Characterization of spike processing and entry mechanisms of seasonal human coronaviruses NL63, 229E and HKU1 (preprint)

Although much has been learned about the entry mechanism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the details of entry mechanisms of seasonal human coronaviruses (HCoVs) remain less well understood. In the present study, we established that 293T cell lines that stably express angiotensin converting enzyme (ACE2), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) support high level transduction of lentiviral pseudoviruses bearing spike proteins of seasonal HCoVs, HCoV-NL63, -229E, or -HKU1, respectively. Our results showed that entry of HCoV-NL63, -229E and -HKU1 pseudoviruses is sensitive to endosomal acidification inhibitors (chloroquine and NH4Cl), indicating virus entry via the endocytosis route. Although HCoV-HKU1 pseudovirus infection requires TMPRSS2 expression on cell surface, endocytosis-mediated HCoV-HKU1 entry requires the serine protease domain but not the serine protease activity of TMPRSS2. We also show that amino acids in the predicted S1/S2 junctions of spike proteins of HCoV-NL63, and -229E are essential for optimal entry but non-essential for spike-mediated entry of HCoV-HKU1. Our findings provide insights into entry mechanism of seasonal HCoVs that may support the development of novel treatment strategies.

Evaluation of association of anti-PEG antibodies with anaphylaxis after mRNA COVID-19 vaccination (preprint)

Background: The mechanism for anaphylaxis following mRNA COVID-19 vaccination has been widely debated; understanding this serious adverse event is important for future vaccines of similar design. A mechanism proposed is type I hypersensitivity (i.e., IgE-mediated mast cell degranulation) to excipient polyethylene glycol (PEG). Using an assay that, uniquely, had been previously assessed in patients with anaphylaxis to PEG, our objective was to compare anti-PEG IgE in serum from mRNA COVID-19 vaccine anaphylaxis case-patients and persons vaccinated without allergic reactions. Secondarily, we compared anti-PEG IgG and IgM to assess alternative mechanisms. Methods: Selected anaphylaxis case-patients reported to U.S. Vaccine Adverse Event Reporting System December 14, 2020 - March 25, 2021 were invited to provide a serum sample. mRNA COVID-19 vaccine study participants with residual serum and no allergic reaction post-vaccination ("controls") were frequency matched to cases 3:1 on vaccine and dose number, sex and 10-year age category. Anti-PEG IgE was measured using a dual cytometric bead assay. Anti-PEG IgG and IgM were measured using two different assays. Laboratorians were blinded to case/control status. Results: All 20 case-patients were women; 17 had anaphylaxis after dose 1, 3 after dose 2. Thirteen (65%) were hospitalized and 7 (35%) were intubated. Time from vaccination to serum collection was longer for case-patients vs controls (post-dose 1: median 105 vs 21 days). Among Moderna recipients, anti-PEG IgE was detected in 1 of 10 (10%) case-patients vs 8 of 30 (27%) controls (p=0.40); among Pfizer-BioNTech recipients, it was detected in 0 of 10 case-patients (0%) vs 1 of 30 (3%) controls (p>0.99). Anti-PEG IgE quantitative signals followed this same pattern. Neither anti-PEG IgG nor IgM was associated with case status with both assay formats. Conclusion: Our results support that anti-PEG IgE is not a predominant mechanism for anaphylaxis post-mRNA COVID-19 vaccination.