HACC-TNF-α-VLP Nanoparticles Facilitate HSP90-Dependent Antigen Cross-Presentation to Enhance CD8⁺ T Cell and Mucosal Immunity Against Foot-and-Mouth Disease

Objective: Current foot-and-mouth disease (FMD) vaccines lack the efficient induction of CD8 + T cell-mediated cellular immunity and mucosal protection, highlighting the need for novel immunomodulatory delivery systems. Methods: HACC-TNF-α-virus-like particles (VLPs) nanoparticles were prepared via the ionic cross-linking method, followed by systematic evaluation of morphological characteristics, particle size distribution, in vitro release behavior, stability, and biotoxicity. In vitro experiments, the prepared nanoparticles were co-cultured with bone marrow-derived dendritic cells (BMDCs), and flow cytometry (FCM) and cytokine array assays were subsequently used to detect the activation status of dendritic cells. The combined application of laser confocal microscopy and FCM was employed to further explore the antigen cross-presentation ability mediated by the nanoparticles. For the in vivo experiments, mice were administered with the nanoparticles via two routes: intranasal immunization and subcutaneous immunization. The levels of specific antibodies in the serum were quantitatively detected using enzyme-linked immunosorbent assay, and lymphocyte proliferation assay, immunohistochemical technology, and FCM were combined to comprehensively evaluate the intensity of CD8 + T cell immune response and mucosal immune effects in mice. Results: HACC-TNF-α-VLPs nanoparticles were successfully prepared, showing spherical morphology (~162.7 nm, − 11.9 mV zeta potential), 84.40% encapsulation efficiency, 12.82% drug loading, and sustained release (75.83% cumulative release on day 4). They were non-cytotoxic to L929 cells at concentrations of ≤ 400 μg/mL. HACC-TNF-α-VLPs nanoparticles promoted BMDCs maturation, facilitated BMDCs endocytosis of FMDV-VLPs, and antigen cross-presentation mediated by heat shock protein 90. Moreover, it can activate CD8 + T cells in vitro via cross-presentation. HACC-TNF-α-VLPs nanoparticles upregulated cytotoxic T cells, tissue-resident memory T cells, sIgA, and systemic FMDV-specific antibodies in vivo. Conclusion: HACC-TNF-α-VLPs nanoparticles exhibit potent immunomodulatory activity by efficiently mediating antigen cross-presentation and play a dual role in both induction of CD8 + T cell responses and mucosal immunity, indicating their potential role in FMD novel vaccine development. Keywords: nanoparticles, foot-and-mouth disease virus-like particles, cross-presentation, CD8 + T cells, tissue-resident memory t cells, dendritic cells