**2.1 Viral gene delivery systems**

Viral-based gene delivery systems are carriers on which modifications are made to transfer therapeutic genes to target cells without creating viral disease. Viral gene carriers such as adenoviruses, adeno-associated viruses, lentiviruses and retroviruses exhibit an effective ability to transfer genetic material to the target cell with high gene transfer efficiency [17–19]. In viral gene delivery systems, the transferred genetic material either remains an episomal element or is integrated into the host chromosome. Desired genetic placement leads to persistent and stable protein expression but increases with the introduction of oncogenic potential mutagenesis [18, 20]. In addition, viral vectors being targeted to specific cell types, the limited availability of DNA, and the laborious and expensive large-scale production have led to a growing disincentive for the development of non-viral gene carriers. The safety risk is lower for non-viral carriers compared to viral gene carriers [18, 21–24].

### **2.2 Non-viral gene delivery systems**

### *2.2.1 Physical delivery systems*

Physical gene transfection delivery systems are consisting of Non-viral gene delivery systems are in which pDNA is usually applied alone, which involves mechanical processes such as microinjection, biojector, pressure and particle bombardment (gene gun), ultrasound, magnetofection, photoporation (laser assisted), hydroporation (hydrodynamic forced), droplet-based microfluidic platforms for in vitro transfection and electrical processes such as electroporation [10, 11, 21, 23, 25]. In addition, ultrasound and microbubble-mediated plasmid DNA uptake is a fast, global and multi-mechanisms involved process [26, 27].

#### *2.2.1.1 Microinjection*

In microinjection, cell membrane or nuclear membrane is penetrated by simple mechanical force using a microneedle diameter of 0.5–5 μm, at a specific and reproducible depth with less physical pain than conventional DNA delivery. This gene delivery system is mainly used to inject DNA constructs in vivo. Application of DNA by this method leads to constant expression of the antigen encoded in the skin. This method can also be used to deliver DNA for a prolonged period of time, similar to the administration of drugs at a constant rate. In the method, usually the stratum corneum and the viable epidermis are breached by microneedles, after which DNA can be delivered into the dermis. There are several different microneedle methods for DNA delivery; solid microneedles can be coated with DNA prior to skin penetration, uncoated microneedles can be used to damage the epidermis prior to application of a transdermal patch containing the DNA of interest, solid microneedles constructed with biopolymers can be coated with DNA such that the needles dissolve upon contact with the fluid in the dermis to release DNA into the skin and hollow microneedles can deliver DNA into the dermis through the needles [28–30]. Quantitative introduction of multiple components into the same cell is an advantage of this technique, while technical skills are required to prevent cell damage [31].

**131**

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

The ballistic DNA delivery or DNA-coated particle bombardment (gene gun) that was first used for gene transfer to plants in 1987 uses heavy metal microparticles (e.g., gold, silver microparticles or tungsten, 1–5 μm in diameter) to hold nucleic acids and penetrate the target cells. Momentum allows penetration of these particles to a few millimeters of the tissue and then cellular DNA release. Gas pressure, particle size and dose frequency are critical factors in determining the degree of tissue damage and penetration effectiveness of the application [18, 28]. This method has various advantages such as safety, high efficiency against parenteral injection, total amount of DNA required for delivery is low, no receptor is required, size of DNA is not a problem and production of DNA-coated metal particles is easy to generate. A major disadvantage is that it induces greater immune responses than microinjection due to tissue damage with intradermal delivery, even in low doses, and also, gene expression is short term and low. This technique is a widely tested method for intramuscular, intradermal and intratumoral genetic immunization. The use of gene gun for gene therapy against various cancers in clinical trials has

This device is commonly used to deliver medications through the skin for intradermal, subcutaneous and intramuscular applications. Usually, CO2 pressure is used to force medications (e.g., vaccine) loaded in the device through a tiny orifice, which creates a high-pressure stream capable of penetrating the skin in the absence of a needle [28–30]. Biojectors have been used to deliver different kinds of vaccines such as DNA vaccine, in preclinical studies and human clinical trials to elicit significantly higher antibody responses and cell-mediated immunity (CMI) to the conventional (needle and syringe) vaccine delivery systems [29]. Because biojector-based delivery systems can increase the uptake of DNA in tissues of the skin and muscle, efficacy of the DNA vaccine is considerably increased. In a phase 1 trial for HIV vaccine, the success demonstrated with biojector used to enhance the efficiency of DNA transfection coupled with the fact that biojectors do not use needles that will most likely lead to the increased use of biojectors for DNA delivery

Electroporation was first studied on the degradation of cell membrane with electric induction in the 1960s. The first reported study is transfection of eukaryotic culture cells through electroporation in 1982 [18, 28]. In many subsequent studies, transfection was performed on animal and plant cells via electroporation [18]. This physical gene delivery technique uses electrical pulse to generate transient pores in the cell plasma membrane allowing efficient transfer of DNA into the cells. Pore formation occurs very rapidly, in approximately 10 ns. The size of the electric pore is estimated to be smaller than 10-nm radius. If the molecule is smaller than the pore size (as in oligonucleotides and chemical compounds), it can be transferred to the cell cytosol through diffusion [18, 28]. This method has been effectively applied in humans in order to enhance gene transfer and tested in several clinical trials such as prostate cancer [28, 30], leukemia [28], colorectal cancer [28], malignant melanoma [28, 30], brain carcinomas [28], Parkinson's disease [28], Alzheimer's

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

also been demonstrated [18, 28–30].

*2.2.1.2 Gen gun*

*2.2.1.3 Biojector*

in the clinic [28–30].

*2.2.1.4 Electroporation*

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

#### *2.2.1.2 Gen gun*

*Immune Response Activation and Immunomodulation*

**2. Delivery systems for DNA vaccine**

Viral-based gene delivery systems are carriers on which modifications are made to transfer therapeutic genes to target cells without creating viral disease. Viral gene carriers such as adenoviruses, adeno-associated viruses, lentiviruses and retroviruses exhibit an effective ability to transfer genetic material to the target cell with high gene transfer efficiency [17–19]. In viral gene delivery systems, the transferred genetic material either remains an episomal element or is integrated into the host chromosome. Desired genetic placement leads to persistent and stable protein expression but increases with the introduction of oncogenic potential mutagenesis [18, 20]. In addition, viral vectors being targeted to specific cell types, the limited availability of DNA, and the laborious and expensive large-scale production have led to a growing disincentive for the development of non-viral gene carriers. The safety risk is lower for non-viral carriers compared to viral gene

Physical gene transfection delivery systems are consisting of Non-viral gene delivery systems are in which pDNA is usually applied alone, which involves mechanical processes such as microinjection, biojector, pressure and particle bombardment (gene gun), ultrasound, magnetofection, photoporation (laser assisted), hydroporation (hydrodynamic forced), droplet-based microfluidic platforms for in vitro transfection and electrical processes such as electroporation [10, 11, 21, 23, 25]. In addition, ultrasound and microbubble-mediated plasmid DNA uptake is a

In microinjection, cell membrane or nuclear membrane is penetrated by simple

mechanical force using a microneedle diameter of 0.5–5 μm, at a specific and reproducible depth with less physical pain than conventional DNA delivery. This gene delivery system is mainly used to inject DNA constructs in vivo. Application of DNA by this method leads to constant expression of the antigen encoded in the skin. This method can also be used to deliver DNA for a prolonged period of time, similar to the administration of drugs at a constant rate. In the method, usually the stratum corneum and the viable epidermis are breached by microneedles, after which DNA can be delivered into the dermis. There are several different microneedle methods for DNA delivery; solid microneedles can be coated with DNA prior to skin penetration, uncoated microneedles can be used to damage the epidermis prior to application of a transdermal patch containing the DNA of interest, solid microneedles constructed with biopolymers can be coated with DNA such that the needles dissolve upon contact with the fluid in the dermis to release DNA into the skin and hollow microneedles can deliver DNA into the dermis through the needles [28–30]. Quantitative introduction of multiple components into the same cell is an advantage of this technique, while technical skills are required to prevent cell

fast, global and multi-mechanisms involved process [26, 27].

**2.1 Viral gene delivery systems**

carriers [18, 21–24].

*2.2.1.1 Microinjection*

**2.2 Non-viral gene delivery systems**

*2.2.1 Physical delivery systems*

**130**

damage [31].

The ballistic DNA delivery or DNA-coated particle bombardment (gene gun) that was first used for gene transfer to plants in 1987 uses heavy metal microparticles (e.g., gold, silver microparticles or tungsten, 1–5 μm in diameter) to hold nucleic acids and penetrate the target cells. Momentum allows penetration of these particles to a few millimeters of the tissue and then cellular DNA release. Gas pressure, particle size and dose frequency are critical factors in determining the degree of tissue damage and penetration effectiveness of the application [18, 28]. This method has various advantages such as safety, high efficiency against parenteral injection, total amount of DNA required for delivery is low, no receptor is required, size of DNA is not a problem and production of DNA-coated metal particles is easy to generate. A major disadvantage is that it induces greater immune responses than microinjection due to tissue damage with intradermal delivery, even in low doses, and also, gene expression is short term and low. This technique is a widely tested method for intramuscular, intradermal and intratumoral genetic immunization. The use of gene gun for gene therapy against various cancers in clinical trials has also been demonstrated [18, 28–30].

## *2.2.1.3 Biojector*

This device is commonly used to deliver medications through the skin for intradermal, subcutaneous and intramuscular applications. Usually, CO2 pressure is used to force medications (e.g., vaccine) loaded in the device through a tiny orifice, which creates a high-pressure stream capable of penetrating the skin in the absence of a needle [28–30]. Biojectors have been used to deliver different kinds of vaccines such as DNA vaccine, in preclinical studies and human clinical trials to elicit significantly higher antibody responses and cell-mediated immunity (CMI) to the conventional (needle and syringe) vaccine delivery systems [29]. Because biojector-based delivery systems can increase the uptake of DNA in tissues of the skin and muscle, efficacy of the DNA vaccine is considerably increased. In a phase 1 trial for HIV vaccine, the success demonstrated with biojector used to enhance the efficiency of DNA transfection coupled with the fact that biojectors do not use needles that will most likely lead to the increased use of biojectors for DNA delivery in the clinic [28–30].

## *2.2.1.4 Electroporation*

Electroporation was first studied on the degradation of cell membrane with electric induction in the 1960s. The first reported study is transfection of eukaryotic culture cells through electroporation in 1982 [18, 28]. In many subsequent studies, transfection was performed on animal and plant cells via electroporation [18]. This physical gene delivery technique uses electrical pulse to generate transient pores in the cell plasma membrane allowing efficient transfer of DNA into the cells. Pore formation occurs very rapidly, in approximately 10 ns. The size of the electric pore is estimated to be smaller than 10-nm radius. If the molecule is smaller than the pore size (as in oligonucleotides and chemical compounds), it can be transferred to the cell cytosol through diffusion [18, 28]. This method has been effectively applied in humans in order to enhance gene transfer and tested in several clinical trials such as prostate cancer [28, 30], leukemia [28], colorectal cancer [28], malignant melanoma [28, 30], brain carcinomas [28], Parkinson's disease [28], Alzheimer's

disease [28] and depression [28]. When the parameters are optimized, this method is equally effective as viral vectors for in vivo application. But, the disadvantage is that it often results in a high incidence of cell death because of high temperature due to high voltage application. And also, transfection of the cells in large regions of the tissues is difficult [18, 28–30].

### *2.2.1.5 Ultrasound*

Ultrasound (US) is a promising tool for gene delivery that has been able to facilitate DNA transfection of cells. US-mediated delivery is of interest due to its potential for repeated application, organ specificity, broad applicability to acoustically accessible organs, low toxicity and low immunogenicity. Different kinds of studies have examined gene transfection in various types of cells in vitro and with various organs and tissues in vivo, including brain [26, 30], cornea [30], pancreas [30], skeletal muscle [26, 30], liver [26, 30], heart [26, 30] and kidney [26, 30]. The advantages are that only acoustic energy is introduced into the cellular environment, which avoids possible safety concerns associated with chemical, viral or other materials introduced and left behind by other methods. Also, US-mediated delivery has been seen in many kinds of cell types and so may be broadly applicable, in contrast to other methods that often require reformulation for specific cell types. Unlike chemical delivery systems, US-mediated DNA uptake is often shown to be non-endocytotic. Acoustic cavitation plays a major role in the cell membrane permeabilization that facilitates DNA uptake. US can possibly deliver plasmid DNA to the periphery of the cell nucleus and facilitate rapid transfection by altering the cytoskeletal network. However, US-based gene transfection studies are still in preclinical trials and have the major challenge of relatively low transfection efficiency compared to optimal complexed chemical formulations and viral gene delivery systems [26, 28, 30].

The use of ultrasonication with the microbubble technique has shown great potential for intracellular gene delivery. The microbubble-cell membrane interaction serves as the key element bridging the acoustic conditions and the endpoint delivery outcomes. However, since the fundamental mechanical question of how plasmid DNA enters the intracellular space mediated by ultrasound and microbubbles is not fully understood, gene transfection efficiency is much lower than the potential for large-scale clinical needs [26, 27]. In one study, the gene transfection of human prostate cancer cell line (DU145) with fluorescently labeled DNA (pDNA gWiz-GFP) was studied after ultrasound exposure. In this process, different sonication conditions have been studied. DNA uptake, location of DNA during its intracellular trafficking and gene transfection efficiency after ultrasound exposure were followed for various periods by confocal microscopy and flow cytometry. As a result, ultrasounds delivered DNA into cell nuclei shortly after sonication and that the rest of the DNA cleared by autophagosomes/autophagolysosomes [26]. Also, ultrasound application combined with microbubbles has shown good potential for gene delivery. In one study, to unveil the detailed intracellular uptake process of plasmid DNA stimulated by ultrasound and microbubbles, the role of microbubbles in this process was investigated. So, targeted microbubbles were used to apply intracellular local stimulation on the cell membrane, and high-speed video microscopic recordings of microbubble dynamics were correlated with post-ultrasound 3D fluorescent confocal microscopic images fixed immediately after the cell. Two ultrasound conditions (high pressure, short pulse and low pressure, long pulse) were chosen to trigger different plasmid DNA uptake routes. Results showed that plasmid DNA uptake evoked by localized acoustically excited microbubbles was a fast (<2 min), global (not limited to the site where microbubbles were attached) and multi-mechanisms involved process [27].

**133**

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

The term adjuvant is derived from the Latin word "adjuvare" which means "help" or "develop." Adjuvants are used to increase the life, quality and degree of the specific immune response developed against the antigens. At the same time, they have low toxicity and are capable of sustaining the immunological activity alone by inhibiting polymer accumulation in the cell [11, 32, 33]. So, they are preferred in vaccinations for newborns or adults. Also, they can stimulate a long-term immune response by reducing the amount of antigen that must be given in a single dose of

Adjuvants are classified according to the source of their constituents, their physicochemical properties or their mechanism of action and are generally grouped into two subheadings. One of them is molecular adjuvants that are immunostimulants (and also genetic adjuvants) (e.g., TLR ligands, cytokines, saponins and bacterial exotoxins that stimulate the immune response) and act directly on the immune system to enhance immune response against antigens. The other one is carrier systems; they are systems that promote vaccine antigens in the most appropriate way to the immune system while also exhibiting controlled release and depot effects, thereby increasing the immune response (e.g., mineral salts, emulsions, liposomes, virosomes, biodegradable polymer micro/nano particles and immune

Cytokines can also be delivered directly with the DNA vaccine, either on the same or on a separate expression plasmid as adjuvant duty. The effects of plasmid encoding cytokines such as interleukin IL-10, IL-12 or IFN-γ together with DNA vaccines have been studied in a variety of animal and disease models, up to clinical trials in humans [38]. Also, various studies describe the usage of plasmids coding for immune-signaling molecules, either as partial or as full-length genes. Many adjuvants function by activating the innate immune system via binding to Toll-like receptors (TLRs). Another innate immune mechanism, which is being explored for improving DNA vaccination, is the sensing of viral infections via pathogen recognition receptors (PRRs). Both proteins detect the presence of viral RNA in the cytosol. Co-delivery adjuvants with the vector coding for antigenic proteins result in significantly higher antibody titers as compared to the non-adjuvanted controls. Other strategies for genetic adjuvants include components of the complement system, protein aggregation domains, chemokines or co-stimulatory molecules. Whereas DNA vectors encoding certain cytokines have already entered clinical testing in humans, studies with many other genetic adjuvants were mostly performed in mice. Therefore, these promising studies should be optimized into powerful strategies to boost DNA vaccines in more complicated animals and humans [38]. Adjuvants can easily be internalized by the antigen-presenting cells (APCs) (macrophages and dendritic cells) because of their size being similar to pathogens (<10 μm) [11, 22, 25, 40]. The internalization and presentation of the adjuvant depends on the chemical and physical properties of the adjuvant system. It has been shown in various studies that the particles with cationic properties are more efficiently taken up by macrophages and dendritic cells [21, 24, 25, 41–43].

The characteristics that should be present in the adjuvants can be listed as follows: stability in an acidic, basic and enzymatic environment; retention of retained antigen or nucleic acid by constant release; systemic and mucosal immunity being effectively induced and balance between effective immunity and immunological tolerance at high doses [9, 44]. Also, the immunogenicity of weak antigens should increase the speed and duration of the immune response, cause only minimal local and systemic side effects, and be capable of a wide variety of vaccination with stability and ease of production. The ability to be effective in the living system of

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

stimulating complexes—ISCOMs) [1, 9, 11, 34–39].

*2.2.2 Chemical adjuvant systems*

vaccine [9, 34].

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

### *2.2.2 Chemical adjuvant systems*

*Immune Response Activation and Immunomodulation*

tissues is difficult [18, 28–30].

*2.2.1.5 Ultrasound*

disease [28] and depression [28]. When the parameters are optimized, this method is equally effective as viral vectors for in vivo application. But, the disadvantage is that it often results in a high incidence of cell death because of high temperature due to high voltage application. And also, transfection of the cells in large regions of the

Ultrasound (US) is a promising tool for gene delivery that has been able to facilitate DNA transfection of cells. US-mediated delivery is of interest due to its potential for repeated application, organ specificity, broad applicability to acoustically accessible organs, low toxicity and low immunogenicity. Different kinds of studies have examined gene transfection in various types of cells in vitro and with various organs and tissues in vivo, including brain [26, 30], cornea [30], pancreas [30], skeletal muscle [26, 30], liver [26, 30], heart [26, 30] and kidney [26, 30]. The advantages are that only acoustic energy is introduced into the cellular environment, which avoids possible safety concerns associated with chemical, viral or other materials introduced and left behind by other methods. Also, US-mediated delivery has been seen in many kinds of cell types and so may be broadly applicable, in contrast to other methods that often require reformulation for specific cell types. Unlike chemical delivery systems, US-mediated DNA uptake is often shown to be non-endocytotic. Acoustic cavitation plays a major role in the cell membrane permeabilization that facilitates DNA uptake. US can possibly deliver plasmid DNA to the periphery of the cell nucleus and facilitate rapid transfection by altering the cytoskeletal network. However, US-based gene transfection studies are still in preclinical trials and have the major challenge of relatively low transfection efficiency compared to optimal complexed chemical formulations and viral gene delivery systems [26, 28, 30]. The use of ultrasonication with the microbubble technique has shown great potential for intracellular gene delivery. The microbubble-cell membrane interaction serves as the key element bridging the acoustic conditions and the endpoint delivery outcomes. However, since the fundamental mechanical question of how plasmid DNA enters the intracellular space mediated by ultrasound and microbubbles is not fully understood, gene transfection efficiency is much lower than the potential for large-scale clinical needs [26, 27]. In one study, the gene transfection of human prostate cancer cell line (DU145) with fluorescently labeled DNA (pDNA gWiz-GFP) was studied after ultrasound exposure. In this process, different sonication conditions have been studied. DNA uptake, location of DNA during its intracellular trafficking and gene transfection efficiency after ultrasound exposure were followed for various periods by confocal microscopy and flow cytometry. As a result, ultrasounds delivered DNA into cell nuclei shortly after sonication and that the rest of the DNA cleared by autophagosomes/autophagolysosomes [26]. Also, ultrasound application combined with microbubbles has shown good potential for gene delivery. In one study, to unveil the detailed intracellular uptake process of plasmid DNA stimulated by ultrasound and microbubbles, the role of microbubbles in this process was investigated. So, targeted microbubbles were used to apply intracellular local stimulation on the cell membrane, and high-speed video microscopic recordings of microbubble dynamics were correlated with post-ultrasound 3D fluorescent confocal microscopic images fixed immediately after the cell. Two ultrasound conditions (high pressure, short pulse and low pressure, long pulse) were chosen to trigger different plasmid DNA uptake routes. Results showed that plasmid DNA uptake evoked by localized acoustically excited microbubbles was a fast (<2 min), global (not limited to the site where microbubbles were attached)

**132**

and multi-mechanisms involved process [27].

The term adjuvant is derived from the Latin word "adjuvare" which means "help" or "develop." Adjuvants are used to increase the life, quality and degree of the specific immune response developed against the antigens. At the same time, they have low toxicity and are capable of sustaining the immunological activity alone by inhibiting polymer accumulation in the cell [11, 32, 33]. So, they are preferred in vaccinations for newborns or adults. Also, they can stimulate a long-term immune response by reducing the amount of antigen that must be given in a single dose of vaccine [9, 34].

Adjuvants are classified according to the source of their constituents, their physicochemical properties or their mechanism of action and are generally grouped into two subheadings. One of them is molecular adjuvants that are immunostimulants (and also genetic adjuvants) (e.g., TLR ligands, cytokines, saponins and bacterial exotoxins that stimulate the immune response) and act directly on the immune system to enhance immune response against antigens. The other one is carrier systems; they are systems that promote vaccine antigens in the most appropriate way to the immune system while also exhibiting controlled release and depot effects, thereby increasing the immune response (e.g., mineral salts, emulsions, liposomes, virosomes, biodegradable polymer micro/nano particles and immune stimulating complexes—ISCOMs) [1, 9, 11, 34–39].

Cytokines can also be delivered directly with the DNA vaccine, either on the same or on a separate expression plasmid as adjuvant duty. The effects of plasmid encoding cytokines such as interleukin IL-10, IL-12 or IFN-γ together with DNA vaccines have been studied in a variety of animal and disease models, up to clinical trials in humans [38]. Also, various studies describe the usage of plasmids coding for immune-signaling molecules, either as partial or as full-length genes. Many adjuvants function by activating the innate immune system via binding to Toll-like receptors (TLRs). Another innate immune mechanism, which is being explored for improving DNA vaccination, is the sensing of viral infections via pathogen recognition receptors (PRRs). Both proteins detect the presence of viral RNA in the cytosol. Co-delivery adjuvants with the vector coding for antigenic proteins result in significantly higher antibody titers as compared to the non-adjuvanted controls. Other strategies for genetic adjuvants include components of the complement system, protein aggregation domains, chemokines or co-stimulatory molecules. Whereas DNA vectors encoding certain cytokines have already entered clinical testing in humans, studies with many other genetic adjuvants were mostly performed in mice. Therefore, these promising studies should be optimized into powerful strategies to boost DNA vaccines in more complicated animals and humans [38].

Adjuvants can easily be internalized by the antigen-presenting cells (APCs) (macrophages and dendritic cells) because of their size being similar to pathogens (<10 μm) [11, 22, 25, 40]. The internalization and presentation of the adjuvant depends on the chemical and physical properties of the adjuvant system. It has been shown in various studies that the particles with cationic properties are more efficiently taken up by macrophages and dendritic cells [21, 24, 25, 41–43].

The characteristics that should be present in the adjuvants can be listed as follows: stability in an acidic, basic and enzymatic environment; retention of retained antigen or nucleic acid by constant release; systemic and mucosal immunity being effectively induced and balance between effective immunity and immunological tolerance at high doses [9, 44]. Also, the immunogenicity of weak antigens should increase the speed and duration of the immune response, cause only minimal local and systemic side effects, and be capable of a wide variety of vaccination with stability and ease of production. The ability to be effective in the living system of

adjuvants depends on its ability to stimulate antigen-presenting cells (dendritic cells and macrophages) and T and B lymphocytes of the natural defense system [11]. There is a need for definitive information on the structure, stability, safety and immunogenicity of the adjuvant to be used in an effective vaccine development process [32, 33]. For this purpose, the choice of adjuvant depends on factors such as antigenic structure, immunization scheme, mode of administration and desired immune response pattern [33].
