Mannosylated polyethylenimine-cholesterol-based nanoparticles for targeted delivery of minicircle DNA vaccine against COVID-19 to antigen-presenting cells.

DNA vaccines can be a potential solution to protect global health, triggering both humoral and cellular immune responses. DNA vaccines are valuable in preventing intracellular pathogen infections, and therefore can be explored against coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2). This work explored different systems based on polyethylenimine (PEI), functionalized for the first time with both cholesterol (CHOL) and mannose (MAN) to deliver parental plasmid (PP) and minicircle DNA (mcDNA) vectors encoding the receptor-binding domain (RBD) of SARS-CoV-2 to antigen-presenting cells (APCs). For comparative purposes, three different systems were evaluated: PEI, PEI-CHOL and PEI-CHOL-MAN. The systems were prepared at various nitrogen-to-phosphate group (N/P) ratios and characterized in terms of encapsulation efficiency, surface charge, size, polydispersity index (PDI), morphology, and stability over time. Moreover, in vitro transfection studies of dendritic cells (JAWS II) and human fibroblast cells were performed. Viability studies assured the biocompatibility of all nanocarriers. Confocal microscopy studies confirmed intracellular localization of systems, resulting in enhanced cellular uptake using PEI-CHOL and PEI-CHOL-MAN systems when compared with the PEI system. Regarding the RBD expression, PEI-CHOL-MAN was the system that led to the highest levels of transcripts and protein expression in JAWS II cells. Furthermore, the nanosystems significantly stimulated pro-inflammatory cytokines production and dendritic cell maturation in vitro. Overall, mannosylated systems can be considered a valuable tool in the delivery of plasmid DNA or mcDNA vaccines to APCs.

Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD.

Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the 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 receptor-binding 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 non-significant lack of fit (p-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, 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 yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.

Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System.

In current times, DNA vaccines are seen as a promising approach to treat and prevent diseases, such as virus infections and cancer. Aiming at the production of a functional and effective plasmid DNA (pDNA) delivery system, four chitosan polymers, differing in the molecular weight, were studied using the design of experiments (DoE) tool. These gene delivery systems were formulated by ionotropic gelation and exploring the chitosan and TPP concentrations as DoE inputs to maximize the nanoparticle positive charge and minimize their size and polydispersity index (PDI) as DoE outputs. The obtained linear and quadratic models were statistically significant (p-value < 0.05) and non-significant lack of fit, with suitable coefficient of determination and the respective optimal points successfully validated. Furthermore, morphology, stability and cytotoxicity assays were performed to evaluate the endurance of these systems over time and their further potential for future in vitro studies. The subsequent optimization process was successful achieved for the delivery systems based on the four chitosan polymers, in which the smallest particle size was obtained for the carrier containing the 5 kDa chitosan (~82 nm), while the nanosystem prepared with the high molecular weight (HMW) chitosan displayed the highest zeta potential (~+26.8 mV). Delivery systems were stable in the formulation buffer after a month and did not exhibit toxicity for the cells. In this sense, DoE revealed to be a powerful tool to explore and tailor the characteristics of chitosan/pDNA nanosystems significantly contributing to unraveling an optimum carrier for advancing the DNA vaccines delivery field.

Methods to improve the immunogenicity of plasmid DNA vaccines.

DNA vaccines have emerged as innovative approaches that have great potential to overcome the limitations of current conventional vaccines. Plasmid DNA vaccines are often safer than other vaccines because they carry only antigen genetic information, are more stable and easier to produce, and can stimulate both humoral and cellular immune responses. Although the results of ongoing clinical trials are very promising, some limitations compromise the immunogenicity of these vaccines. Thus, this review describes different strategies that can be explored to improve the immunogenicity of plasmid DNA vaccines, including the optimization of the plasmid vector backbone, the use of different methods for vaccine delivery, the use of alternative administration routes and the inclusion of adjuvants. In combination, these improvements could lead to the successful clinical use of plasmid DNA vaccines.

Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine.

DNA vaccines still represent an emergent area of research, giving rise to continuous progress towards several biomedicine demands. The formulation of delivery systems to specifically target mannose receptors, which are overexpressed on antigen presenting cells (APCs), is considered a suitable strategy to improve the DNA vaccine immunogenicity. The present study developed binary and ternary carriers, based on polyethylenimine (PEI), octa-arginine peptide (R8), and mannose ligands, to specifically deliver a minicircle DNA (mcDNA) vaccine to APCs. Systems were prepared at various nitrogen to phosphate group (N/P) ratios and characterized in terms of their morphology, size, surface charge, and complexation capacity. In vitro studies were conducted to assess the biocompatibility, cell internalization ability, and gene expression of formulated carriers. The high charge density and condensing capacity of both PEI and R8 enhance the interaction with the mcDNA, leading to the formation of smaller particles. The addition of PEI polymer to the R8-mannose/mcDNA binary system reduces the size and increases the zeta potential and system stability. Confocal microscopy studies confirmed intracellular localization of targeting systems, resulting in sustained mcDNA uptake. Furthermore, the efficiency of in vitro transfection can be influenced by the presence of R8-mannose, with great implications for gene expression. R8-mannose/PEI/mcDNA ternary systems can be considered valuable tools to instigate further research, aiming for advances in the DNA vaccine field.

Minicircle DNA Vaccine Purification and E7 Antigen Expression Assessment.

Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.

The Performance of Minicircle DNA Versus Parental Plasmid in p53 Gene Delivery Into HPV-18-Infected Cervical Cancer Cells.

Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), this small vector is able to sustain high expression rates throughout time. Thus, this work describes the construction, production, and purification of mcDNA-p53 and its precursor parental plasmid (PP)-p53 for a comparative study of both DNA vectors in the growth suppression of human papillomavirus (HPV)-18-infected cervical cancer cells. First, live cell imaging and fluorescence microscopy studies allowed to understand that mcDNA-p53 vector was able to enter cell nuclei more rapidly than PP-p53 vector, leading to a transfection efficiency of 68% against 34%, respectively. Then, p53 transcripts and protein expression assessment revealed that both vectors were able to induce transcription and the target protein expression. However, the mcDNA-p53 vector performance stood out, by demonstrating higher p53 expression levels (91.65 ± 2.82 U/mL vs. 74.75 ± 4.44 U/mL). After assuring the safety of both vectors by viability studies, such potential was confirmed by proliferation and apoptosis assays. These studies confirmed the mcDNA-p53 vector function toward cell cycle arrest and apoptosis in HPV-18-infected cervical cancer cells. Altogether, these results suggest that the mcDNA vector has a more promising and efficient role as a DNA vector than conventional pDNA, opening new investigation lines for cervical cancer treatment in the future.

Biosynthesis and isolation of gellan polysaccharide to formulate microspheres for protein capture.

In the present work, the biosynthesis of the gellan gum exopolysaccharide from Sphingomonas paucimobilis fermentation was improved to 24 g/L, by increasing the oxygen content (1:5). FTIR and NMR data indicated the following procedures to recover gellan from culture medium: filtration; washing with acetone and ether (200 mL each one); permeate dissolution with distilled water; precipitation with acetonitrile (1:3). Gellan microsphere formulation by water-in-oil emulsion was optimized through central composite design to minimize the diameter (≈ 300 µm), and reinforced with different counter-ions. The optimal point was reached with 1.27% gellan concentration, 750 rpm and 92.54 °C of oil temperature. Microspheres with nickel as counter-ion revealed the most satisfactory results from semi-optical microscopy, SEM and EDX analysis. The manipulation of ionic strength or pH on the binding and elution steps allowed capturing different model proteins, exploring electrostatic or affinity interactions with these microspheres.