consent. The BIDMC Institutional Review Board approved this study (#2021P000344) as part of a parent biorepository study (#2020P000361). Participants were asked to provide their vaccine, symptom, and testing history, as well as their race and ethnicity based on specified categories they could select multiple race categories. Participants also self-referred from flyers posted on social media. Participants were provided contact information for the Beth Israel Deaconess Medical Center (BIDMC) study team for recruitment and informed consent. To recruit participants who were part of the outbreak investigation of the breakthrough infections in Provincetown, Massachusetts, the MA DPH and Boston Public Health Commission (BPHC) provided information about this immunologic study to individuals by telephone or email. We measured peripheral and mucosal antibody responses as well as cellular immune responses in vaccinated individuals with or without SARS-CoV-2 breakthrough infection. ![]() We recruited vaccinated individuals who were part of the MA DPH outbreak investigation or enhanced surveillance and who tested positive or negative for COVID-19 by nasopharyngeal swabs to participate in a detailed immunologic study at Beth Israel Deaconess Medical Center (BIDMC) in Boston, Massachusetts. Centers for Disease Control and Prevention (CDC) to reinstate indoor masking recommendations for fully vaccinated individuals. This outbreak was the first known large cluster of infections with the SARS-CoV-2 delta variant in a highly vaccinated population, and it prompted the U.S. Viral sequencing revealed that over 90% of cases sequenced were the SARS-CoV-2 delta variant, and viral loads in nasal swabs were similar in vaccinated and unvaccinated individuals 1. Most cases were mildly or moderately symptomatic. Approximately 74% of cases occurred in individuals who were fully vaccinated with the BNT162b2 (Pfizer/BioNTech), mRNA-1273 (Moderna), or Ad26.COV.2 (Johnson & Johnson) COVID-19 vaccines 2- 4. A large cluster of COVID-19 infections were reported to the Massachusetts Department of Public Health (MA DPH) after the Jweekend in Provincetown, Barnstable County, Massachusetts 1.
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