A rapid approach for linear epitope vaccine profiling reveals unexpected epitope tag immunogenicity

Antibody epitope profiling is essential for assessing the robustness of vaccine-induced immune responses, particularly while in development. Despite advancements in computational tools, high throughput experimental epitope validation remains an important step. Here, we describe a readily accessible method for rapid linear epitope profiling using phage-displayed oligo pools in combination with Nanopore deep sequencing. We applied this approach to TeeVax3, a Group A Streptococcus vaccine candidate, to investigate the antibody response generated in a pre-clinical rabbit model and assess antigen immunogenicity. Surprisingly, we found a strong bias in antibody binding response towards the N-terminal epitope tag used for purification. These tags are widely reported to have low immunogenicity and are frequently left uncleaved in pre-clinical studies. We further confirmed that the observed immune response against the epitope tag dominated even the conformational binding response and, using synthetic peptides, narrowed the epitope down to a set of 10 residues inclusive of the Histidine residues. Our findings highlight the importance of epitope-tag removal in pre-clinical studies and demonstrate the utility of rapid nanopore sequencing for early-stage vaccine evaluation.