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Vaccine Development Strategies and the Current Status of COVID-19 Vaccines
Published in Debmalya Barh, Kenneth Lundstrom, COVID-19, 2022
Mohsen Akbarian, Kenneth Lundstrom, Elrashdy M. Redwan, Vladimir N. Uversky
The first DNA-based vaccines were already developed in 1983. DNA vaccines are based on plasmid DNA expressing antigens for the vaccine targets in transfected host cells. DNA plasmids are relatively stable and can replicate independently in host cells. However, low transfection efficacy and the need for delivery to the nucleus has hampered their efficacy as vaccine vectors. To address these issues, electroporation, jet injection, gene gun, and nanoparticle technologies have been applied. Numerous types of nanoparticles, such as lipid and polymer nanoparticles, lipid-polymer hybrid nanoparticles, DNA-polymer complexes, nanoparticles coated with polymeric materials and gold, and protein-DNA complexed nanoparticles have been formulated for improved DNA delivery [48]. MERS-CoV S and N proteins have been expressed from DNA plasmid as vaccine antigens [49]. Effective and specific immunogenic responses, including the production of γ-interferon, IL-2, CD4+, CD8+, and IL-2, and the induction of cytotoxic T lymphocytes have been reported in animal studies. One of the most important advantages is that production of plasmid DNA in bacteria is rapid and inexpensive [50]. Other important advantages are the possibility of combining DNA-based vaccines with other vaccine platforms (such as first generation vaccines), excellent heat and shelf-life stability, ease of DNA sequence engineering and reduced safety risk compared to viral vectors [48]. However, limited immune responses caused by low transfection efficiency of DNA vaccines is a disadvantage [19].
HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
The anticipated yield of plasmid DNA using the methods described above is approximately 300 μg from a 200-ml starting culture. As the final plasmid DNA pellet is dissolved in 0.2 ml TE buffer, the concentration of DNA should be approximately 1.5 ug/pl If cesium chloride-ethidium bromide density gradient purification has been used, adjust the concentration of plasmid DNA to 1.5 μg/μl.
The Regulatory Process and Gene Therapy 1
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
At a CBER-sponsored workshop, December 11-13, 1995, on well-characterized biotechnology products, a breakout session on plasmid DNA products was held. The recommendation of this session was that plasmid DNA products be considered eligible for designation as "well-characterized." A "well-characterized product" would have well-defined physicochemical characteristics, could be manufactured reproducibly, and would not require a separate establishment license or lot release submission to CBER if licensed. Note that a product can be considered well-characterized by virtue of its structure and mode of manufacture and testing, and yet the product is not eligible for licensure unless data collected under an IND demonstrate safety or efficacy in an appropriate patient population.
Suitability of transiently expressed antibodies for clinical studies: product quality consistency at different production scales
Published in mAbs, 2022
Sara Rodriguez-Conde, Sophie Inman, Viv Lindo, Leanne Amery, Alison Tang, Uche Okorji-Obike, Wenjuan Du, Berend-Jan Bosch, Paul J. Wichgers Schreur, Jeroen Kortekaas, Isabel Sola, Luis Enjuanes, Laura Kerry, Katharina Mahal, Martyn Hulley, Olalekan Daramola
Traditionally, transient gene expression (TGE) has been the technology used for production of therapeutic glycoproteins at early drug development stages because it allows for rapid production of high-quality material.1 This technology involves introducing plasmid DNA, which encodes the protein of interest, into mammalian cells. The cells then express the recombinant protein over a limited period of time, typically up to 14–21 days. Several methods are used to transfer plasmid DNA into mammalian cells for TGE. Some of the most common chemical agents used for transfection are calcium phosphate, polyethyleneimines (PEIs) and cationic lipids. In particular, PEIs are frequently used due to the high transfection efficiency and relatively low cost compared to lipid-based reagents. An alternative to chemical-based transfection is the use of electroporation methods, such as the MaxCyte® STXTM flow electroporation system. Using this approach, Steger et al. were able to produce 3.5 g of antibody from less than 3 L of culture.2 Although currently this technology has been tested only at shake flask culture scale, it has the potential to be scaled up to several liters in bioreactors.
The baculovirus expression vector system: a modern technology for the future of influenza vaccine manufacturing
Published in Expert Review of Vaccines, 2022
Claudia Maria Trombetta, Serena Marchi, Emanuele Montomoli
Even DNA vaccine seems to be a promising technology in development since the 1990’. These vaccines do not require the growth of live virus, are temperature stable, noninfectious, non-replicating and the production process is rapid and cheap. The great advantage is that the target sequences of clinical isolates can be used as soon as available and are able to induce both humoral and cellular immune responses. The route of administration is critical and different devices have been evaluated, such as patches, gene-gun, and electroporation. The main concerns are with regard to safety and the potential integration of the plasmid DNA into host genome, the development of anti-DNA antibodies resulting in auto-immune disease and antibiotic resistance [108–113]. However, a phase 1 randomized clinical trial in children and adolescents priming with trivalent DNA vaccine and boosting with trivalent IIV provided evidence that the strategy is safe and well tolerated [114]. So far, no DNA vaccines have been approved for use in humans.
TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins
Published in Journal of Receptors and Signal Transduction, 2018
Radka Moravcova, Barbora Melkes, Jiri Novotny
HEK293 cells were transfected with the above-mentioned plasmid using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Two weeks posttransfection, the cells were sorted on a BD Influx TM cell sorter (BD Biosciences, Franklin Lakes, NJ), and thereupon grown for 6 weeks in the presence of geneticin (800 μg/ml). This resulted in the isolation of several cell clones harboring the introduced plasmid DNA. One of these clones stably expressing TRHR-YFP, designated TRY-1, was used for all subsequent experiments. The expression level of TRHR-YFP in these cells (as assessed by immunoblotting) was comparable to endogenous expression of TRH receptors in pituitary GH1 cells. The specificity of TRHR antibody (Santa Cruz Biotechnology, Santa Cruz, CA; sc-11574) used for this purpose was confirmed elsewhere [14].