In the 1990s, the in vivo transfection of APCs in mice to induce primary adaptive immune responses initiated the development of DNA vaccines (Hobernik & Bros, 2018). Similarly, DNA administered directly to the skin and muscles of mice generated immune responses to the encoded antigen (Coban et al., 2013; Weiner, 2008). This led to the development and approval of the first veterinary DNA vaccine against Equine West Nile virus (WNV) in 2005. DNA vaccines enter cell nuclei where they are transcribed into RNA, which is then translated into the corresponding antigenic protein(s) in the cytoplasm (Lee & Nguyen, 2015). The antigens can be secreted to generate an antibody response and are also processed by the proteasome, leading to peptide presentation on cell surface MHC molecules and recognition by T cell receptors on naïve CD4 or CD8 T cells. Presentation can either be directly on the surface of transfected cells (somatic cells or professional APCs, e.g., dendritic cells) or cross-presentation following phagocytosis of transfected somatic cells by APCs (Ghaffarifar, 2018; Khan, 2013; Sun et al., 2021).

DNA vaccines also trigger innate immune responses: an unidentified cytosolic DNA sensor binds to transfected plasmids and triggers the TBK1 STING pathway. In this process, type I interferons are produced, which act as an adjuvant (Sun et al., 2021). DNA vaccines are easy and relatively inexpensive to produce, and have a longer shelf life (Hobernik & Bros, 2018). To date, only a few veterinary DNA vaccines have been approved . They include vaccines against Equine WNV (De Filette et al., 2012); infectious hematopoietic necrosis (IHNV) in salmon (Corbeil et al., 2000); canine melanoma (Liao et al., 2006); and the CLYNAVTM vaccine for Atlantic salmon (European Food Safety Authority et al., 2017). A conditional approval by the United States Department of Agriculture (USDA) has been given for the first commercial DNA vaccine against the highly pathogenic H5N1 influenza in chickens (Redding & Weiner, 2009).

 Beyond prophylaxis, DNA can also be employed in therapeutic cancer vaccines. In this regard, personalized antitumor DNA vaccines can be made directly from biopsies, or synthesized following sequencing of tumor DNA and identification of potential neoepitopes (Lee et al., 2018). This topic is explored in more detail in the chapter on approaches to cancer vaccination. Other applications for this approach include the treatment of autoimmune and allergic diseases (Gutowska-Owsiak & Ogg, 2017; Wraith et al., 2003).

 Historically, DNA vaccines have evoked low levels of immunogenicity, illustrated by the first clinical trials of vaccines for Influenza, HIV, malaria, and cancer (Weiner, 2008). Furthermore, immune responses generated in primate and large animal models are lower in comparison to those generated in small animals (Weiner, 2008). Various approaches have been taken to boost host cellular and humoral response to DNA vaccines (Coban et al., 2013; Weiner, 2008).

The efficiency of DNA vaccines can be improved by vector optimization, the introduction of nanocarriers and the attachment of virus-derived nuclear localization sequences. Vector optimization strategies include the use of APC-specific promoters, terminator sequences, inclusion of multiple antigen sequences, and the use of molecular adjuvants (Ghaffarifar, 2018; Lee et al., 2018). Nanocarriers can prevent extracellular DNA degradation, enhance endo/lysosomal escape of DNA, and enable APC targeting (Ghaffarifar, 2018). The use of specific localization sequences in the vaccine facilitates the entry of DNA into the nucleus for integration into the genome (Ghaffarifar, 2018; Karunamoorthi, 2014).

 A major concern surrounding DNA vaccines is the possibility that foreign DNA might integrate to aberrant sites in the human genome leading to potential pathologies, including transformation to a cancerous phenotype. In the first two decades of the 21st century, DNA vaccines against HCV, malaria, Influenza, HIV-1, and cytomegalovirus have undergone various stages of human clinical testing (Weiner, 2008)

References
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2- Coban, C., Kobiyama, K., Jounai, N., Tozuka, M., & Ishii, K. J. (2013). DNA vaccines: A sim ple DNA sensing matter? Human Vaccines & Immunotherapeutics., 9(10), 2216 2221. Available from https://doi.org/10.4161/hv.25893, Epub 2013 Aug 2. Available from 23912600.

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