Development of a minicircular DNA vaccine against COVID-19

• Autor(a) principal: Ventura, Cathy Lopes
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/13046
• topic: Covid-19; Maximização do Mcdna; Quitosano; Sistemas de Entrega; Vacinas de Dna; Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas

Nucleic acid vaccines have proven to be a promising technology in the fight against global threats such as coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptorbinding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value < 0.05) and presented a nonsignificant lack of fit (p-value > 0.05) with a suitable coefficient of determination. The production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO2 in the fermentation step during 5 h and 30% pO2 in the induction step, with 0.01% L-arabinose throughout 5 h. The application of delivery systems improves the DNA vaccines efficacy and allows their targeting when functionalized with specific ligands. In this work were explored two chitosan (Ch) polymers to formulate different Ch-TPP/R8 and R8-mannose based nanosystems for the delivery of a new mcDNA vaccine against COVID-19, encoding the receptor-binding domain (RBD) gene of severe acute respiratory syndrome coronavirus (SARS-CoV-2). For this purpose, different ratios of TPP, R8 and R8-mannose were evaluated. All systems were formulated using the ionotropic gelation technique and their size, surface charge, encapsulation efficiency and stability were subsequently evaluated. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) were also performed to ascertain the functional groups on the surface of the nanoparticles and their shape and morphology, respectively. Two cell lines, human fibroblasts (h-Fibro) and immature dendritic cells (JAWS II) were used in in vitro studies to evaluate the compatibility, transfection efficiency and gene expression of formulated systems. The 3-[4,5- dimethylthiazol2-yl]-2,5-diphenyltetrazolium (MTT) assays showed the biosafety of all Ch-based nanosystems. Subsequently, confocal microscopy studies were performed on dendritic cells (JAWSii), to verify the difference in internalization of non-mannosylated and mannosylated systems in APCs. Systems functionalized with R8-mannose showed better internalization into the cells.