Pseudovirus-Based Neutralization Assays as Customizable and Scalable Tools for Serological Surveillance and Immune...

Area of Science:

• Virology
• Immunology
• Assay Development

Background:

• Neutralizing antibodies (nAbs) are critical for assessing SARS-CoV-2 protection and vaccine efficacy.
• The plaque reduction neutralization test (PRNT) is the gold standard but lacks high-throughput capability.
• Efficient and scalable methods are needed to monitor population immunity and vaccine responses.

Purpose of the Study:

• To standardize and compare in-house and commercial pseudovirus-based neutralization (PBN) assays against the gold standard PRNT.
• To evaluate the performance, reproducibility, and scalability of PBN assays for SARS-CoV-2.
• To profile immune responses in vaccinated individuals against different SARS-CoV-2 variants using PBN.

Main Methods:

• Developed and standardized an in-house PBN assay using VSV-ΔG pseudoviruses (Wuhan, Omicron BA.1) and NanoLuc reporter.
• Utilized a commercial PBN assay (Integral Molecular) with lentiviral pseudoviruses (Wuhan, Omicron XBB.1.5/XBB.1.9) and GFP/Renilla reporters.
• Compared PBN assays with PRNT using 600 serum samples from vaccinated individuals across various time points.

Main Results:

• Both PBN assays demonstrated strong correlation with PRNT, with commercial assay showing superior reproducibility and scalability.
• The in-house PBN assay offers a cost-effective, BSL-2 compatible alternative.
• Large-scale analysis revealed significant differences in neutralization between Wuhan and Omicron XBB variants, with an approximately eightfold reduction against Omicron.
• Identified antibody waning and antigenic drift, and classified responders using k-means clustering.

Conclusions:

• Standardized PBN assays provide a robust and scalable framework for SARS-CoV-2 neutralization testing.
• PBN assays are suitable for large-scale immune surveillance, vaccine response monitoring, and tracking viral evolution.
• These assays facilitate the classification of immune responses and highlight the impact of viral variants on neutralizing antibody activity.