Evaluation of Simultaneous Production Strategies for Adenoviral Vector-Based SARS-CoV-2 Multivalent Vaccines via an Expanded Allele-specific Competitive Blocker PCR Quantification Method.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, developing multivalent vaccines becomes crucial to ensure broader immunogenicity against the emerging variants. Traditional multivalent vaccines are produced by combining separately formulated monovalent vaccines in equal proportions, which necessitates precisely quantifying each component. However, the minimal differences in SARS-CoV-2 spike protein sequences among variants pose challenges for immunological quantification methods. In this study, we established an allele-specific competitive blocker polymerase chain reaction (ACB-PCR)-based quantification method to accurately determine the composition of a multivalent adenoviral vector-based SARS-CoV-2 vaccine. By designing primers tailored to each variant and integrating a blocker for target-specific amplification, we achieved high accuracy in distinguishing closely related spike protein variants. Additionally, we explored coinfection and cotransfection strategies as alternative approaches for the simultaneous production of multivalent vaccines. Our results indicated that both these methods maintained antigen composition within a percentage error of approximately 10%, thereby supporting their feasibility for large-scale vaccine manufacturing. This study provides a robust molecular quantification tool for multivalent vaccine analysis and highlights efficient coproduction strategies that could enhance vaccine manufacturing scalability and responsiveness to emerging SARS-CoV-2 variants.