Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain.

Previous work employing five SARS-CoV-2 spike protein receptor-binding domain (RBD) constructs, comprising versions originally developed by Mt. Sinai or the Ragon Institute and later optimized in-house, revealed potential heterogeneity which led to questions regarding variable seropositivity assay performance. Each construct was subjected to N-deglycosylation and subsequent intact mass analysis, revealing significant deviations from predicted theoretical mass for all five proteins. Complementary tandem MS/MS analysis revealed the presence of an additional pyroGlu residue on the N-termini of the two Mt. Sinai RBD constructs, as well as on the N-terminus of the full-length spike protein from which they were derived, thus explaining the observed mass shift and definitively establishing the spike protein N-terminal sequence. Moreover, the observed mass additions for the three Ragon Institute RBD constructs were identified as variable N-terminal cleavage points within the signal peptide sequence employed for recombinant expression. To resolve this issue and minimize heterogeneity for further seropositivity assay development, the best-performing RBD construct was further optimized to exhibit complete homogeneity, as determined by both intact mass and tandem MS/MS analysis. This new RBD construct has been validated for seropositivity assay performance, is available to the greater scientific community, and is recommended for use in future assay development.

A review of alternative promoters for optimal recombinant protein expression in baculovirus-infected insect cells.

Generating recombinant proteins in insect cells has been made possible via the use of the Baculovirus Expression Vector System (BEVS). Despite the success of many proteins via this platform, some targets remain a challenge due to issues such as cytopathic effects, the unpredictable nature of co-infection and co-expressions, and baculovirus genome instability. Many promoters have been assayed for the purpose of expressing diverse proteins in insect cells, and yet there remains a lack of implementation of those results when reviewing the landscape of commercially available baculovirus vectors. In advancing the platform to produce a greater variety of proteins and complexes, the development of such constructs cannot be avoided. A better understanding of viral gene regulation and promoter options including viral, synthetic, and insect-derived promoters will be beneficial to researchers looking to utilize BEVS by recruiting these intricate mechanisms of gene regulation for heterologous gene expression. Here we summarize some of the developments that could be utilized to improve the expression of recombinant proteins and multi-protein complexes in insect cells.