Development and Qualification of a VSV-N IgG ELISA to Assess Vector-Directed Humoral Immunity in VSV-Vectored Vaccine Studies.

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.