Abstract

Background/Objectives: Vesicular Stomatitis Virus (VSV)-vectored vaccines represent a versatile platform for the development of vaccines against infectious diseases. Replication-competent recombinant VSV vectors in which the native glycoprotein (G) is replaced with a heterologous antigen (rVSVΔG) are widely used and have demonstrated clinical success. In addition to antigen-specific responses, these vaccines induce humoral immunity directed against vector components, which primarily reflects vector exposure and contributes to the overall characterization of vaccine-induced immunity. Standardized assays for quantifying such vector-directed responses are therefore of increasing importance. Methods: We developed and qualified a quantitative enzyme-linked immunosorbent assay (ELISA) for the detection of human IgG antibodies against VSV-Nucleoprotein (N). Assay development included optimization of antigen coating, blocking conditions, and detection reagents. A 10-point standard curve was established using pooled human serum, and assay performance was evaluated by assessing dynamic range, sensitivity, cut point, dilutional linearity, precision, robustness, and sample stability. Results: The optimized assay utilized a coating concentration of 2 μg/mL VSV-N antigen (100 ng/well) and 1% casein as the blocking buffer. The assay demonstrated a dynamic range of 0.33–41.66 arbitrary units per milliliter (AU/mL) with excellent curve fit (R2 > 0.996). The cut point was established at an OD450 of 0.286. Precision across intra-assay, inter-assay, and inter-operator evaluations met predefined acceptance criteria (≤25% CV). The assay was robust across different plate washers and readers and maintained performance following up to three freeze–thaw cycles. Conclusions: This study describes a robust and reproducible ELISA for quantifying anti-VSV-N IgG responses. The assay provides a fit-for-purpose tool for assessing vector-directed humoral immunity and supports standardized immunogenicity evaluations across VSV-vectored vaccine studies.

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