**2.5.1 Genetic adjuvants**

Genetic adjuvants are molecules such as cytokines, chemokines and co-stimulatory factors that may be cloned into the DNA plasmid vaccine and expressed *in vivo*. These adjuvants can be encoded on the same vector expressing the antigen or inserted into a separate vector and co-injected with the vaccine. This method provides adjuvant activity at the site of antigen production, with lasting effect from transfected cells. Among various factors, cytokines are highly preferred as genetic adjuvants because they act on cells involved in the host defense and can be used to modulate immune responses. Co-delivery of cytokines in DNA vaccine formulations has been used extensively for a wide range of infectious diseases such as malaria, leishmania, schistosoma to enhance the T cell mediated responses (Ivory and Chadee 2004). One of the earliest cytokines to be incorporated into a DNA vaccine was IL-2, a well known T-cell growth factor included in several immunotherapy protocols. Addition of this cytokine to a plasmid vaccination vector resulted in enhanced antibody responses in low responder mice against malaria (Good et al. 1988) and increased production of antibodies directed against the complementary determining hypervariable region 3 of the Ig heavy chain in human B-cell lymphoma (Rinaldi et al. 2001). IL-2

epitopes can be presented to T cells by professional APCs. However, recent advances in the immunological research suggest that most, if not all, adjuvants enhance T and B cell responses by engaging components of the innate immune system, rather than by exerting direct effects on the lymphocytes. In DNA vaccinations, adjuvants are used to achieve qualitative alteration of the immune response. Adjuvants confer to DNA or to subunit vaccines the ability to promote an immune response which might not occur in their absence. Here we describe certain classes of adjuvants most widely used in DNA vaccinations. A

**Class of adjuvants Adjuvant name Nature of the adjuvant** 

Cytokine Cytokine

Cytokine

Peptides

Lipid derivative Oil in water emulsion Liposomal formulation Saponin Oligodeoxynucleotides

> Mineral salt Mineral salt

Interleukin-2 Interleukin-12 Granulocyte Monocyte-Colony Stimulating Factor

T-helper epitopes of toxins

Monophosphoryl Lipid A AS02 AS01 QS-21 CpG-DNA

Aluminium Phosphate Aluminium Hydroxide

Genetic adjuvants are molecules such as cytokines, chemokines and co-stimulatory factors that may be cloned into the DNA plasmid vaccine and expressed *in vivo*. These adjuvants can be encoded on the same vector expressing the antigen or inserted into a separate vector and co-injected with the vaccine. This method provides adjuvant activity at the site of antigen production, with lasting effect from transfected cells. Among various factors, cytokines are highly preferred as genetic adjuvants because they act on cells involved in the host defense and can be used to modulate immune responses. Co-delivery of cytokines in DNA vaccine formulations has been used extensively for a wide range of infectious diseases such as malaria, leishmania, schistosoma to enhance the T cell mediated responses (Ivory and Chadee 2004). One of the earliest cytokines to be incorporated into a DNA vaccine was IL-2, a well known T-cell growth factor included in several immunotherapy protocols. Addition of this cytokine to a plasmid vaccination vector resulted in enhanced antibody responses in low responder mice against malaria (Good et al. 1988) and increased production of antibodies directed against the complementary determining hypervariable region 3 of the Ig heavy chain in human B-cell lymphoma (Rinaldi et al. 2001). IL-2

summary of the adjuvants described bellow is presented in **Table 2.**

**Genetic adjuvants** 

**Adjuvants targeting Pattern Recognition Receptors** 

**Aluminium-based compounds** 

**2.5.1 Genetic adjuvants** 

Table 2. Adjuvants used in vaccination with naked DNA.

contributed to increase the efficacy of a DNA vaccine against a simian immunodeficiency virus when it was fused with the immunoglobulin Fc fragment, resulting in augmentation of the cytokine half-life (Barouch et al. 2000). However, the use of IL-2 is now being limited by the emerging evidence that this cytokine can play a major role in maintaining self-tolerance and in supporting survival of CD25+ CD4+ regulatory T cells (T-regs) (Bayer et al. 2005; Setoguchi et al. 2005).

IL-12 is another cytokine used in DNA vaccination. It acts on T and NK cells by inducing the generation of CTLs through T-helper 1 cell activation and IFN-γ production. The beneficial effect of IL-12 in pre-clinical experimental tumour models suggested the possibility of using IL-12 as an anti-tumour agent in clinical trials. Despite some toxicity associated with certain doses of IL-12 when administered as a drug in patients affected by melanoma and colon cancer, some clinical responses were observed; this indicates that IL-12 can be used in clinical protocols of cancer therapy where a toxic effect of the cytokine could be acceptable (Atkins et al. 1997; Gollob et al. 2003). Granulocyte/macrophage colony-stimulatory factor (GM-CSF) is probably the most attractive adjuvant for DNA vaccines for its ability to recruit antigen-presenting cells to the site where antigen synthesis occurs as well as for its capacity to stimulate DC maturation. Plasmid DNA vaccines were constructed fusing GM-CSF to the S antigen of Hepatitis B Virus (HBV) to vaccinate HBV-transgenic mice. This fusion construct worked well in conferring protection from the HBV to both normal and transgenic mice (Qing et al. 2010). In another study the utility of GM-CSF as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) was evaluated in the vaccination of a murine model. Mouse co-inoculation with a vector expressing GM-CSF enhanced the protective immunity against PrV infection. This immunity was caused by the induction of increased humoural and cellular immunity in response to PrV antigen (Yoon et al. 2006). A DNA vaccine encoding the GM-CSF gene and a DNA vaccine encoding the H1N1 influenza (A/New Caledonia/20/99) HA antigen were co-administered by particle-mediated epidermal delivery in Rhesus Macaques. After three immunizations the DNA vaccines were shown to significantly enhance both the systemic and mucosal immunogenicity of the HA influenza vaccine (Loudon et al. 2010).

Among genetic adjuvants the pathogen-derived immune-enhancing proteins are noteworthy for their ability to stimulate the immune system when they are fused with target antigens. Modified bacterial toxins, such as anthrax, diphteria and pertussis toxins, are being used in vaccination as effective carriers to deliver foreign epitopes which stimulate protective CTL responses in mammalian cells (Ballard et al. 1996; Carbonetti et al. 1999). However, the ability of modified toxins to activate the host immune system does not reside only in the delivery effect exerted on the fused antigen (Stevenson et al. 2004).

The tetanus toxin Fragment C (FrC) is one of the widely used genetic adjuvant as a fusion partner for foreign antigens. This protein was found to increase the immunogenicity of the Schistosoma mansoni glutathione S-transferase antigen when administered as genetic fusion in a live Aro-attenuated vaccine strain of Salmonella (Khan et al. 1994b) and similar results were obtained when a vaccine construct consisting of a portion of P28 glutathione Stransferase was administering intravenously as C-terminal fusion to tetanus toxin FrC in a live Aro-attenuated vaccine strain of Salmonella (Khan et al. 1994a). In cancer vaccination, a domain of the tetanus toxin FrC fused to a single antigenic determinant was demonstrated able to induce an anti-tumoural CTL mediated response in vaccinated mice (Rice et al. 2002). Likewise, in vaccination against B cell lymphoma, DNA vaccines containing the idiotypic determinants of the Ig variable region provided protective immunity against the tumour

DNA Vaccination by Electrogene Transfer 181

lipopolysaccharide (LPS), CpG-containing oligonucleotides (CpG), and peptidoglycans. LPS is a Gram-negative membrane molecule consisting of a hydrophilic polysaccharide and a lipophilic phospholipid (lipid A). The lipophilic portion of LPS is such a potent stimulus on the pro-inflammatory cytokine production that it can lead to septic shock (Heine et al. 2001) whereas monophosphoryl lipid A (MPL) is a lipid A derivative included in many adjuvant formulations with good adjuvanticity and lower toxicity (Ismaili et al. 2002). MPL was the first TLR ligand approved for human use in the hepatitis B vaccine, Fendrix (GlaxoSmithKline Biologicals, Rixensart, Belgium) (Baldridge and Crane 1999). The adjuvanticity of these adjuvant molecules depends on their ability to bind and activate the TLR4. As a result, many TLR agonists or lipid A mimetics displaying TLR4-dependent immunostimulating functions, have been synthesized and proposed as new adjuvants (Johnson et al. 1999). AS02 is an oil-in-water emulsion containing MPL and QS-21, a saponin-derived immunostimulator, induces strong antibody and Th1 responses. AS02 is being evaluated in clinical trials in vaccines against malaria, human papillomavirus (HPV), HBV, tuberculosis, and HIV (Vandepapeliere et al. 2007). Smilarly to AS02, AS01 is a liposomal formulation containing MPL that induces potent humoural and cell-mediated responses, including cytotoxic T lymphocyte responses, and is being evaluated in clinical trials of vaccine against

In bacterial DNA there is a high frequency of unmethylated CpG dinucleotide sequences in comparison to the human genome which makes perception of plasmids as "foreign" elements by the human host. This is the only characteristic that confers a certain level of immunogenicity to plasmid DNA vaccines since the CpG unmethylated sequence is recognized by the Toll Like Receptor 9 preferentially expressed on the host APCs (Liu and Ulmer 2005). CpG-DNA is used as immunostimulatory potentiator in both pre-clinical peptide and DNA vaccination trials leading to activation of innate immunity and cytokine-dependent promotion of the Th-1 response. The CpG motifs, present in bacterial DNA, consist of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines. During an infection, the release of unmethylated CpG-DNA from bacteria serves as a danger signal stimulating the immune system of the host (Krieg 2002). Bacterial DNA and synthetic unmethylated CpG oligodeoxynucleotides trigger an immunostimulatory cascade that culminates in maturation, differentiation and proliferation of several immune cells creating a pro-inflammatory and Th1-biased environment. The immunostimulatory activity of this CpG-DNA is species-specific. As a result, sequences specific for the host are designed and optimized for selective TLR9 binding. In particular, *in vivo* CpG-DNA half-life has been improved by replacing phosphodiester CpG-DNA with a nuclease-resistant phosphothioate oligodeoxynucleotide although it may induce immune reaction leading to an anti-DNA immune response (Ciafre et al. 1995). Addition of the immunostimulatory CpG-DNA to peptide vaccines, tilts the balance towards a Th-1 immune response, which is often accompanied by a significant increase in IgG2a production in comparison to IgG1. The general effect of CpG-DNA addition is augmentation of the antibody serum titer against antigens and production of Th-1 cytokines such as IFN-γ (Klinman et al. 1999). By contrast, in DNA plasmid vaccination, the immunomodulating effect of bacterial CpG oligodeoxynucleotides on T-helper cell balance, shows great variability. Such variability

malaria.

often depends on the route of immunisation.

when expressed as single-chain variable fragment (sc-Fv) fused to tetanus toxin FrC (King et al. 1998). Reproducible data from several published papers, show that the high immunogenicity of the tetanus toxin FrC depends on two main attributes; 1) a conformational sequence-dependent effect ; 2) the presence of promiscuous T-helper epitopes within the protein (Umland et al. 1997). Our group has analysed the sequence of the tetanus toxin FrC, and has identified numerous T-helper epitopes in the protein domain: 1) the universal p30 T-helper epitope (FNNFTVSFWLRVPKVSASHLE aa 947-967), a strong promiscuous immunogenic T-helper epitope consisting of at least three distinct overlapping helper peptides, each of which is presented in association with multiple HLA class II alleles [42]; 2) the p21TT helper epitope (IREDNNITLKLDRCNN aa 1064-1079); 3) the p23TT epitope (VSIDKFRIFCKALNPK aa 1084-1099), 4) the pGINGKA epitope (PGINGKAIHLVNNESSE aa 916-932) [47]; 5) the p32TT epitope (LKFIIKRYTPNNEID aa 1173-1188); 6) the pGQI epitope (GQIGNDPNRDIL aa 1273-1284 (Chiarella et al. 2007). Since CD4+ T-helper cells support both cell and humoural immunity, it seems that the antigen fusion to promiscuous T-helper peptides contributes to the activation of these lymphocytes. The result is enhancement of the immune response mediated by T-helper cells and this might explain the strong potency of the tetanus toxin FrC domain as vaccine adjuvant. Specific T-cell epitopes of FrC that are universally immunogenic, have also been widely exploited in peptide vaccination as they have been demonstrated to enhance the humoural immune response. In particular, T-helper epitopes were successfully used as vaccine carriers to induce humoural response against polysaccharide antigens when used in form of string-of-beads (Baraldo et al. 2005). This approach is based on the concept that response to the subset of antigens and epitopes, and not to the whole organism, can be sufficient for host protection. Furthermore, the availability of bioinformatic tools and softwares for prediction of the antigen binding to the human MHC molecules helps in the design of DNA vaccines. In multi-epitope vaccination, more than one CTL epitope belonging to a certain antigen of a specific disease can be linked to a series of promiscuous MHC-II binding T-helper epitope, to generate a string-of-beads vaccine, with or without intervening spacers. In several reports, vaccination with DNA constructs consisting of a single promiscuous T-helper epitope fused to antigen determinants has proved to be effective in stimulating a strong immune response when weakly immunogenic CTL epitopes are either co-injected or chemically linked to the T-helper sequence (Tymciu et al. 2004). Synthetic universal pan HLA-DR-binding T helper epitopes such as the PADRE were conceived and they were successfully used in making DNA vaccines against infectious diseases. PADRE is a synthetic universal peptide that binds to the more common HLA-DR molecules of the human population. Its efficacy in increasing immunogenicity of CTL and B epitopes was demonstrated to be higher (Alexander et al. 1994). The sequence of PADRE has been deduced from the core sequence of the ovalbumin master T-helper peptide (aa 323-339) and adapted for binding to the more representative human MHC class II molecules (del Guercio et al. 1997).

#### **2.5.2 Adjuvants targeting pattern recognition receptors**

Traditional vaccines based on live attenuated pathogens and inactivated whole pathogens have been extremely successful in preventing many common infectious diseases. The potent immunogenicity of such vaccines depends on the presence of "endogenous adjuvants" which are simply molecular portions of the pathogenic agent. *i.e.* now defined with the name of Pathogen Associated Molecular Pattern (PAMPs). These are

when expressed as single-chain variable fragment (sc-Fv) fused to tetanus toxin FrC (King et al. 1998). Reproducible data from several published papers, show that the high immunogenicity of the tetanus toxin FrC depends on two main attributes; 1) a conformational sequence-dependent effect ; 2) the presence of promiscuous T-helper epitopes within the protein (Umland et al. 1997). Our group has analysed the sequence of the tetanus toxin FrC, and has identified numerous T-helper epitopes in the protein domain: 1) the universal p30 T-helper epitope (FNNFTVSFWLRVPKVSASHLE aa 947-967), a strong promiscuous immunogenic T-helper epitope consisting of at least three distinct overlapping helper peptides, each of which is presented in association with multiple HLA class II alleles [42]; 2) the p21TT helper epitope (IREDNNITLKLDRCNN aa 1064-1079); 3) the p23TT epitope (VSIDKFRIFCKALNPK aa 1084-1099), 4) the pGINGKA epitope (PGINGKAIHLVNNESSE aa 916-932) [47]; 5) the p32TT epitope (LKFIIKRYTPNNEID aa 1173-1188); 6) the pGQI epitope (GQIGNDPNRDIL aa 1273-1284 (Chiarella et al. 2007). Since CD4+ T-helper cells support both cell and humoural immunity, it seems that the antigen fusion to promiscuous T-helper peptides contributes to the activation of these lymphocytes. The result is enhancement of the immune response mediated by T-helper cells and this might explain the strong potency of the tetanus toxin FrC domain as vaccine adjuvant. Specific T-cell epitopes of FrC that are universally immunogenic, have also been widely exploited in peptide vaccination as they have been demonstrated to enhance the humoural immune response. In particular, T-helper epitopes were successfully used as vaccine carriers to induce humoural response against polysaccharide antigens when used in form of string-of-beads (Baraldo et al. 2005). This approach is based on the concept that response to the subset of antigens and epitopes, and not to the whole organism, can be sufficient for host protection. Furthermore, the availability of bioinformatic tools and softwares for prediction of the antigen binding to the human MHC molecules helps in the design of DNA vaccines. In multi-epitope vaccination, more than one CTL epitope belonging to a certain antigen of a specific disease can be linked to a series of promiscuous MHC-II binding T-helper epitope, to generate a string-of-beads vaccine, with or without intervening spacers. In several reports, vaccination with DNA constructs consisting of a single promiscuous T-helper epitope fused to antigen determinants has proved to be effective in stimulating a strong immune response when weakly immunogenic CTL epitopes are either co-injected or chemically linked to the T-helper sequence (Tymciu et al. 2004). Synthetic universal pan HLA-DR-binding T helper epitopes such as the PADRE were conceived and they were successfully used in making DNA vaccines against infectious diseases. PADRE is a synthetic universal peptide that binds to the more common HLA-DR molecules of the human population. Its efficacy in increasing immunogenicity of CTL and B epitopes was demonstrated to be higher (Alexander et al. 1994). The sequence of PADRE has been deduced from the core sequence of the ovalbumin master T-helper peptide (aa 323-339) and adapted for binding to the more representative human MHC class II molecules (del

Guercio et al. 1997).

**2.5.2 Adjuvants targeting pattern recognition receptors** 

Traditional vaccines based on live attenuated pathogens and inactivated whole pathogens have been extremely successful in preventing many common infectious diseases. The potent immunogenicity of such vaccines depends on the presence of "endogenous adjuvants" which are simply molecular portions of the pathogenic agent. *i.e.* now defined with the name of Pathogen Associated Molecular Pattern (PAMPs). These are lipopolysaccharide (LPS), CpG-containing oligonucleotides (CpG), and peptidoglycans. LPS is a Gram-negative membrane molecule consisting of a hydrophilic polysaccharide and a lipophilic phospholipid (lipid A). The lipophilic portion of LPS is such a potent stimulus on the pro-inflammatory cytokine production that it can lead to septic shock (Heine et al. 2001) whereas monophosphoryl lipid A (MPL) is a lipid A derivative included in many adjuvant formulations with good adjuvanticity and lower toxicity (Ismaili et al. 2002). MPL was the first TLR ligand approved for human use in the hepatitis B vaccine, Fendrix (GlaxoSmithKline Biologicals, Rixensart, Belgium) (Baldridge and Crane 1999). The adjuvanticity of these adjuvant molecules depends on their ability to bind and activate the TLR4. As a result, many TLR agonists or lipid A mimetics displaying TLR4-dependent immunostimulating functions, have been synthesized and proposed as new adjuvants (Johnson et al. 1999). AS02 is an oil-in-water emulsion containing MPL and QS-21, a saponin-derived immunostimulator, induces strong antibody and Th1 responses. AS02 is being evaluated in clinical trials in vaccines against malaria, human papillomavirus (HPV), HBV, tuberculosis, and HIV (Vandepapeliere et al. 2007). Smilarly to AS02, AS01 is a liposomal formulation containing MPL that induces potent humoural and cell-mediated responses, including cytotoxic T lymphocyte responses, and is being evaluated in clinical trials of vaccine against malaria.

In bacterial DNA there is a high frequency of unmethylated CpG dinucleotide sequences in comparison to the human genome which makes perception of plasmids as "foreign" elements by the human host. This is the only characteristic that confers a certain level of immunogenicity to plasmid DNA vaccines since the CpG unmethylated sequence is recognized by the Toll Like Receptor 9 preferentially expressed on the host APCs (Liu and Ulmer 2005). CpG-DNA is used as immunostimulatory potentiator in both pre-clinical peptide and DNA vaccination trials leading to activation of innate immunity and cytokine-dependent promotion of the Th-1 response. The CpG motifs, present in bacterial DNA, consist of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines. During an infection, the release of unmethylated CpG-DNA from bacteria serves as a danger signal stimulating the immune system of the host (Krieg 2002). Bacterial DNA and synthetic unmethylated CpG oligodeoxynucleotides trigger an immunostimulatory cascade that culminates in maturation, differentiation and proliferation of several immune cells creating a pro-inflammatory and Th1-biased environment. The immunostimulatory activity of this CpG-DNA is species-specific. As a result, sequences specific for the host are designed and optimized for selective TLR9 binding. In particular, *in vivo* CpG-DNA half-life has been improved by replacing phosphodiester CpG-DNA with a nuclease-resistant phosphothioate oligodeoxynucleotide although it may induce immune reaction leading to an anti-DNA immune response (Ciafre et al. 1995). Addition of the immunostimulatory CpG-DNA to peptide vaccines, tilts the balance towards a Th-1 immune response, which is often accompanied by a significant increase in IgG2a production in comparison to IgG1. The general effect of CpG-DNA addition is augmentation of the antibody serum titer against antigens and production of Th-1 cytokines such as IFN-γ (Klinman et al. 1999). By contrast, in DNA plasmid vaccination, the immunomodulating effect of bacterial CpG oligodeoxynucleotides on T-helper cell balance, shows great variability. Such variability often depends on the route of immunisation.

DNA Vaccination by Electrogene Transfer 183

Cationic Solid Lipid Nanoparticles (SLNs) have been recently proposed as alternative carriers for DNA delivery, due to many technological advantages such as large-scale production from substances generally recognized as safe, good storage stability and possibility of steam sterilization and lyophilisation. Cationic lipids are amphiphilic molecules composed of one or two fatty acid side chains (acyl) or alkyl, a linker and a hydrophilic amino group. The hydrophobic part can be cholesterol-derived moieties. In aqueous media, cationic lipids are assembled into a bilayer vesicular-like structure (liposomes). Liposomes/DNA complex is usually termed a lipoplex (Bolhassani et al. 2011). The future success of cationic SLNs for administration of genetic material will depend on their ability to efficiently cross the physiological barriers, selectively targeting a specific cell

In order to address accelerating micro-projectiles into intact cells or tissues, is generally used a biolistic apparatus described in patent US6004287 (Loomis 1999). Application of this strategy to DNA vaccines resulted in the invention of a new DNA delivery technology that made it possible to move naked DNA plasmid into target cells on an accelerated particle carrier. This specific delivery system is based on the use of the gene gun device that, under pressurized helium, is capable of delivering plasmid DNA-coated gold beads to the epidermal layer of skin as described in patent US6436709 (Lin 2002). Because the DNA carrier is introduced directly into the skin cells, delivery of plasmid DNA vaccines using this strategy reduces the amount of DNA needed to induce immune responses. Robust immunogenicity has been shown in many different preclinical models and in clinical trials predominantly for infectious diseases (Fuller et al. 2006). In contrast to intramuscular or intradermal injection by needle, the gene gun delivery system releases plasmid DNA directly into the cells of the epidermis (Yang et al. 1990). Intradermal injection is becoming increasingly popular, as the dense network of antigen-presenting cells in the skin, absent in muscle, provides a favourable environment for induction of antigen uptake. This network of Langerhans cells (LCs) can help in the priming of both cellular and humoural immune responses. Importantly, direct transfection of Langerhans cells is carried out with very small doses of plasmid DNA (i.e. 1-10 μg), suggesting that minimum amounts of vector are required to induce the immune response. The advantage of using low doses of plasmid DNA is particularly attractive for prophylactic vaccines against infectious diseases, where a simple and rapid delivery is the main pre-requisite. Gene gun delivery has recently been used with success in a trial against the influenza virus, inducing sero-protective levels of antibody and it has been used in trials against HBV and HIV infections (Fuller et al. 2006). A further implementation of the biolistic delivery was obtained also by creating improved injection device suitable for application in human tissues. Patent US6730663 describes a flexible multi-needle injector device with a wide surface area as well as a modified injector device to be used for injection through an endoscopic device. Such a method leads to a deep

The use of electric pulses as a safe tool to deliver therapeutic molecules to tissues and organs has been rapidly developed over the last decade. This technology leads to a transient increase in the permeability of cell membranes when exposed to electric field pulses. This

type in vivo and expressing therapeutic genes (Bondi and Craparo 2010).

**2.6.2 Cationic lipids/liposomes** 

**2.6.3 Biolistic particle delivery** 

injection of DNA within tissues (Hennighausen 2004).

**2.6.4 Electropermeabilization** 
