A Pseudovirus-Based Neutralization Assay for SARS-CoV-2 Variants: A Rapid, Cost-Effective, BSL-2–Based High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation

Abstract

Neutralizing antibody responses from COVID-19 vaccines are pivotal in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Effective COVID-19 vaccines and assays measuring neutralizing antibodies against emerging variants (i.e., XBB.1.5, XBB.1.16, and XBB.2.3) are needed. The use of biosafety level (BSL)-3 laboratories for live virus assays results in higher costs and a longer turnaround time; therefore, a BSL-2–based pseudovirus neutralization assay (PNT) was developed. The pseudoviruses were produced by cotransfecting cells with plasmids encoding a lentiviral backbone-expressing luciferase reporter; non-surface proteins for lentiviral production; and ancestral or Omicron (BA.1 and BA.5) SARS-CoV-2 spike (S) proteins. The PNT was developed and optimized in dose and kinetics experiments. The representative serum samples (COVID-19–convalescent or NVX-CoV2373–vaccinated participants enrolled in the 2019nCoV-101 trial) demonstrated a wide dynamic range. The neutralization data showed robust correlation with validated anti-recombinant spike IgG levels and angiotensin-converting enzyme 2 inhibition titers (ancestral). This assay is suitable for measurement of the neutralization ability in clinical samples from individuals infected with SARS-CoV-2 or immunized with a COVID-19 vaccine. The results suggest that this PNT provides a lower cost, high-throughput, rapid turnaround alternative to BSL-3–based microneutralization assays and enables the discovery and development of effective vaccines against emerging variants.

Validation of a Pseudovirus Neutralization Assay for Severe Acute Respiratory Syndrome Coronavirus 2: A High-Throughput Method for the Evaluation of Vaccine Immunogenicity

“…The PNT assay provides a high-throughput, robust, and cost-effective method for the Our previous publication [10] was an FFP method paper, whereas this manuscript focuses on method validation. The principle of the assay mentioned in both manuscripts is the same.…”

“…Along similar lines, the adaptability of the validation method to forward-drifted variants is based on the availability of serum samples with reasonably high titers to establish the ULoQ. Other limitations of the PNT assay, already mentioned in our previous publication [ 10 ], include the need for special critical reagents (such as pseudoviruses expressing the S protein), the permissive cell lines, and the fact that the pseudovirus may not mimic the distribution/presentation of antigens presented on the live virus.…”

“…A PNT assay was developed to assess virus-specific neutralizing antibodies against SARS-CoV-2 in human serum [ 10 ]. To perform the assay, serum samples were heat-inactivated in a 56 °C water bath for 30 min prior to the assay.…”

“…Microorganisms 2024, 12, x FOR PEER REVIEW 9 of 12 the whole-virus and pseudovirus neutralization assays and therefore indicate no significant difference in the neutralizing antibody results (Figure 4a,b) from either of the assays. Our previous publication [10] was an FFP method paper, whereas this manuscript focuses on method validation. The principle of the assay mentioned in both manuscripts is the same.…”