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

Abstract

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.

Keywords

Human papillomavirus 16 / E7 oncoprotein / Heat shock protein 27 / Green fluorescent protein / Exosome / Immunos-timulatory effects

ACKNOWLEDGEMENTS

F. Rezaei was supported by Pasteur Institute of Iran to pursue her study in the Ph.D. thesis.

AUTHORS CONTRIBUTIONS

F.R. performed the experiments and wrote the manuscript. A.A., F.F., S.M.S. and A.B. analysed and validated the data, and revised the manuscript. All authors approved the manuscript.

FUNDING

This research received no external funding.

CONFLICTS OF INTEREST DISCLOSURE

The authors declare they have no conflicts of interest.

INFORMED CONSENT

Not applicable.

ETHICS STATEMENT

In vitro studies were conducted in strict adherence to approved protocols and in compliance with the highest standards of animal care at the Pasteur Institute of Iran. This study was conducted in accordance with ethics code IR.PII.REC.1400.025.

ETHICS APPROVAL

The Publication Ethics Committee of the Sciedu Press. The journal’s policies adhere to the Core Practices established by the Committee on Publication Ethics (COPE).

PROVENANCE AND PEER REVIEW

Not commissioned; externally double-blind peer reviewed.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available in the manuscript.

DATA SHARING STATEMENT

Not applicable.

OPEN ACCESS

This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).

COPYRIGHTS

Copyright for this article is retained by the author(s), with first publication rights granted to the journal.

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