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- Development of a minicircular DNA vaccine against COVID-19Publication . Ventura, Cathy Lopes; Sousa, Ângela Maria Almeida de; Costa, Diana Rita BarataNucleic 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.
- The Effectiveness of Therapeutic Vaccines for the Treatment of Cervical Intraepithelial Neoplasia 3: A Systematic Review and Meta-AnalysisPublication . Ventura, Cathy; Luís, Ângelo; Soares, Christiane P.; Venuti, Aldo; Paolini, Francesca; Pereira, L.; Sousa, ÂngelaCervical cancer (CC) is a disease that affects many women worldwide, especially in low-income countries. The human papilloma virus (HPV) is the main causative agent of this disease, with the E6 and E7 oncoproteins being responsible for the development and maintenance of transformed status. In addition, HPV is also responsible for the appearance of cervical intraepithelial neoplasia (CIN), a pre-neoplastic condition burdened by very high costs for its screening and therapy. So far, only prophylactic vaccines have been approved by regulatory agencies as a means of CC prevention. However, these vaccines cannot treat HPV-positive women. A search was conducted in several databases (PubMed, Scopus, Web of Science, and ClinicalTrials.gov) to systematically identify clinical trials involving therapeutic vaccines against CIN 3. Histopathological regression data, immunological parameters, safety, DNA clearance, and vaccine efficacy were considered from each selected study, and from the 102 articles found, 8 were selected based on the defined inclusion criteria. Histopathological regression from CIN 3 to CIN < 1 was 22.1% (95% CI: 0.627–0.967; p-value = 0.024), showing a vaccine efficacy of 23.6% (95% CI; 0.666–0.876; p-value < 0.001). DNA clearance was assessed, and the risk of persistent HPV DNA was 23.2% (95% CI: 0.667–0.885; p-value < 0.001). Regarding immunological parameters, immune responses by specific T-HPV cells were more likely in vaccinated women (95% CI: 1.245–9.162; p-value = 0.017). In short, these studies favored the vaccine group over the placebo group. This work indicated that therapeutic vaccines are efficient in the treatment of CIN 3, even after accounting for publication bias.