**3. Molecular adjuvants and Bcl-xL anti-apoptotic protein**

Molecular adjuvants can be defined as plasmids expressing cytokines, chemokines or co-stimulatory molecules which can be co-administered with the antigenic DNA vaccine plasmid [103] or vaccine plasmid can be constructed as a bicistronic vector system. The magnitude of immune response after DNA vaccination is very closely related to: (i) the source of Ag presentation, (ii) the immunological properties of the DNA itself and (iii) the role of cytokines in eliciting the immune responses [104]. Thus, with the cells transfected by molecular adjuvant, encoding plasmids secrete the adjuvant into the surrounding region stimulating both local antigen-presenting cells (APCs) and cells in the draining lymph node, especially dendritic cells. The examples of molecular adjuvants as cytokines: GM-CSF (granulocyte-macrophage colony stimulating factor), M-CSF, IFN-γ, IL-2, IL-4, IL-7 and IL-8, IL-10, IL-12, IL-15, IL-18; as chemokines: IL-8, MCP-1 (monocyte chemoattractant protein 1), MIP-1a (macrophage inflammatory protein), RANTES (CCL5); and as co-stimulatory proteins: CD40L, CD80/86, ICAM-1 (intercellular adhesion molecule 1) [103]. In addition, ligands of pattern recognition receptors (PRRs) are described as molecular adjuvants. There are 13 TLR genes (TLR1–TLR13). TLR3 and TLR9 recognize dsRNA and ssDNA, respectively, and their ligands have been shown to act as molecular adjuvants. Poly(I:C) is a classical TLR3 ligand, and CpG is a TLR 9 ligand, showing molecular adjuvant properties via increasing cytotoxic T-cell responses [105]. Bcl-xL anti-apoptotic protein was also described as molecular adjuvant. Inhibiting the apoptosis of antigen-presenting dendritic cells, the cytotoxic T-cell responses (CD8+ T cells) are increased due to the longer survival of the dendritic cells [106]. In addition, rather than co-transfection, expression of Bcl-xL in a bicistronic vector further enhances CD8+ T-cell responses compared to co-transfection [107, 108].

In our previous studies, pIRESEGFP/Bcl-xL is a bicistronic vector bearing CMV (cytomegalovirus) promoter and IRES (internal ribosomal entry site) used as a backbone for DNA vaccination studies. Bcl-xL anti-apoptotic protein in frame with eGFP (enhanced green florescence protein) as a molecular adjuvant was also encoded by the plasmid. It's shown that Bcl-xL anti-apoptotic protein rescued cells from serum deprivation, doxorubicin, camptothecin and staurosporine induced apoptosis [71, 109], induced prolonged expression of the antigen of interest in expressed under CMV promoter that facilitates an increased CD8+ T cell response in DNA vaccination studies encoding foot and mouth disease multi-epitopes [4] and Toxoplasma gondii, SporoSAG antigen [5].

### **4. Conclusion**

In this chapter, we have discussed DNA vaccines, several widely used and emerging gene delivery systems to increase efficacy of DNA vaccines, characterization of these systems and cellular uptake of DNA vaccines yet to be tested in the clinic in the future. Also, as means of molecular adjuvants, several agents are in

**141**

in the future.

*Current State of the Art in DNA Vaccine Delivery and Molecular Adjuvants: Bcl-xL…*

consideration like chemokines, cytokines, co-stimulatory molecules, PPR ligands

Nanotechnology offers new strategies in formulating better adjuvants for DNA vaccines. However, they are very stable and the long-term cytotoxic effects in the body appear to be a potential problem. In order to remove this problem, most effective surface and content modifications of nanoparticles studied are being made. The relatively short history of the use of nanoparticles has led to a lack of understanding of the safety profile of human use. For this reason, many studies are being carried out in this regard today. If a safe profile can be shown as a result of these studies, this new vaccine delivery system will be considered to be an effective method, which will be widely used. In addition, nanoparticle-based DNA vaccines are seen as a strategy for future single-dose applications and the need for needle-free vaccines, as they enhance cell transfection efficiency and immunogenicity and enable targeting strategies.

In future studies, the development of nanoparticle-based gene delivery systems

for different purposes will continue to be critical. Modification of toxicity and immunogenicity problems of viral vectors, enhancement of transfection efficiency as much as possible for non-viral vectors, enhancement of vector targeting and specificity, regulation of gene expression and identification of synergies between gene-based agents and other cancer therapies are promising studies. Nevertheless, the safe and efficient transport of plasmid DNA to initiate immunological responses remains an important barrier to human DNA vaccination. The development of new non-viral strategies for DNA vaccines has to continue to serve as biological insight and clinic-related methods. Specific concerns include difficulties with transfection of dendritic cells. This includes methods that target strong antigen signaling, antigen-presenting cell uptake and lymph node transduction without sacrificing biocompatibility. Carriers must deliver the genetic load specifically to the target tissue, while protecting the genetic material from metabolic and immune pathways. DNA transfection of cells in vitro/in vivo studies requires overcoming both extracellular and intracellular barriers to gene transport from cell plasma membrane which is the barrier of intracellular DNA uptake and hinders DNA trafficking in the cytoplasm, and also into the cell nucleus that is nuclear envelope. Therefore, gene delivery methods including viral, non-viral, physical, chemical and molecular systems should facilitate DNA delivery across these barriers and into the nucleus to enable transcription without any degradation very quickly. Gene delivery systems are so important that besides the characterization of these systems by various methods such as SEM, TEM, AFM and FT-IR, the examination of their cellular uptake by various techniques like confocal complex fluorescence microscopy and flow cytometry and the development of study done in this area are extremely important

*DOI: http://dx.doi.org/10.5772/intechopen.82203*

and anti-apoptotic proteins.

#### *Current State of the Art in DNA Vaccine Delivery and Molecular Adjuvants: Bcl-xL… DOI: http://dx.doi.org/10.5772/intechopen.82203*

consideration like chemokines, cytokines, co-stimulatory molecules, PPR ligands and anti-apoptotic proteins.

Nanotechnology offers new strategies in formulating better adjuvants for DNA vaccines. However, they are very stable and the long-term cytotoxic effects in the body appear to be a potential problem. In order to remove this problem, most effective surface and content modifications of nanoparticles studied are being made. The relatively short history of the use of nanoparticles has led to a lack of understanding of the safety profile of human use. For this reason, many studies are being carried out in this regard today. If a safe profile can be shown as a result of these studies, this new vaccine delivery system will be considered to be an effective method, which will be widely used. In addition, nanoparticle-based DNA vaccines are seen as a strategy for future single-dose applications and the need for needle-free vaccines, as they enhance cell transfection efficiency and immunogenicity and enable targeting strategies.

In future studies, the development of nanoparticle-based gene delivery systems for different purposes will continue to be critical. Modification of toxicity and immunogenicity problems of viral vectors, enhancement of transfection efficiency as much as possible for non-viral vectors, enhancement of vector targeting and specificity, regulation of gene expression and identification of synergies between gene-based agents and other cancer therapies are promising studies. Nevertheless, the safe and efficient transport of plasmid DNA to initiate immunological responses remains an important barrier to human DNA vaccination. The development of new non-viral strategies for DNA vaccines has to continue to serve as biological insight and clinic-related methods. Specific concerns include difficulties with transfection of dendritic cells. This includes methods that target strong antigen signaling, antigen-presenting cell uptake and lymph node transduction without sacrificing biocompatibility. Carriers must deliver the genetic load specifically to the target tissue, while protecting the genetic material from metabolic and immune pathways.

DNA transfection of cells in vitro/in vivo studies requires overcoming both extracellular and intracellular barriers to gene transport from cell plasma membrane which is the barrier of intracellular DNA uptake and hinders DNA trafficking in the cytoplasm, and also into the cell nucleus that is nuclear envelope. Therefore, gene delivery methods including viral, non-viral, physical, chemical and molecular systems should facilitate DNA delivery across these barriers and into the nucleus to enable transcription without any degradation very quickly. Gene delivery systems are so important that besides the characterization of these systems by various methods such as SEM, TEM, AFM and FT-IR, the examination of their cellular uptake by various techniques like confocal complex fluorescence microscopy and flow cytometry and the development of study done in this area are extremely important in the future.

*Immune Response Activation and Immunomodulation*

images [26, 27, 100–102].

co-transfection [107, 108].

and Toxoplasma gondii, SporoSAG antigen [5].

efficiency. Cellular uptake of nucleic acid-loaded delivery systems and their localization in 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro cell culture models and also in vivo models are studied by multi-labeling 3D confocal fluorescence microscopy, flow cytometry, overlaid bright field fluorescence microscopy based on GFP expressions, luciferase assays and fluorescence

Molecular adjuvants can be defined as plasmids expressing cytokines, chemokines or co-stimulatory molecules which can be co-administered with the antigenic DNA vaccine plasmid [103] or vaccine plasmid can be constructed as a bicistronic vector system. The magnitude of immune response after DNA vaccination is very closely related to: (i) the source of Ag presentation, (ii) the immunological properties of the DNA itself and (iii) the role of cytokines in eliciting the immune responses [104]. Thus, with the cells transfected by molecular adjuvant, encoding plasmids secrete the adjuvant into the surrounding region stimulating both local antigen-presenting cells (APCs) and cells in the draining lymph node, especially dendritic cells. The examples of molecular adjuvants as cytokines: GM-CSF (granulocyte-macrophage colony stimulating factor), M-CSF, IFN-γ, IL-2, IL-4, IL-7 and IL-8, IL-10, IL-12, IL-15, IL-18; as chemokines: IL-8, MCP-1 (monocyte chemoattractant protein 1), MIP-1a (macrophage inflammatory protein), RANTES (CCL5); and as co-stimulatory proteins: CD40L, CD80/86, ICAM-1 (intercellular adhesion molecule 1) [103]. In addition, ligands of pattern recognition receptors (PRRs) are described as molecular adjuvants. There are 13 TLR genes (TLR1–TLR13). TLR3 and TLR9 recognize dsRNA and ssDNA, respectively, and their ligands have been shown to act as molecular adjuvants. Poly(I:C) is a classical TLR3 ligand, and CpG is a TLR 9 ligand, showing molecular adjuvant properties via increasing cytotoxic T-cell responses [105]. Bcl-xL anti-apoptotic protein was also described as molecular adjuvant. Inhibiting the apoptosis of antigen-presenting dendritic cells, the cytotoxic T-cell responses (CD8+ T cells) are increased due to the longer survival of the dendritic cells [106]. In addition, rather than co-transfection, expression of Bcl-xL in a bicistronic vector further enhances CD8+ T-cell responses compared to

In our previous studies, pIRESEGFP/Bcl-xL is a bicistronic vector bearing CMV (cytomegalovirus) promoter and IRES (internal ribosomal entry site) used as a backbone for DNA vaccination studies. Bcl-xL anti-apoptotic protein in frame with eGFP (enhanced green florescence protein) as a molecular adjuvant was also encoded by the plasmid. It's shown that Bcl-xL anti-apoptotic protein rescued cells from serum deprivation, doxorubicin, camptothecin and staurosporine induced apoptosis [71, 109], induced prolonged expression of the antigen of interest in expressed under CMV promoter that facilitates an increased CD8+ T cell response in DNA vaccination studies encoding foot and mouth disease multi-epitopes [4]

In this chapter, we have discussed DNA vaccines, several widely used and emerging gene delivery systems to increase efficacy of DNA vaccines, characterization of these systems and cellular uptake of DNA vaccines yet to be tested in the clinic in the future. Also, as means of molecular adjuvants, several agents are in

**3. Molecular adjuvants and Bcl-xL anti-apoptotic protein**

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**4. Conclusion**
