(M1330-01-07) Critical Insights into Method Qualification for Virus-Like Particle (VLP) Blockade Assays to Human Norovirus

Purpose: Moderna’s Norovirus vaccine candidates mRNA-1403 and mRNA-1405, have demonstrated strong immunogenicity in early-phase clinical trials, characterized using in-house developed porcine gastric mucin (PGM)-based histo-blood group antigen (HBGA) blockade assays. These assays utilize virus-like particles (VLPs) as surrogates to evaluate the ability of serum antibodies to block VLP binding to HBGA receptors. In this abstract, we present the assay qualification results and discuss the key challenges encountered during assay development, qualification, and sample testing.

Methods: To support the mRNA-1403/1405 clinical programs, the Moderna Clinical Biomarkers Lab developed and optimized five genotype-specific assays. Methodologically, Microplates are coated with PGM serving as the ligand for VLPs. Serum samples are serial diluted and pre-incubated with the VLPs to allow specific antibodies to bind. The VLP-serum mixture is then transferred to the PGM-coated plates; if antibodies block VLP-HBGA interaction, a reduction in VLP binding to the plate occurs. Bound VLPs are detected using a strain specific anti-norovirus antisera followed by an HRP-conjugated secondary antibody, with signal measured by absorbance. Blockade titers (BT50) are calculated based on the serum dilution that results in a 50% reduction in VLP binding relative to control wells. A fit-for-purpose approach was used to qualify the HBGA blockade assays, which involves evaluating key performance for its intended use. Linearity is assessed by testing serial dilutions of positive samples to confirm proportional response across a defined range, while relative accuracy is evaluated by comparing measured blockade titers to expected values. Intra- and inter-assay precision are determined by analyzing samples within and across runs on multiples days by multiple analysts. Sample stability is verified by testing aliquots stored under various conditions over time (e.g., room temperature, freeze-thaw cycle). Specificity is confirmed by showing the assay ability to differentiate between homologous and heterologous Norovirus VLPs and irrelevant antigens. Robustness is evaluated by varying assay conditions (e.g., incubation time, temperature) to assess performance consistency, while ruggedness tests the assay capacity and reliability under routine usage.

Results: To support the mRNA-1403/1405 clinical programs, the Moderna Clinical Biomarkers Laboratory developed and optimized five genotype-specific Norovirus VLP blockade assays. Key qualification parameters for each of the five strains are summarized. Key Challenges and Strategies During Method Qualification: Interference from Goat Serum used in assay buffer. Goat serum is commonly used as a component in assay buffer in Norovirus HBGA blockade assays. However, we observed that some Lots of goat serum contain pre-existing neutralizing antibodies that directly neutralize certain Norovirus genotypes. To mitigate this, a goat serum-free assay was developed to maintain assay specificity and accuracy. Workflow Optimization: The assay process was enhanced by optimizing the plate layout and partially automating the workflow. These improvements increased throughput and consistency across runs. Extension of Analytical Range: After the first interim analysis data report during Phase 1 sample testing, five high-titer study samples were further evaluated for accuracy, precision, and linearity. The data supported an extension of the Upper Limit of Quantitation (ULOQ) thus enhancing the assay's analytical measurement range. These collective efforts ensured that the assays are analytically robust and suitable for clinical implementation.

Conclusion: The development and qualification of Norovirus genotype-specific VLP-based HBGA blockade assays have played a critical role in supporting the clinical evaluation of Moderna’s mRNA-1403 and mRNA-1405 vaccine candidates. These assays meet fit-for-purpose assay qualification criteria and demonstrate robust precision, accuracy, and specificity across genotypes. In addition, workflow optimization and analytical range expansion enabled high-throughput and reliable data delivery, with over 18,000 results delivered. Collectively, these efforts establish the assay platform as a robust immunogenicity tool for early-phase clinical development and lay a solid foundation for its continued use in later-stage trials

References: 1. U.S. Department of Health and Human Services, Food and Drug Administration. Bioanalytical Method Validation Guidance for Biomarkers Guidance for Industry, January 2025
2. ICH guideline, M10 on bioanalytical method validation and study sample analysis. November 2022
3. Dessy F, Sonderegger I, Wagner L, et al. Harmonization of Vaccine Ligand Binding Assays Validation. Bioanalysis. 2024;16(19-20):1067-1091.
4. Lindesmith, Lisa C et al. Particle conformation regulates antibody access to a conserved GII.4 norovirus blockade epitope. Journal of virology vol. 88,16 (2014): 8826-42

Table 1. Key qualification parameters