In silico studies of green fluorescent protein-tagged Hsp27-HPV16 E7 fusion protein and evaluation of cytokine secretion from antigen-presenting cells exposed to the fusion protein-carrying tumor-derived exosomes

Objective: Exosome (Exo)-based therapies have attracted considerable interest due to their potential as carriers for therapeutic molecules and their capacity to elicit anti-tumor immune responses. The objective of this study was to engineer TC-1 tumor cell line-derived exosomes with GFP-tagged heat shock protein (Hsp) 27-human papillomavirus (HPV)16 E7 fusion protein and evaluation of cytokine secretion from antigen-presenting cells (APCs: macrophages and dendritic cells) exposed to the engineered exosomes in vitro.Methods: In this study, different in silico methods were employed to evaluate the Hsp27-E7 and Hsp27-E7-GFP fusion proteins as potential vaccine candidates. Regarding to the in silico data, the Hsp27-E7-GFP fusion gene was subcloned into pCDH lentiviral vector for production of lentivirions harboring the Hsp27-E7-GFP fusion protein in eukaryotic cells. Subsequently, the TC-1 tumor cells were transduced with these lentivirions to isolate the engineered exosomes (i.e., Exo-Hsp27-E7-GFP) using the ExoQuick-TCTM kit and their characterization using physicochemical methods. Finally, the secretion of key cytokines (IFN-γ, TNF-α, and IL-10) was evaluated through incubation of antigen-presenting cells (APCs) with the engineered Exo-Hsp27-E7-GFP using enzyme-linked immunosorbent assay (ELISA).Results: Our in silico data showed that both the Hsp27-E7 and Hsp27-E7-GFP constructs were soluble and non-allergenic, and exhibited strong interaction with TLR4. Indeed, the linkage of GFP did not affect the physicochemical properties, and interaction of Hsp27-E7 with the immune receptors. Moreover, western blot analysis confirmed the presence of Hsp27-E7-GFP fusion protein in the isolated exosomes. The Exo-Hsp27-E7-GFP could significantly enhance the secretion of TNF-α and IL-10 from APCs compared to Exo and Exo-GFP, as well.Conclusions: These engineered vesicles derived from tumor cells demonstrate the capacity to induce effective immunity, suggesting their potential as a promising strategy in the development of cell-free vaccine candidates.