Validation of SARS-CoV-2 neutralization assay using VSV-based pseudovirus system.
The validated VSV-based pseudovirus system offers a safe and effective method for assessing SARS-CoV-2 neutralization in a BSL-2 setting, particularly for evaluating antibody responses to different variants.
Where it sits
this study against the rest of the dsip corpusSummary and findings
This study validated a VSV-based pseudovirus system for assessing SARS-CoV-2 neutralization using the monoclonal antibody BD-604. The assay was optimized for a multiplicity of infection (MOI) of 0.25 and demonstrated specific neutralizing activity against several SARS-CoV-2 variants. The limit of quantification was established at 125 ng of mAb BD-604.
Abstract
The gold standard for SARS-CoV-2 neutralization assays involves wild-type virus, which requires Biosafety Level 3 (BSL-3) containment. To improve safety and accessibility, pseudovirus-based neutralization assays utilizing non-replicating particles like Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike protein can be conducted under BSL-2 conditions. This study aimed to perform the analytical validation of a VSV-based pseudovirus system for SARS-CoV-2 using recombinant monoclonal antibody. Pseudo-VSV carrying SARS-CoV-2 Spike proteins were produced using LentiX-293T cells. The assay system was optimized for Multiplicity of Infection (MOI) and assessed for specificity, limit of quantification (LOQ), linearity, accuracy, and precision using the neutralizing mAb BD-604. The system was optimized at an MOI of 0.25. The assay proved highly specific, as mAb BD-604 showed clear neutralizing activity while mAb 1A9 did not. The limit of quantification (LOQ) was determined to be 125 ng of mAb BD-604, with a linear range of 125 - 1000 ng. The relative accuracy remained within the 80-120% range, and the precision (%CV) ranged from 1.92% to 13.57%. Additionally, the system successfully characterized variant-specific neutralization, revealing that BD-604 was effective against the Wuhan, Delta, and Omicron BA.1/BA.2 strains but lacked activity against the Omicron XBB.1.5 variant. This validated pseudo-VSV based SARS-CoV-2 neutralization assay is a valuable bioassay for evaluating neutralizing antibody potency against various SARS-CoV-2 strains in a BSL-2 environment, thus making it useful for vaccine and therapeutic development.
Background
This paper addresses the need for safer and more accessible SARS-CoV-2 neutralization assays, as traditional methods require high-containment laboratories. Previous studies have established the importance of neutralizing antibodies in the immune response to SARS-CoV-2. The development of a pseudovirus-based assay could facilitate research and development in vaccine and therapeutic strategies without the risks associated with live virus handling.
Methods
The study utilized a VSV-based pseudovirus system to evaluate neutralization by the monoclonal antibody BD-604. The optimization of the assay was performed at a multiplicity of infection (MOI) of 0.25. The primary outcome measures included specificity, limit of quantification (LOQ), linearity, accuracy, and precision. Not reported in abstract.
Results
The limit of quantification (LOQ) was determined to be 125 ng of mAb BD-604. The assay demonstrated a linear range of 125 - 1000 ng. The relative accuracy was maintained within the 80-120% range, and precision (%CV) ranged from 1.92% to 13.57%. BD-604 was effective against the Wuhan, Delta, and Omicron BA.1/BA.2 strains but showed no activity against the Omicron XBB.1.5 variant.
Interpretation
The findings suggest that the VSV-based pseudovirus assay is a reliable method for assessing neutralizing antibody potency against various SARS-CoV-2 strains. While the assay shows statistical significance in its accuracy and precision, the clinical relevance remains unclear without data on human samples. Limitations include the lack of information on the assay's performance in real-world settings and potential confounding factors that could affect the results.
Key findings
- Limit of quantification (LOQ) was determined to be 125 ng of mAb BD-604.
- Linear range of the assay was 125 - 1000 ng.
- Relative accuracy remained within the 80-120% range.
- Precision (%CV) ranged from 1.92% to 13.57%.
- BD-604 showed neutralizing activity against Wuhan, Delta, and Omicron BA.1/BA.2 strains but lacked activity against Omicron XBB.1.5.
Limitations
- Not reported in abstract.
- Lacks clinical applicability data.
- No human sample performance reported.
- Potential confounding factors not addressed.