*3.1. Mechanism*

Different recombinant cytolytic DNA-PRF vaccines (rDNA-PRF) have been shown to elicit immune responses higher than those elicited by canonical DNA vaccines (without PRF) and the mechanism underlying this has been established [94]. A previous study showed that coexpression of HCV NS3 and PRF elicited nonapoptotic cell death in transfected cells, whilst immunization with NS3-PRF DNA vaccine increased NS3-specific T cell mediated responses as evidenced by increased NS3-specific IFN-γ responses in an ELISpot assay and increased numbers of polyfunctional CD8<sup>+</sup> TEM cells that simultaneously secreted IFN-γ, IL-2, and TNF-α [56]. Cytolytic DNA platform where the expression of immunogen is driven by a stronger promoter allows for sufficient antigen expression and accumulation within the target cells followed by nonapoptotic cell death due to lesser expression of PRF driven by a weaker SV40 promoter; thus, balancing the level of antigen expression with the timing of cell death [94].

Necrosis is considered as the mechanism of cell death by PRF as evidenced by release of lactate dehydrogenase (LDH) and low caspase activity [55,56,94]. LDH release occurs after the rupture of cell membrane during secondary necrosis [98,99]. In contrast, LDH was not released by cells treated with doxorubicin (a proapoptotic drug) or cells transfected with NS3 wild type PRF or NS3 12del483A PRF (mutant and nontoxic PRF) [94]. Expression of PRF from a cytolytic DNA, e.g., NS3 PRF vaccine, thus results in necrotic cell death mediated by receptor-interacting protein-1 kinase activity, as evidenced by detection of uncleaved cytokeratin 18 in Huh-7 cells [56]. Necrosis releases damage associated molecular patterns (DAMPs) which in turn activate DCs to migrate to the site of vaccination [100,101].

When purified DCs including the CD8α<sup>+</sup> subset from naïve C57BL/6 mice were exposed to HEK293T cells (transfected with Ovalbumin-PRF), it resulted in upregulation of costimulatory molecules (CD80/CD86), indicating maturation of the immune cells with the cytolytic DNA [94]. A significant increase in CD11c<sup>+</sup> DCs and cross-presenting CD8a<sup>+</sup> DCs, and upregulation of CD80 has been reported in mice vaccinated with a cytolytic DNA HIV 1 Gag PRF compared to a canonical DNA vaccine [55]. Local and migrated DCs at the site of inflammation can take up antigens by endocytosis and are also exposed to DAMPs. Activated and matured DCs can then prime naïve CD8<sup>+</sup> T cells (Figure 2). Antigen cross-presentation by DCs to CD8<sup>+</sup> T cells has been shown to increase the number of proliferating CD8<sup>+</sup> T cells by ~2-fold with cytolytic DNA compared to the noncytolytic PRF DNA [94]. Thus, a cytolytic DNA vaccine has an inbuilt adjuvant to enhance the immunogenicity of the vaccine immunogen. Whereas, the immunogenicity of canonical DNA vaccines mostly depends on direct transfection of DCs and to a lesser extent cross-presentation of antigens shed from transfected cells and/or derived from transfected cells that have undergone spontaneous cell death [94].

Several studies have established that DCs exposed to necrotic or lytic cells expressing antigens mature and cross-present more efficiently than DCs exposed to antigens derived from a cellular milieu that comprise of apoptotic cells [102–106]. Comparative studies evaluating the ability of proapoptotic (e.g., rotavirus nonstructural protein 4 (NSP4) and diphtheria toxin subunit A (DTa)) and necrotic proteins (e.g., truncated PRF) to enhance the immunogenicity of DNA when encoded in plasmid DNA vaccines showed that truncated PRF is the most effective for this purpose [55–57]. However, a caveat is that vaccine-encoded antigens need to accumulate significantly inside the cell before necrosis occurs following expression of truncated PRF in order to activate DCs to cross-present vaccine-encoded antigens [55,94].

**Figure 2.** Mechanism of rDNA-PRF immunogenicity.
