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DNA Vaccine

 

Nucleic acid-based diagnostic and therapeutic platforms are promising tools that are replacing protein-based platforms due to their unique properties, such as thermostability, resistance to denaturation, and simple storage. A good vaccine platform should be rapid, simple to develop, reproducible, thermostable, and manufacturable with reduced development costs and risks. The DNA platform addresses many of these challenges.

 

DNA vaccines are DNA vectors such as bacterial plasmids, minicircular DNA, or linear, covalently closed expression constructs that contain at least one eukaryotic expression cassette encoding the antigen of interest. An expression cassette typically consists of a eukaryotic promoter/enhancer, antigen gene, and poly(A) signal sequence, which are essential for antigen expression in eukaryotic cells such as muscle cells. DNA vaccines have shown convincing safety and immunogenicity in preclinical studies. Several DNA vaccines are currently approved for veterinary use in both large animals (such as horses) and small animals (such as chickens). India, based on the safety, immunogenicity and efficacy results of the ZyCoV-D Phase 3 trial, granted it emergency use authorization, which is also a milestone in the development of DNA vaccines. Clinical trials of DNA vaccines for West Nile virus (WNV), Ebola and Marburg viruses, and SARS-CoV-2 have shown that antibodies are produced in humans weeks after immunization. But there are also many cases of poor immunogenicity in clinical trials. Target antigens and optimization of construction, formulation, and delivery methods appear to be key factors in the immunogenicity of DNA vaccines.

 

In the past few years, many advances have been made in the field of DNA vaccines. Advances in DNA construction, delivery and administration routes, and the use of molecular adjuvants have enhanced the immunogenicity of DNA vaccines. DNA immunization promises to revolutionize the field of vaccines. DNA vaccines are less expensive to manufacture and store, making them ideal candidates for global vaccinations, even in low-income countries.

 

Plasmid DNA is produced by genetically modified bacteria, usually Escherichia coli (E. coli). Production of plasmid DNA following Good Manufacturing Practice (GMP) at preclinical and clinical scale requires careful development of optimal and economical commercial processes. Bacterial cells are grown under fermentative conditions, usually in a defined or minimal cell culture medium consisting of chemically defined substances such as glucose or glycerol as a carbon source, salts, vitamins, etc. After fermentation, bacterial cells are harvested by centrifugation or microfiltration. Cell lysis is then performed using chemical, physical or mechanical methods. Cell lysis produces a lysate containing cell debris, plasmid DNA, and soluble impurities. Clarification techniques such as tangential flow filtration are used to remove solids from lysates. Contaminants (e.g., host proteins, endotoxin, RNA, genomic DNA, linear and open circular forms of plasmid DNA). Purified plasmid DNA is formulated with excipients and adjuvants and filtered through sterile filters.

 

Minicircle DNA was generated by inducing intramolecular recombination of the parental plasmid in E.Coli. For example, the expression of recombinases, such as φC31 integrase, and restriction enzymes (REs), such as I-SceI, is induced by the arabinose-inducible gene expression system. Recombinases mediate site-specific recombination between their recognition sequences, resulting in two distinct circular DNA molecules (i) a minicircular DNA containing a eukaryotic expression cassette and (ii) a mini-plasmid (MP), MP can be specifically degraded by the induced RE. Minicircle DNA can then be extracted, purified, and formulated similarly to the method used for DNA plasmids.


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