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Dalinda Eusebio, Speaker at Vaccine Research Conference
CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal
Title : Targeted delivery of a minicircle DNA vaccine against COVID-19 to antigen-presenting cells using mannosylated polyethylenimine-cholesterol-based nanoparticles


Coronavirus disease (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has resulted in 775 million confirmed cases and approximately 7 million deaths reported by the World Health Organization (WHO) as of 31 March 2024. Nucleic acid vaccines have emerged as a novel approach to induce efficient and safe immune responses against COVID-19. DNA vaccines have advantages over mRNA vaccines, given their superior stability, simpler manufacturing process, and cost-effective manufacturing and storage, qualifying them as desirable candidates for global vaccination efforts, particularly in low-income countries. Minicircle DNA (mcDNA) presents a safer alternative to conventional plasmid DNA, as it lacks bacterial-derived sequences that are often associated with safety concerns. In this work, different systems based on polyethylenimine (PEI) were investigated for delivering parental plasmid (PP) and mcDNA vectors encoding the receptor-binding domain (RBD) of SARS-CoV-2 spike protein to antigen-presenting cells (APCs). Three polymeric systems were evaluated: PEI alone, PEI functionalized with cholesterol (PEI-CHOL), and PEI functionalized with both cholesterol and mannose ligand (PEI-CHOL-MAN). Different ratios of protonatable nitrogen groups (N) in the polymer to anionic phosphate groups (P) in DNA were investigated, and nanocomplexes were characterized in terms of DNA encapsulation efficiency, surface charge, size, and polydispersity index (PDI). Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed the successful formation of PEI complexes and confirmed DNA encapsulation. Scanning Electron Microscopy (SEM) verified that the formulated nanosystems exhibited a spherical shape and homogenous structure. Stability assays demonstrated that the formulated nanosystems were effective in protecting the DNA vector after incubation with cell culture media, trypsin, and 10% FBS. Decomplexation assays confirmed that DNA was able to maintain its integrity and supercoiled conformation after being released from the nanocomplexes. In addition, in vitro transfection studies were conducted using immature dendritic cells (JAWS II) and human fibroblast cells (hFibro). Viability studies assured the safety of all nanocarriers, nevertheless, nanosystems functionalized with cholesterol seem to reduce the inherent cytotoxicity associated with PEI. Fluorescence confocal microscopy studies conducted in JAWS II cells confirmed the intracellular localization of nanosystems, demonstrating enhanced cellular uptake with PEI-CHOL and PEI-CHOL-MAN compared to PEI alone. Regarding RBD expression, cells transfected with the mcDNA vector exhibited higher levels of RBD transcripts compared to those transfected with the PP vector. Furthermore, the PEI-CHOL-MAN system formulated with the mcDNA-RBD vector displayed higher levels of both transcripts and proteins in JAWS II cells. These findings suggest that mannose ligands facilitated the specific recognition by mannose receptors, which are overexpressed on the surface of APCs, in contrast to cells lacking this receptor (hFibro). Additionally, dendritic cell maturation was successfully induced by the nanosystems, resulting in a significant increase in pro-inflammatory cytokine production. This study highlights the potential for improving DNA vaccine efficacy by combining the mcDNA vector with mannosylated polymeric delivery systems. This innovative approach shows promise for enhancing immune responses against SARS-CoV-2, contributing to the fight against COVID-19, while also holding promise for addressing other infectious diseases.


Dalinda Eusébio completed her BSc in Biotechnology in 2016 and her MSc in Biomedical Sciences in 2018 at the University of Beira Interior, Portugal. Currently, she is pursuing her Ph.D. Degree in Biomedicine at the Health Sciences Research Centre of the University of Beira Interior (CICS-UBI) in collaboration with The University of Texas at Austin, supported by a Ph.D. fellowship from the Portuguese Foundation for Science and Technology (FCT). Her research focuses on pharmaceutical nanobiotechnology, specifically on the production, purification, and targeted delivery of the innovative minicircle DNA vector for gene therapy and DNA vaccine applications.