(24e) Rapid Population-Wide Screening for Immune Escape of Viral Pathogens

As viral diseases such as influenza, COVID-19, and norovirus become increasingly prevalent, demand for treatments and vaccines against them is on the rise. However, even as vaccinations become more widespread, there is concern that emerging virus strains may escape vaccine- and infection-induced immunity. Mutations in SARS-CoV-2 have caused first generation COVID vaccines to lose efficacy against Omicron variants, and antigenic drift in influenza viruses can result in endemics and potential pandemics despite annual vaccinations. Developing effective treatments against viral diseases requires monitoring these pathogens as they mutate and circulate, making their surveillance within the population a public health need. However, rapidly screening large portions of the population using conventional means is demanding in terms of efficiency and cost. Here we demonstrate the use of a high-throughput, chip-based platform that is scalable to more population-wide surveillance, using SARS-CoV-2 as a model virus.

Pseudovirus surrogates are ideal for studying interactions of SARS-CoV-2 with cell-surface receptors for viral entry. We produced SARS-CoV-2 pseudovirus constructs in Lenti-X™ 293T cells by transfecting with a reporter plasmid expressing EGFP, a packaging plasmid, and the plasmid expressing the SARS-CoV-2 spike glycoprotein. The collected cell supernatants undergo a 2-step concentration protocol, which results in titers as high as 6.0 x 10⁶ TU/mL of pseudovirus particles. These particles are then used in neutralization assays using human ACE2-expressing 293T cells on a 532-micropillar-microwell chip platform, with volumes as low as 1 µL per microwell. The pillar chip is seeded with ACE2 293T cells and stamped onto a well chip containing pseudovirus and different concentrations of test compounds followed by imaging after 48 h. We have successfully used the 532-chip system to determine IC₅₀ values of antibodies such as CC12.14 (1.7 µg/mL) and CC12.30 (0.16 µg/mL), and tested patient-derived sera samples against pseudovirus bearing the spike protein of the wild-type and BA.4/5 Omicron variants of SARS-CoV-2. Interestingly, some sera showed inhibition potential against BA.4/5 even though this variant began circulating a year after the sera samples were collected, indicating the presence of cross-reactivity against distinct SARS-CoV-2 spike protein sequences. Going forward, this approach could be applied to dried blood spots from finger pricks of patients and volunteers to monitor antibody-induced immunity against a wide variety of pathogens, paving the way for broad surveillance screening for immune escape of viral pathogens.