A high-throughput SARS-CoV-2 pseudovirus multiplex neutralization assay.

Evaluating the neutralizing antibody titer following SARS-CoV-2 vaccination is essential in defining correlates of protection. We describe an assay that uses single-cycle vesicular stomatitis virus (VSV) pseudoviruses linking a fluorophore with a spike (S) from a variant of concern (VOC). Using two fluorophores linked to two VOC S, respectively, allows us to determine the neutralization titer against two VOCs in a single run. This is a generalizable approach that saves time, samples, and run-to-run variability. For complete details on the use and execution of this protocol, please refer to Sievers et al. (2022).1.

A high throughput SARS-CoV-2 pseudovirus multiplex neutralization assay

Evaluating the neutralizing antibody titer following SARS-CoV-2 vaccination is essential in defining correlates of protection. We describe an assay that uses single-cycle vesicular stomatitis virus (VSV) pseudoviruses linking a fluorophore with a spike (S) from a variant of concern (VOC). Using two fluorophores linked to two VOC S, respectively, allows us to determine the neutralization titer against two VOCs in a single run. This is a generalizable approach that saves time, samples and run-to-run variability. Graphical Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Antibodies elicited by SARS-CoV-2 infection or mRNA vaccines have reduced neutralizing activity against Beta and Omicron pseudoviruses.

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that have mutations associated with increased transmission and antibody escape have arisen over the course of the current pandemic. Although the current vaccines have largely been effective against past variants, the number of mutations found on the Omicron (B.1.1.529) spike protein appear to diminish the protection conferred by preexisting immunity. Using vesicular stomatitis virus (VSV) pseudoparticles expressing the spike protein of several SARS-CoV-2 variants, we evaluated the magnitude and breadth of the neutralizing antibody response over time in individuals after infection and in mRNA-vaccinated individuals. We observed that boosting increases the magnitude of the antibody response to wild-type (D614), Beta, Delta, and Omicron variants; however, the Omicron variant was the most resistant to neutralization. We further observed that vaccinated healthy adults had robust and broad antibody responses, whereas responses may have been reduced in vaccinated pregnant women, underscoring the importance of learning how to maximize mRNA vaccine responses in pregnant populations. Findings from this study show substantial heterogeneity in the magnitude and breadth of responses after infection and mRNA vaccination and may support the addition of more conserved viral antigens to existing SARS-CoV-2 vaccines.

Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity.

A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19.