T Cell Activating Thermostable Self-Assembly Nanoscaffold Tailored for Cellular Immunity Antigen Delivery

Antigen delivery based on non-virus-like particle self-associating protein nanoscffolds, such as Aquifex aeolicus lumazine synthase (AaLS), is limited due to the immunotoxicity and/or premature clearance of antigen-scaffold complex resulted from triggering unregulated innate immune responses. Here, using rational immunoinformatics prediction and computational modeling, we screen the T epitope peptides from thermophilic nanoproteins with the same spatial structure as hyperthermophilic icosahedral AaLS, and reassemble them into a novel thermostable self-assembling nanoscaffold RP_T that can specifically activate T cell-mediated immunity. Tumor model antigen ovalbumin T epitopes and the severe acute respiratory syndrome coronavirus 2 receptor-binding domain are loaded onto the scaffold surface through the SpyCather/SpyTag system to construct nanovaccines. Compared to AaLS, RP_T -constructed nanovaccines elicit more potent cytotoxic T cell and CD4⁺ T helper 1 (Th1)-biased immune responses, and generate less anti-scaffold antibody. Moreover, RP_T significantly upregulate the expression of transcription factors and cytokines related to the differentiation of type-1 conventional dendritic cells, promoting the cross-presentation of antigens to CD8⁺ T cells and Th1 polarization of CD4⁺ T cells. RP_T confers antigens with increased stability against heating, freeze-thawing, and lyophilization with almost no antigenicity loss. This novel nanoscaffold offers a simple, safe, and robust strategy for boosting T-cell immunity-dependent vaccine development.