**Meet the editor**

Guy Huynh Thien Duc is Research Director Emeritus from the CNRS (Centre National de la Recherche Scientifique). Medicine student and Master's degree of Science, he started his career in the Pasteur Institute and the Sorbonne, Faculty of Science in Paris, where he prepared his Ph.D in the field of Immunopathology. Thereafter, as researcher in the CNRS, he has been mainly involved

in the fundamental aspects of Immunology, focusing on Transplantation Immunity and Immunomodulation. In the last two decades, his work at the University Paris XI, southern of Paris, presently inside the structure of INSERM U-1014, Groupe hospitalier Paul-Brousse-Villejuif, is essentially devoted to Immunology, its fundamental aspects and Cancer Immunotherapy.

Contents

**Preface VII**

**Section 2 Immunomodulation 73**

**Stimulation 75**

Karine Breckpot

Moorman

**and Deviation 121** Andrew D. Foey

Chapter 1 **Cationic Nanostructures for Vaccines 3** Ana Maria Carmona-Ribeiro

**Biomedical Applications 45**

Campo and Alfredo De Ioannes

**and Facilitating Infection 105**

Chapter 2 **Mollusk Hemocyanins as Natural Immunostimulants in**

María Inés Becker, Sergio Arancibia, Fabián Salazar, Miguel Del

Joeri J. Pen, Joeri. L. Aerts, Thérèse Liechtenstein, David Escors and

Jeddidiah Griffin, Marie Moulton, Rabab Elmezayen and Jonathan

Chapter 3 **Manipulating Immune Regulatory Pathways to Enhance T Cell**

Chapter 4 **Negative Immunomodulators – Blunting Immunostimulation**

Chapter 5 **Macrophages — Masters of Immune Activation, Suppression**

Chapter 6 **Immunostimulatory Effects of Triggering TLR3 Signaling**

**Pathway — Implication for Cancer Immunotherapy 151** Mohamed Labib Salem, Said M. Hammad, Mohamed R. Elshanshory, Mohamed A. Attia and Abdel-Aziz A. Zidan

**Section 1 Vaccines 1**

### Contents

#### **Preface XI**


Joeri J. Pen, Joeri. L. Aerts, Thérèse Liechtenstein, David Escors and Karine Breckpot


#### **Section 3 Local Immunity 175**


Preface

related to:

plants.

The immune system plays a central role against pathogens that is seen further, extend‐ ing beyond this context in recognizing alloantigens, modified transformed antigens ex‐ pressed on tumoral cells, but unfortunately also self-antigens. As such, the immune response is involved in a large number of clinical settings, among which, protection against pathogenic organisms (bacteria, viruses), transformed tumoral cells and, finally, recognition and reaction against allergens and autoantigens. Therefore, beside the as‐ pect relative to inducing generation of neutralizing protective antibodies and cytotoxic T lymphocytes against pathogens, there is need to consider the resistance developed by some pathogens to the traditional immune effector agents. Moreover, the immune re‐ sponse could also be acerbated in response to allergens and/or targeting the organism's own components leading to allergy and autoimmune diseases, respectively. Thus, along with its stimulation and activation, the immune response has to be tightly con‐ trolled and regulated. In this context, the multifaceted immune response is analyzed in the present book in several review chapters, treating a number of representative cases in which the immune response is, on one hand, activated against pathogens and, on the other hand, involved in pathologic settings, leading to allograft rejection, allergy and autoimmunity. The regulatory mechanisms in which the immune system can be modu‐ lated for rendering its effector components more efficient and not harmful to the organ‐ ism is also dissected in translational purposes in cancer immunotherapy, local

immunity against bacteria and viruses, as well as in allergy and autoimmunity.

naling Pathway — Implication for Cancer Immunotherapy.

In this regard, the book "Immune Response Activation", is divided into four sections

**Vaccines**, with chapters concerning: 1) Cationic Nanostructures for Vaccines, and 2) Mollusk Hemocyanins as Natural Immunostimulants in Biomedical Applications.

**Immunomodulation**, with chapters: 1) Manipulating Immune Regulatory Pathways to Enhance T Cell Stimulation ; 2) Negative Immunomodulators – Blunting Immunosti‐ mulation and Facilitating Infection ; 3) Macrophages — Masters of Immune Activation, Suppression and Deviation, and 4) Immunostimulatory Effects of Triggering TLR3 Sig‐

**Local Immunity**, with chapters : 1) Immune Regulation of Chlamydia trachomatis In‐ fections of the Female Genital Tract, and 2) The Roles of Invariant NKT Cells in Bowel Immunity — Suppression of Tumor Progression and Rejection of Intestinal Trans‐

**Section 4 Autoimmune Disregulation 247**

#### Chapter 9 **Immunostimulation by Silica Particles and the Development of Autoimmune Dysregulation 249** Suni Lee, Hiroaki Hayashi, Megumi Maeda, Hidenori Matsuzaki, Naoko Kumagai-Takei, Ying Chen, Kozo Urakami, Masayasu Kusaka, Yasumitsu Nishimura and Takemi Otsuki

### Preface

**Section 3 Local Immunity 175**

**VI** Contents

**Female Genital Tract 177**

**Transplants 227**

**Section 4 Autoimmune Disregulation 247**

Chapter 7 **Immune Regulation of Chlamydia trachomatis Infections of the**

Louise M. Hafner, Trudi A. Collet and Danica K. Hickey

Chapter 9 **Immunostimulation by Silica Particles and the Development of**

Kusaka, Yasumitsu Nishimura and Takemi Otsuki

Suni Lee, Hiroaki Hayashi, Megumi Maeda, Hidenori Matsuzaki, Naoko Kumagai-Takei, Ying Chen, Kozo Urakami, Masayasu

**Suppression of Tumor Progression and Rejection of Intestinal**

Chapter 8 **The Roles of Invariant NKT Cells in Bowel Immunity —**

Tatsuaki Tsuruyama and Wulamujiang Aini

**Autoimmune Dysregulation 249**

The immune system plays a central role against pathogens that is seen further, extend‐ ing beyond this context in recognizing alloantigens, modified transformed antigens ex‐ pressed on tumoral cells, but unfortunately also self-antigens. As such, the immune response is involved in a large number of clinical settings, among which, protection against pathogenic organisms (bacteria, viruses), transformed tumoral cells and, finally, recognition and reaction against allergens and autoantigens. Therefore, beside the as‐ pect relative to inducing generation of neutralizing protective antibodies and cytotoxic T lymphocytes against pathogens, there is need to consider the resistance developed by some pathogens to the traditional immune effector agents. Moreover, the immune re‐ sponse could also be acerbated in response to allergens and/or targeting the organism's own components leading to allergy and autoimmune diseases, respectively. Thus, along with its stimulation and activation, the immune response has to be tightly con‐ trolled and regulated. In this context, the multifaceted immune response is analyzed in the present book in several review chapters, treating a number of representative cases in which the immune response is, on one hand, activated against pathogens and, on the other hand, involved in pathologic settings, leading to allograft rejection, allergy and autoimmunity. The regulatory mechanisms in which the immune system can be modu‐ lated for rendering its effector components more efficient and not harmful to the organ‐ ism is also dissected in translational purposes in cancer immunotherapy, local immunity against bacteria and viruses, as well as in allergy and autoimmunity.

In this regard, the book "Immune Response Activation", is divided into four sections related to:

**Vaccines**, with chapters concerning: 1) Cationic Nanostructures for Vaccines, and 2) Mollusk Hemocyanins as Natural Immunostimulants in Biomedical Applications.

**Immunomodulation**, with chapters: 1) Manipulating Immune Regulatory Pathways to Enhance T Cell Stimulation ; 2) Negative Immunomodulators – Blunting Immunosti‐ mulation and Facilitating Infection ; 3) Macrophages — Masters of Immune Activation, Suppression and Deviation, and 4) Immunostimulatory Effects of Triggering TLR3 Sig‐ naling Pathway — Implication for Cancer Immunotherapy.

**Local Immunity**, with chapters : 1) Immune Regulation of Chlamydia trachomatis In‐ fections of the Female Genital Tract, and 2) The Roles of Invariant NKT Cells in Bowel Immunity — Suppression of Tumor Progression and Rejection of Intestinal Trans‐ plants.

#### XII Preface

**Autoimmune Disregulation**, with the chapter : Immunostimulation by Silica Particles and the Development of Autoimmune Dysregulation.

#### **Guy Huynh Thien Duc**

**Vaccines**

**Section 1**

Research Director Emeritus from the CNRS (Centre National de la Recherche Scientifique), University Paris XI, INSERM U-1014-Groupe Hospitalier Paul-Brousse, France

#### **Acknowledgments**

We thank Chaobin Zhu for his assistance.

**Section 1**

### **Vaccines**

**Autoimmune Disregulation**

VIII Preface

, with the chapter

and the Development of Autoimmune Dysregulation.

: Immunostimulation by Silica Particles

Research Director Emeritus from the CNRS (Centre National de la Recherche Scientifique),

We thank Chaobin Zhu for his assistance.

INSERM U-1014-Groupe Hospitalier Paul-Brousse,

**Guy Huynh Thien Duc**

University Paris XI,

**Acknowledgments**

France

**Chapter 1**

**Cationic Nanostructures for Vaccines**

Additional information is available at the end of the chapter

Cationic lipid bilayers, particles, polysaccharides and a variety of hybrid nanostructures provide adequate matrixes for supporting antigens such as peptides, proteins, DNA or oligonucleotides on model surfaces (latex, silica, silicon wafers, self-assembled monolayers, metals, polymers, insoluble drugs, biological cells and viruses).Particulate vaccines are currently an area receiving a high level of attention [1-3]. Particles deliver both antigen and adjuvant into the same population of antigen presenting cells limiting both the systemic distribution of the adjuvant and its potential toxicity [1]. Biological particlesrepresented by live or attenuated bacterial vacines, engineered biological vectors and virus-like particles are often less safe than synthetic particulates for which quality control and validation in vaccine development and production are more rapid [2]. While developing novel particulate vaccines, particle size and charge do matter [2]. Virus-sized particles (20–200 nm) are usually taken up by endocytosis via clathrin-coated vesicles, caveolae or their independent receptors [4], and preferentially ingested by dendritic cells (DC). Larger sized particles such as bacteria (500– 5000 nm) are predominantly taken up by phagocytosis, and primarily ingested by macro‐ phages. All particles used in vaccine formulations are consequently internalized efficiently by antigen presenting cells by one or a combination of the quoted mechanisms [5, 6]. Particles with diameters smaller than 500 nm, in particular the nanometric ones with sizes in the 40– 100 nm range are more eficient to promote CD8 and CD4 type 1 T cell responses than those with diameters above 500 nm, although the latter could induce good type 2 and antibody responses [5]. Cationic microparticles are effectively taken up both by macrophages and dendritic cells since electrostatic attraction promotes particle binding and subsequent inter‐ nalization. Cationized polymeric particles carrying antigen significantly enhanced both antibody production and cytotoxic T cells at low antigen dose [7] and induced maturation of dendritic cells [8, 9]. As a consequence particles for vaccines should be positive and available over a range of sizes. Cationic particles of aluminium compounds, identified as having

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ana Maria Carmona-Ribeiro

http://dx.doi.org/10.5772/57543

**1. Introduction**

#### **Chapter 1**

### **Cationic Nanostructures for Vaccines**

#### Ana Maria Carmona-Ribeiro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57543

#### **1. Introduction**

Cationic lipid bilayers, particles, polysaccharides and a variety of hybrid nanostructures provide adequate matrixes for supporting antigens such as peptides, proteins, DNA or oligonucleotides on model surfaces (latex, silica, silicon wafers, self-assembled monolayers, metals, polymers, insoluble drugs, biological cells and viruses).Particulate vaccines are currently an area receiving a high level of attention [1-3]. Particles deliver both antigen and adjuvant into the same population of antigen presenting cells limiting both the systemic distribution of the adjuvant and its potential toxicity [1]. Biological particlesrepresented by live or attenuated bacterial vacines, engineered biological vectors and virus-like particles are often less safe than synthetic particulates for which quality control and validation in vaccine development and production are more rapid [2]. While developing novel particulate vaccines, particle size and charge do matter [2]. Virus-sized particles (20–200 nm) are usually taken up by endocytosis via clathrin-coated vesicles, caveolae or their independent receptors [4], and preferentially ingested by dendritic cells (DC). Larger sized particles such as bacteria (500– 5000 nm) are predominantly taken up by phagocytosis, and primarily ingested by macro‐ phages. All particles used in vaccine formulations are consequently internalized efficiently by antigen presenting cells by one or a combination of the quoted mechanisms [5, 6]. Particles with diameters smaller than 500 nm, in particular the nanometric ones with sizes in the 40– 100 nm range are more eficient to promote CD8 and CD4 type 1 T cell responses than those with diameters above 500 nm, although the latter could induce good type 2 and antibody responses [5]. Cationic microparticles are effectively taken up both by macrophages and dendritic cells since electrostatic attraction promotes particle binding and subsequent inter‐ nalization. Cationized polymeric particles carrying antigen significantly enhanced both antibody production and cytotoxic T cells at low antigen dose [7] and induced maturation of dendritic cells [8, 9]. As a consequence particles for vaccines should be positive and available over a range of sizes. Cationic particles of aluminium compounds, identified as having

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

immunostimulatory properties more than 70 years ago, remain the only type of adjuvant licensed world-wide [3]. Oil-in-water emulsions or virosomes were licensed in some countries in compositions for influenza [10] or hepatitis B vaccines [11]. However, both of these adjuvants are characterised by inducing humoral immune response and are thus effective in elevating serum antibody titers whereas their ability to elicit cell-mediated immune response is limited [12]. For vaccines against influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. For vaccines against tuberculosis, a cellular response seems to be the sole effector mechanism required for protection [13]. Among the cationic compounds used to produce cationic nanostructures to present antigens for vaccines formulations, some cationic compounds appear to be of special relevance.They are the cationic and synthetic lipid named dioctadecyldimethylammonium bromide (DODAB), the cationic polysaccharide named chitosan (CH) and its derivatives with pH-independent positive charge and the biocompatible cationic polymeric nanostructures.This chapter presents and critically evaluates these three types of cationic nanostructures.

BF-or are dispersions of cationic particles with controllable nature and size as obtained after covering silica or polystyrene sulfate latex (PSS) with a cationic DODAB bilayer (Figure 1).

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 5

**Figure 1.** Cationic nanostructures and particles based on the cationic lipid DODAB useful to present antigens (repro‐ duced with permission from [27]).The DODAB bilayer fragments are usually obtained at low ionic strength by sonica‐ tion with a macrotip.Particles acting as supports for the cationic bilayer can be organic or inorganic. Reprinted from Vaccine, 27/42, Nilton Lincopan,Noelí M. Espíndola,Adelaide J. Vaz,Maria Helena B. da Costa,Eliana Faquim-Mauro,Ana M. Carmona-Ribeiro, Novel immunoadjuvants based on cationic lipid: Preparation, characterization and

DODAB BF may interact with proteins both via the hydrophobic effect and the electrostatic attraction at low ionic strength. This interaction has been properly characterized by means of dynamic light scattering for sizing, zeta-potential analysis and evaluation of immunoadjuvant activity *in vivo*. The model antigens employed with DODAB-based cationic nanostructures were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from *Taenia crassiceps* cysticerci (18/14-*Tcra*) or a recombinant heat-shock protein from *Mycobacterium leprae* [27]. Antigen choices were due to different reasons. BSA adsorption at interfaces [28] and, specifi‐ cally, onto large DODAB vesicles [29] is well described and has been useful to prevent nonspecific binding in immunoassays, biosensing and proteomics applications [28,30].The purified 18 kDa/14 kDa antigens from *Taenia crassiceps* cisticerci are proteins specific for this parasite found as circulating antigens and often employed in immunodiagnosis;they can be obtained from *in-vitro* cultures of *T. crassiceps* cysticerci in hybridoma media from vesicles budding from cysts contain the excretory/secretory (ES) antigens [31,32]. Human and pig infections by *T. solium* and *crassiceps*, respectively, represent an important health problem, with socio-economical repercussions affecting many countries in Latin America, Asia and Africa

activity *in vivo*, 5760-5771.Copyright 2009, with permission from Elsevier.

#### **2. Cationic nanostructures based on DODAB**

DODAB is a cationic lipid which can be dispersed ultrasonically in aqueous solution using a macrotip probe [14]. Thereby, bilayer vesicles are simultaneously obtained and disrupted yielding nano-sized bilayer disks or cationic bilayer fragments (BF) stabilized by electrostatic repulsion at low ionic strength [14,15].DODAB BF have previously been used as antimicrobial agents [16] or in the production of lipid-covered particles such as bilayer-coated silica [17] or latex [18]. These cationic, bilayer-covered latex or silica particles where the bilayer is solely composed of DODAB were recently employed to present antigens to the immune system with better results than alum as adjuvants for induction of cellular immune responses [19, 20]. The open DODAB BF in aqueous dispersions are different from their mother vesicles presenting the following features: (i) osmotic non-responsiveness of the dispersion indicative of absence of inner vesicle compartment [21]; (ii) discoidal shape with disks exhibiting one bilayer thickness as visualised by means of transmission electronic microscopy (TEM) after electronic staining the nanostructures [22]; (iii) cryo-TEM micrographs performed without any staining [23]; (iv) fluid and solid state coexistence and complex formation with oppositely charged surfactant [24]; (v) solubilization of hydrophobic drugs at the borders of DODAB bilayer fragments, which does not occur for DODAB closed bilayer vesicles [25]. These bilayer fragments have more fluid environments at their edges that are absent in closed bilayer systems such as vesicles or liposomes. Due to its cylindrical molecular shape, DODAB molecules selfassemble in aqueous solutions as bilayers instead of micelles as shown in the seventies by transmission electron microscopy [26]. Supramolecular assemblies of DODAB bilayer frag‐ ments by themselves or after interaction with supporting particles have been combined with different model antigens in separate and tested as immunoadjuvants. The cationic nanoadju‐ vants with DODAB BF are either reduced to a single-component, nanosized system-DODAB BF-or are dispersions of cationic particles with controllable nature and size as obtained after covering silica or polystyrene sulfate latex (PSS) with a cationic DODAB bilayer (Figure 1).

immunostimulatory properties more than 70 years ago, remain the only type of adjuvant licensed world-wide [3]. Oil-in-water emulsions or virosomes were licensed in some countries in compositions for influenza [10] or hepatitis B vaccines [11]. However, both of these adjuvants are characterised by inducing humoral immune response and are thus effective in elevating serum antibody titers whereas their ability to elicit cell-mediated immune response is limited [12]. For vaccines against influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. For vaccines against tuberculosis, a cellular response seems to be the sole effector mechanism required for protection [13]. Among the cationic compounds used to produce cationic nanostructures to present antigens for vaccines formulations, some cationic compounds appear to be of special relevance.They are the cationic and synthetic lipid named dioctadecyldimethylammonium bromide (DODAB), the cationic polysaccharide named chitosan (CH) and its derivatives with pH-independent positive charge and the biocompatible cationic polymeric nanostructures.This chapter presents and critically evaluates these three

DODAB is a cationic lipid which can be dispersed ultrasonically in aqueous solution using a macrotip probe [14]. Thereby, bilayer vesicles are simultaneously obtained and disrupted yielding nano-sized bilayer disks or cationic bilayer fragments (BF) stabilized by electrostatic repulsion at low ionic strength [14,15].DODAB BF have previously been used as antimicrobial agents [16] or in the production of lipid-covered particles such as bilayer-coated silica [17] or latex [18]. These cationic, bilayer-covered latex or silica particles where the bilayer is solely composed of DODAB were recently employed to present antigens to the immune system with better results than alum as adjuvants for induction of cellular immune responses [19, 20]. The open DODAB BF in aqueous dispersions are different from their mother vesicles presenting the following features: (i) osmotic non-responsiveness of the dispersion indicative of absence of inner vesicle compartment [21]; (ii) discoidal shape with disks exhibiting one bilayer thickness as visualised by means of transmission electronic microscopy (TEM) after electronic staining the nanostructures [22]; (iii) cryo-TEM micrographs performed without any staining [23]; (iv) fluid and solid state coexistence and complex formation with oppositely charged surfactant [24]; (v) solubilization of hydrophobic drugs at the borders of DODAB bilayer fragments, which does not occur for DODAB closed bilayer vesicles [25]. These bilayer fragments have more fluid environments at their edges that are absent in closed bilayer systems such as vesicles or liposomes. Due to its cylindrical molecular shape, DODAB molecules selfassemble in aqueous solutions as bilayers instead of micelles as shown in the seventies by transmission electron microscopy [26]. Supramolecular assemblies of DODAB bilayer frag‐ ments by themselves or after interaction with supporting particles have been combined with different model antigens in separate and tested as immunoadjuvants. The cationic nanoadju‐ vants with DODAB BF are either reduced to a single-component, nanosized system-DODAB

types of cationic nanostructures.

4 Immune Response Activation

**2. Cationic nanostructures based on DODAB**

**Figure 1.** Cationic nanostructures and particles based on the cationic lipid DODAB useful to present antigens (repro‐ duced with permission from [27]).The DODAB bilayer fragments are usually obtained at low ionic strength by sonica‐ tion with a macrotip.Particles acting as supports for the cationic bilayer can be organic or inorganic. Reprinted from Vaccine, 27/42, Nilton Lincopan,Noelí M. Espíndola,Adelaide J. Vaz,Maria Helena B. da Costa,Eliana Faquim-Mauro,Ana M. Carmona-Ribeiro, Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity *in vivo*, 5760-5771.Copyright 2009, with permission from Elsevier.

DODAB BF may interact with proteins both via the hydrophobic effect and the electrostatic attraction at low ionic strength. This interaction has been properly characterized by means of dynamic light scattering for sizing, zeta-potential analysis and evaluation of immunoadjuvant activity *in vivo*. The model antigens employed with DODAB-based cationic nanostructures were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from *Taenia crassiceps* cysticerci (18/14-*Tcra*) or a recombinant heat-shock protein from *Mycobacterium leprae* [27]. Antigen choices were due to different reasons. BSA adsorption at interfaces [28] and, specifi‐ cally, onto large DODAB vesicles [29] is well described and has been useful to prevent nonspecific binding in immunoassays, biosensing and proteomics applications [28,30].The purified 18 kDa/14 kDa antigens from *Taenia crassiceps* cisticerci are proteins specific for this parasite found as circulating antigens and often employed in immunodiagnosis;they can be obtained from *in-vitro* cultures of *T. crassiceps* cysticerci in hybridoma media from vesicles budding from cysts contain the excretory/secretory (ES) antigens [31,32]. Human and pig infections by *T. solium* and *crassiceps*, respectively, represent an important health problem, with socio-economical repercussions affecting many countries in Latin America, Asia and Africa [31]. The 18 kDa-hsp protein is a heat-shock protein of *M. leprae* displaying pronounced immunogenicity and considered able to induce proliferation of peripheral blood mononuclear cells and T-cell lines from *M. leprae* vaccinated subjects [33] andavailable at large amounts for studies on vaccine formulation; its overexpression and scaling-up in *Saccharomyces cerevisae* have already been described and the recombinant protein can be produced in large scale [34,35]. Nanostructured cationic adjuvant/antigen complexes based on DODAB were charac‐ terized over a range of adjuvant and antigen concentrations. Figure 2 shows the effect of antigen and adjuvant concentration on physical properties of the dispersions. Stable cationic nanostructures are available over a limited range of adjuvant and antigen concentrations which clearly depend on antigen nature and are different for different antigens.

The humoral and cellular immune responses induced by stable cationic adjuvant/antigen complexes were evaluated in mice from determination of antigen-specific-IgG antibody in serum by ELISA, delayed type hypersensitivity (DH) reactions from footpad swelling tests and cytokines analysis. The results evidence the good colloid stability of the complexes, complete absence of toxicity in mice (i.e. local or general reactions) and their potential utility to induce Th 1 immune response at reduced doses of cationic and toxic DODAB lipid. Possibly due to its chemical stability and low cost when compared to other natural or synthetic lipids, DODAB use as an immunoadjuvant started more than forty years ago [36,37]. This was well before the bilayer nature of DODAB self-assembly in water solution had been described [26]. DODAB as an effective immunoadjuvant has been intensively investigated aiming at subunit vaccine design [38-41]. A major problem of liposomal formulations based on DODAB has been the usually high concentration employed, namely 1-10 mM DODAB [38-41]. DODAB is a cationic lipid and as such, cytotoxic [14-16] requiring dose minimization for administration in vivo. Using the DODAB bilayer fragments to present antigens only 0.1 mM DODAB was employed [27]. The large cellular imune response achieved might be related to the total surface area available for antigen association, which is much larger in the BF dispersion than in closed, large and sometimes multibilayered liposomes. Moreover, the hydrophobic interaction possibly available from BF edges could be an additional and powerful driving force for antigen adsorption and presentation. The second advantage of the bilayer fragments besides their large surfaces area is their size. Depending on sonication power and time plus composition of the dispersing medium, which determine colloid stability, DODAB BF/antigens complexes may have their sizes reduced to and stabilized at a few tenths of nanometers acting similarly to solid inert beads of nanometric size (40-50 nm). These beads turned out to be effective for antigen delivery to antigen-presenting cells (APC), generating potent and combined humoral and CD8+T cell immunity [3]. They are also expected to be optimal for dendritic cells uptake since their size is inside the range of particle diameters (500 nm and below) for optimal dendritic cells uptake of antigens and elicitation of an adequate cellular response [2,5,42]. In reconstituted pig gastric mucus, sub-200 nm particulates from poly (D, L-lactic-co-glycolic) acid and DODAB condensing DNA on particles surface exhibited improved transport rates, stability in mucus, and ability to transfect cells [43]. Silica/DODAB, latex/DODAB and DODAB BF (Figure 1) are available over the sub-200 nm range of sizes thus presenting potential also for design of mucosal vaccines. The third advantage of the bilayer fragments is the absence of depots at the injection site [27]. These depots have been reported for other DODAB formula‐

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 7

tions at concentrations much higher than 0.1 mM [37,41,44].

Consistently with electrostatic forces between negatively charged antigens and positively charged DODAB BF, cationic bilayer fragments readily adsorb BSA, 18-14/*Tcra* and the hsp-18 kDa recombinant protein.Rapid and extensive adsorption of BSA, anti-BSA or ovalbumin onto large DODAB liposomes was indeed reported previously [29,45,46].On the other hand, bacteria, fungus and eukaryotic cells were also found to adsorb DODAB vesicles and bilayer fragments with high affinity at low ionic strength [14-16], so that cationic DODAB liposomes readily adsorb antigen, such as ovalbumin, and bind avidly to dendritic cells [41], thereby enhancing antigen uptake. Delivery of antigen to cells by immediate contact with the cell surface via electrostatic interaction followed by the induction of active uptake seems to be the mechanism behind the ability of DODAB liposomes or bilayer fragments to act as immunoad‐ juvants. Sizes and zeta-potentials for assemblies of antigen and cationic lipid based adjuvants

**Figure 2.** Effect of time and DODAB concentration on the zeta-average diameter of DODAB/BSA (A) or DODAB BF/ 18/14-Tcra complexes (B). In (A), the kinetics were obtained upon adding DODAB BF at a final concentration of 0.005 ((▷), 0.01 (△), 0.02 (◁), 0.05 (○), 0.5 (◇), 0.8 (□), and 1.0 mM DODAB (◁) to 0.5 mg/mL BSA. In (B), the kinetics were obtained upon adding DODAB BF at a final concentration of 0.0005 (□), 0.001 (○), 0.005 (▷), 0.01(▽), 0.05(◇), and 0.1 mM DODAB (△) to 0.05 mg/mL 18/14-Tcra. The effect of [DODAB] on zeta-potential of complexes with 0.5 mg/mL BSA (C) or 0.05 mg/mL 18/14-Tcra (D) were obtained after 1 h interaction at 25 °C in 1 mM NaCl.Reproduced with permission from reference [27].Reprinted from Vaccine, 27/42, Nilton Lincopan,Noelí M. Espíndola,Adelaide J. Vaz,Maria Helena B. da Costa,Eliana Faquim-Mauro,Ana M. Carmona-Ribeiro,Novel immunoadjuvants based on cati‐ onic lipid: Preparation, characterization and activity in vivo,5760-5771.Copyright 2009, with permission from Elsevier.

The humoral and cellular immune responses induced by stable cationic adjuvant/antigen complexes were evaluated in mice from determination of antigen-specific-IgG antibody in serum by ELISA, delayed type hypersensitivity (DH) reactions from footpad swelling tests and cytokines analysis. The results evidence the good colloid stability of the complexes, complete absence of toxicity in mice (i.e. local or general reactions) and their potential utility to induce Th 1 immune response at reduced doses of cationic and toxic DODAB lipid. Possibly due to its chemical stability and low cost when compared to other natural or synthetic lipids, DODAB use as an immunoadjuvant started more than forty years ago [36,37]. This was well before the bilayer nature of DODAB self-assembly in water solution had been described [26]. DODAB as an effective immunoadjuvant has been intensively investigated aiming at subunit vaccine design [38-41]. A major problem of liposomal formulations based on DODAB has been the usually high concentration employed, namely 1-10 mM DODAB [38-41]. DODAB is a cationic lipid and as such, cytotoxic [14-16] requiring dose minimization for administration in vivo. Using the DODAB bilayer fragments to present antigens only 0.1 mM DODAB was employed [27]. The large cellular imune response achieved might be related to the total surface area available for antigen association, which is much larger in the BF dispersion than in closed, large and sometimes multibilayered liposomes. Moreover, the hydrophobic interaction possibly available from BF edges could be an additional and powerful driving force for antigen adsorption and presentation. The second advantage of the bilayer fragments besides their large surfaces area is their size. Depending on sonication power and time plus composition of the dispersing medium, which determine colloid stability, DODAB BF/antigens complexes may have their sizes reduced to and stabilized at a few tenths of nanometers acting similarly to solid inert beads of nanometric size (40-50 nm). These beads turned out to be effective for antigen delivery to antigen-presenting cells (APC), generating potent and combined humoral and CD8+T cell immunity [3]. They are also expected to be optimal for dendritic cells uptake since their size is inside the range of particle diameters (500 nm and below) for optimal dendritic cells uptake of antigens and elicitation of an adequate cellular response [2,5,42]. In reconstituted pig gastric mucus, sub-200 nm particulates from poly (D, L-lactic-co-glycolic) acid and DODAB condensing DNA on particles surface exhibited improved transport rates, stability in mucus, and ability to transfect cells [43]. Silica/DODAB, latex/DODAB and DODAB BF (Figure 1) are available over the sub-200 nm range of sizes thus presenting potential also for design of mucosal vaccines. The third advantage of the bilayer fragments is the absence of depots at the injection site [27]. These depots have been reported for other DODAB formula‐ tions at concentrations much higher than 0.1 mM [37,41,44].

[31]. The 18 kDa-hsp protein is a heat-shock protein of *M. leprae* displaying pronounced immunogenicity and considered able to induce proliferation of peripheral blood mononuclear cells and T-cell lines from *M. leprae* vaccinated subjects [33] andavailable at large amounts for studies on vaccine formulation; its overexpression and scaling-up in *Saccharomyces cerevisae* have already been described and the recombinant protein can be produced in large scale [34,35]. Nanostructured cationic adjuvant/antigen complexes based on DODAB were charac‐ terized over a range of adjuvant and antigen concentrations. Figure 2 shows the effect of antigen and adjuvant concentration on physical properties of the dispersions. Stable cationic nanostructures are available over a limited range of adjuvant and antigen concentrations

**Figure 2.** Effect of time and DODAB concentration on the zeta-average diameter of DODAB/BSA (A) or DODAB BF/ 18/14-Tcra complexes (B). In (A), the kinetics were obtained upon adding DODAB BF at a final concentration of 0.005 ((▷), 0.01 (△), 0.02 (◁), 0.05 (○), 0.5 (◇), 0.8 (□), and 1.0 mM DODAB (◁) to 0.5 mg/mL BSA. In (B), the kinetics were obtained upon adding DODAB BF at a final concentration of 0.0005 (□), 0.001 (○), 0.005 (▷), 0.01(▽), 0.05(◇), and 0.1 mM DODAB (△) to 0.05 mg/mL 18/14-Tcra. The effect of [DODAB] on zeta-potential of complexes with 0.5 mg/mL BSA (C) or 0.05 mg/mL 18/14-Tcra (D) were obtained after 1 h interaction at 25 °C in 1 mM NaCl.Reproduced with permission from reference [27].Reprinted from Vaccine, 27/42, Nilton Lincopan,Noelí M. Espíndola,Adelaide J. Vaz,Maria Helena B. da Costa,Eliana Faquim-Mauro,Ana M. Carmona-Ribeiro,Novel immunoadjuvants based on cati‐ onic lipid: Preparation, characterization and activity in vivo,5760-5771.Copyright 2009, with permission from Elsevier.

which clearly depend on antigen nature and are different for different antigens.

6 Immune Response Activation

Consistently with electrostatic forces between negatively charged antigens and positively charged DODAB BF, cationic bilayer fragments readily adsorb BSA, 18-14/*Tcra* and the hsp-18 kDa recombinant protein.Rapid and extensive adsorption of BSA, anti-BSA or ovalbumin onto large DODAB liposomes was indeed reported previously [29,45,46].On the other hand, bacteria, fungus and eukaryotic cells were also found to adsorb DODAB vesicles and bilayer fragments with high affinity at low ionic strength [14-16], so that cationic DODAB liposomes readily adsorb antigen, such as ovalbumin, and bind avidly to dendritic cells [41], thereby enhancing antigen uptake. Delivery of antigen to cells by immediate contact with the cell surface via electrostatic interaction followed by the induction of active uptake seems to be the mechanism behind the ability of DODAB liposomes or bilayer fragments to act as immunoad‐ juvants. Sizes and zeta-potentials for assemblies of antigen and cationic lipid based adjuvants depend on cationic lipid and antigen concentrations. Adjuvant-antigen stability around sizes that are close to the one of adjuvants themselves indicates that the proteins readily adsorb and stabilize them. The adjuvants also stabilized the proteins acting as important dispersing nanocarriers able to induce remarkable degree of protein disaggregation by attaching the proteins either electrostatically or hydrophobically to their structure. At [DODAB] ≤ 0.1 mM and 0.001-0.05 mg/mL of antigen concentration, DODAB based adjuvant /antigen assemblies are cationic, well-dispersed, colloidally stable and immunogenic combining the advantages of low DODAB dose, low cost, controllable sizes for optimal dendritic cells uptake, high chemical stability, ability to incorporate multiple antigens and minimization of toxicity. Their perform‐ ance is remarkably superior to the one of alum as adjuvant regarding Th1 mediated responses. In contrast to alum or cationic liposomes at 1-10 mM of cationic lipid, local or systemic adverse effects in mice were completely absent at 0.1-0.01mM DODAB.

In viral infections, Il-12 enhances the cytotoxic activity of natural killer cells so that NK cellmediated killing of virus-infected cells eliminates the reservoir of infection. In this respect, vaccination against the dengue virus is urgently needed in tropical or neotropical regions of the planet and some recombinant DNA vaccines expressing membrane and envelope of viral proteins have been proposed [49]. Possibly the cationic adjuvants available from our group would properly enhance the required Th-1 response for a more effective vaccination against

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 9

Another possible application for the novel adjuvants might be in immunotherapy for tumors. This approach is based in augmentation of host immunity to tumors with tumor vaccines. Immune responses that are able of killing tumor cells consist of CTLs, NK cells, and activated macrophages and these may be actively enhanced by vaccination with tumor cells or antigens, administration of tumors modified to express high levels of cytokines that stimulate T cell proliferation and differentiation, and systemic administration of cytokines [49]. The induction of T cell responses in tumors depends on processing and presentation of tumor antigens to T cells by professional antigen-presenting cells (APCs) which might internalize the tumor antigen adsorbed onto the novel cationic adjuvants. These APCs may stimulate CD8+T cells and CD4+helper T lymphocytes to differentiate for recognition and killing of tumor cells.

Naïve CD4+T cells may differentiate into distinct subsets, such as Th1 and Th2 cells in response to different antigens. For example, the enhancement in production of IL-10 and Il-13 by lymphonode cells elicited by the antigens of *Taenia crassiceps* presented by the DODAB BF adjuvant can be appreciated in Figure 3 [27]. These cytokines are typically associated with responses to allergens and parasites such as helminths and mediate differentiation of CD4+-T cells into Th2 cells [50]. Consistently, low levels of these cytokines were elicited by the *M. leprae* antigen presented by the novel adjuvants (Figure 3). Responses were indeed different for the helminthes and the bacteria antigens and very antigen-specific as they should be [27]. The size, charge and hydrophobic features of DODAB BF led to novel applications in solubi‐ lization of hydrophobic drugs [25,51,52], production of biomimetic particles from bilayer coverage of silica [17] or polystyrene particles [53] and design of vaccines [27].Recently, BF was also combined with oligonucleotides [54]. Since synthetic oligonucleotides can inhibit the replication of the Rous sarcoma virus [55], antisense oligonucleotides have been considered a great promise as therapeutic agents and several oligonucleotide-based formulations have reached the clinical trial phase [56,57]. Antisense oligonucleotides have also been extensively used in research on gene expression and function [58-60], vaccine formulation [61], allergy [62] and cancer therapeutics [63].Major obstacles as their degradation by nucleases and poor delivery to the target cells [60,64] suggest the essential role of suitable carriers able to protect oligonucleotides in the biological milieu [60,63–65].There are peculiar features for the interac‐ tion between BF and oligonucleotides in comparison to other electrolytes.Effects of salt, dAMP or poly (dA) concentration on BF size and zeta-potential are shown on Figure 4 taken from reference [54]. From 0 to 0.25 mM salt, Dz and zeta-potentials decreased with salt concentration possibly due to massive phosphate anion binding. From 0.25 to 2.5 mM of divalent salt, Dz increased but zeta-potential remained approximately constant and low (Fig. 4A and D).Dz and zeta-potential decreased with dAMP concentration (0– 2.5 mM) (Fig. 4B and E). At 0.05 mM poly (dA) and 0.5 mM DODAB, extensive BF aggregation and/or fusion took place as depicted from large Dz (N 500 nm) (Fig. 4C) and zero of zeta-potential (Fig. 4F). The screening of DODAB

dengue.

An important component of the early innate immune response to viruses and bacteria is the secretion of cytokines, which mediate many of the effector functions of innate immunity. IL-10 is an inhibitor of activated macrophages and dendritic cells and is an example of negative feedback regulation because it is produced by macrophages to inhibit their function. This cytokine also inhibits the production of IL-12 and expression of class II major histocompati‐ bility (MHC) molecules. IL-12 is also secreted by macrophages and dendritic cells inducing T cells differentiation into Th1 and natural killer (NK) cells with increased IFN-gamma synthesis and cytotoxic activity. IL-12 and IFN-gamma are the most important cytokines in innate responses to intracellular bacteria such as *Mycobacterium leprae* or tuberculosis [13]. Figure 3 shows the high levels of IL-12 and IFN-gamma induced by the novel cationic adjuvants while presenting the hsp-18 kDa of *M. leprae* to lymphonode cells suggesting a possible application of the novel adjuvants for the design of subunit vaccines against intracelular bacteria. As in DH, adaptive immunity against intracellular bacteria is principally cell mediated and consists of activation of macrophages by CD4+T cells as well as killing of infected cells by CD8+cyto‐ toxic T lymphocytes (CTL).

On basis of IL-12 enhancement of IFN-gamma production and development of Th1 cells, this interleukin itself has been used as a vaccine adjuvant for many infections that are combated by cell-mediated immunity, e.g. leishmaniasis [47]. Subunit vaccines against protozoa that survive within macrophages require as principal defense mechanism cell-mediated immunity, particularly directed to macrophage activation by Th1 cell-derived cytokines. Leishmaniasis mucocutaneous and disseminated is caused by *Leishmania donovani* and CD4+Th1 cells are required to activate macrophages to kill phagocytosed parasites. Resistance to the infection is associated with activation of Leishmania-specific Th1 CD4+T cells which produce IFN-gamma and thereby activate macrophages to destroy intracellular parasites. Conversely, activation of Th2 cells by the protozoan results in increased parasite survival and exarcerbation of lesions because of the macrophage-suppressive actions of Th2 cytokines [47]. Other significant example is the protective role played by CD8+T cells in immunity to the hepatic stages of malaria. These effects may be mediated by direct killing of sporozoite-infected hepatocytes or indirectly by the secretion of IFN-gamma and activation of hepatocytes to produce nitric oxide and other agents that kill parasites. IL-12 induces resistance to sporozoite challenge in rodents and nonhuman primates, presumably by stimulating IFN-gamma production [48].

In viral infections, Il-12 enhances the cytotoxic activity of natural killer cells so that NK cellmediated killing of virus-infected cells eliminates the reservoir of infection. In this respect, vaccination against the dengue virus is urgently needed in tropical or neotropical regions of the planet and some recombinant DNA vaccines expressing membrane and envelope of viral proteins have been proposed [49]. Possibly the cationic adjuvants available from our group would properly enhance the required Th-1 response for a more effective vaccination against dengue.

depend on cationic lipid and antigen concentrations. Adjuvant-antigen stability around sizes that are close to the one of adjuvants themselves indicates that the proteins readily adsorb and stabilize them. The adjuvants also stabilized the proteins acting as important dispersing nanocarriers able to induce remarkable degree of protein disaggregation by attaching the proteins either electrostatically or hydrophobically to their structure. At [DODAB] ≤ 0.1 mM and 0.001-0.05 mg/mL of antigen concentration, DODAB based adjuvant /antigen assemblies are cationic, well-dispersed, colloidally stable and immunogenic combining the advantages of low DODAB dose, low cost, controllable sizes for optimal dendritic cells uptake, high chemical stability, ability to incorporate multiple antigens and minimization of toxicity. Their perform‐ ance is remarkably superior to the one of alum as adjuvant regarding Th1 mediated responses. In contrast to alum or cationic liposomes at 1-10 mM of cationic lipid, local or systemic adverse

An important component of the early innate immune response to viruses and bacteria is the secretion of cytokines, which mediate many of the effector functions of innate immunity. IL-10 is an inhibitor of activated macrophages and dendritic cells and is an example of negative feedback regulation because it is produced by macrophages to inhibit their function. This cytokine also inhibits the production of IL-12 and expression of class II major histocompati‐ bility (MHC) molecules. IL-12 is also secreted by macrophages and dendritic cells inducing T cells differentiation into Th1 and natural killer (NK) cells with increased IFN-gamma synthesis and cytotoxic activity. IL-12 and IFN-gamma are the most important cytokines in innate responses to intracellular bacteria such as *Mycobacterium leprae* or tuberculosis [13]. Figure 3 shows the high levels of IL-12 and IFN-gamma induced by the novel cationic adjuvants while presenting the hsp-18 kDa of *M. leprae* to lymphonode cells suggesting a possible application of the novel adjuvants for the design of subunit vaccines against intracelular bacteria. As in DH, adaptive immunity against intracellular bacteria is principally cell mediated and consists of activation of macrophages by CD4+T cells as well as killing of infected cells by CD8+cyto‐

On basis of IL-12 enhancement of IFN-gamma production and development of Th1 cells, this interleukin itself has been used as a vaccine adjuvant for many infections that are combated by cell-mediated immunity, e.g. leishmaniasis [47]. Subunit vaccines against protozoa that survive within macrophages require as principal defense mechanism cell-mediated immunity, particularly directed to macrophage activation by Th1 cell-derived cytokines. Leishmaniasis mucocutaneous and disseminated is caused by *Leishmania donovani* and CD4+Th1 cells are required to activate macrophages to kill phagocytosed parasites. Resistance to the infection is associated with activation of Leishmania-specific Th1 CD4+T cells which produce IFN-gamma and thereby activate macrophages to destroy intracellular parasites. Conversely, activation of Th2 cells by the protozoan results in increased parasite survival and exarcerbation of lesions because of the macrophage-suppressive actions of Th2 cytokines [47]. Other significant example is the protective role played by CD8+T cells in immunity to the hepatic stages of malaria. These effects may be mediated by direct killing of sporozoite-infected hepatocytes or indirectly by the secretion of IFN-gamma and activation of hepatocytes to produce nitric oxide and other agents that kill parasites. IL-12 induces resistance to sporozoite challenge in rodents

and nonhuman primates, presumably by stimulating IFN-gamma production [48].

effects in mice were completely absent at 0.1-0.01mM DODAB.

toxic T lymphocytes (CTL).

8 Immune Response Activation

Another possible application for the novel adjuvants might be in immunotherapy for tumors. This approach is based in augmentation of host immunity to tumors with tumor vaccines. Immune responses that are able of killing tumor cells consist of CTLs, NK cells, and activated macrophages and these may be actively enhanced by vaccination with tumor cells or antigens, administration of tumors modified to express high levels of cytokines that stimulate T cell proliferation and differentiation, and systemic administration of cytokines [49]. The induction of T cell responses in tumors depends on processing and presentation of tumor antigens to T cells by professional antigen-presenting cells (APCs) which might internalize the tumor antigen adsorbed onto the novel cationic adjuvants. These APCs may stimulate CD8+T cells and CD4+helper T lymphocytes to differentiate for recognition and killing of tumor cells.

Naïve CD4+T cells may differentiate into distinct subsets, such as Th1 and Th2 cells in response to different antigens. For example, the enhancement in production of IL-10 and Il-13 by lymphonode cells elicited by the antigens of *Taenia crassiceps* presented by the DODAB BF adjuvant can be appreciated in Figure 3 [27]. These cytokines are typically associated with responses to allergens and parasites such as helminths and mediate differentiation of CD4+-T cells into Th2 cells [50]. Consistently, low levels of these cytokines were elicited by the *M. leprae* antigen presented by the novel adjuvants (Figure 3). Responses were indeed different for the helminthes and the bacteria antigens and very antigen-specific as they should be [27].

The size, charge and hydrophobic features of DODAB BF led to novel applications in solubi‐ lization of hydrophobic drugs [25,51,52], production of biomimetic particles from bilayer coverage of silica [17] or polystyrene particles [53] and design of vaccines [27].Recently, BF was also combined with oligonucleotides [54]. Since synthetic oligonucleotides can inhibit the replication of the Rous sarcoma virus [55], antisense oligonucleotides have been considered a great promise as therapeutic agents and several oligonucleotide-based formulations have reached the clinical trial phase [56,57]. Antisense oligonucleotides have also been extensively used in research on gene expression and function [58-60], vaccine formulation [61], allergy [62] and cancer therapeutics [63].Major obstacles as their degradation by nucleases and poor delivery to the target cells [60,64] suggest the essential role of suitable carriers able to protect oligonucleotides in the biological milieu [60,63–65].There are peculiar features for the interac‐ tion between BF and oligonucleotides in comparison to other electrolytes.Effects of salt, dAMP or poly (dA) concentration on BF size and zeta-potential are shown on Figure 4 taken from reference [54]. From 0 to 0.25 mM salt, Dz and zeta-potentials decreased with salt concentration possibly due to massive phosphate anion binding. From 0.25 to 2.5 mM of divalent salt, Dz increased but zeta-potential remained approximately constant and low (Fig. 4A and D).Dz and zeta-potential decreased with dAMP concentration (0– 2.5 mM) (Fig. 4B and E). At 0.05 mM poly (dA) and 0.5 mM DODAB, extensive BF aggregation and/or fusion took place as depicted from large Dz (N 500 nm) (Fig. 4C) and zero of zeta-potential (Fig. 4F). The screening of DODAB

charges by Na2HPO4, followed by a decrease in electrostatic repulsion between fragments, could be responsible for DODAB BFs aggregation and/or fusion. DLS data also showed that diameters increase upon addition of Na2HPO4 concentrations above 0.5mM (Figure 4A), and this diameter increase up to 400 nm is related to a decrease in the zeta-potential of the fragments (Figure 4D). The charge screening of DODAB charged heads by Na2HPO4 explains the decrease in zeta-potential (Figure 4D) as well as the tighter bilayer packing represented by higher mean phase transition temperature for the bilayer [54]. Addition of dAMP leads to a decrease in diameter and zeta-potential of the assemblies (Figures 4B and E). It was shown that DODAB bilayer fragments are able to order dAMP molecules on their surface, causing the dAMP bases to stack [68]. In this case, bulky moieties of dAMP would be exposed at the fragments surface representing a steric hindrance to fragments aggregation and/or fusion which would also contribute to colloid stabilization in dispersion. For polynucleotides such as poly(dA), charge neutralization leads to flocculation whereas charge overcompensation upon increasing poly(dA) concentration leads to colloidal restabilization due to electrostatic repulsion (Figures 4C and F) [54]. This behavior has been often described for polyelectrolytes interacting with particles of opposite charge [67,68]. Beyond the neutralization point, the system regains stability due to charge overcompensation. This phenomenon was observed only for the electrolyte poly (dA) and not for the electrolytes Na2HPO4 and dAMP, which are unable to completely neutralize the bilayer [54], as shown in Figure 4. Colloid instability induced by oligonucleotide or salt could be associated with bilayer fusion [54]. In contrast, mononucleo‐ tide neither reduced colloid stability over the low range of concentrations tested nor caused

0.00 0.05 0.10 0.15 0.20 0.25

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 11


0.0 0.5 1.0 1.5 2.0 2.5

**Figure 4.** Effect of [Na2HPO4], [dAMP] or [poly (dA)] concentrations on the zeta-average diameter (A, B and C) and zeta-potential of DODAB BF at 0.5 mM DODAB (D, E and F).Reprinted from reference [54].Reprinted from Biochimica et Biophysica Acta (BBA)-Biomembranes, 1808/3, Julio H.K. Rozenfeld,Tiago R. Oliveira,M. Teresa Lamy,Ana M. Carmo‐ na-Ribeiro, Interaction of cationic bilayer fragments with a model oligonucleotide, 649-655.Copyright 2011, with per‐

[dAMP] (mM) [poly(dA)] (mM) [Na2

30

0.0 0.5 1.0 1.5 2.0 2.5

HPO4 ] (mM) 40

50

70

80

90

200

D E F

400

600

A B C

BF fusion [54].

mission from Elsevier.

z(mV)

100

200

Dz (nm) 300

400

**Figure 3.** Quantitative analysis of cytokines secreted by lymph node cells from DODAB/18/14-Tcra and Al(OH)3/18/14-Tcra immunized mice (on the left column, A-D) or from 18kDa-hsp, DODAB/18kDa-hsp, PSS/DODAB/ 18kDa-hsp, SiO2/DODAB/18 kDa-hsp and Al(OH)3/18kDa-hsp immunized mice (on the right column, E-F). Cells from lymph nodes of BALB/c mice previously immunized with 10 μg of 18/14-*Tcra* administered alone, in DODAB BF or in Al(OH)3 were *in vitro* stimulated with medium, 160 μg/ml of 18/14-*Tcra* or 2.5 µg/mL of ConA for 48 hours and the supernatants collected for cytokine analysis by sandwich kit enzyme-linked immunosorbent assay (ELISA). The results were expressed as mean of the cytokine concentration of two distinct assays ± standard deviation. Limits of detection are shown as horizontal dashed lines. Similarly, cells from lymph nodes of BALB-c mice previously immunized with 15 μg of 18 kDa-hsp from *M. leprae* administered alone or in DODAB BF, PSS/DODAB, silica/DODAB or Al (OH)3 were *in vitro* stimulated with medium, 250 μg/ml of 18 kDa-hsp or 2.5 µg/mL of ConA for 48 hours before following cytokines analysis as above (reproduced with permission from reference [27]). Reprinted from Vaccine, 27/42, Nilton Lincopan, Noelí M. Espíndola, Adelaide J. Vaz,Maria Helena B. da Costa, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity *in vivo*, 5760-5771.Copyright 2009, with permission from Elsevier.

charges by Na2HPO4, followed by a decrease in electrostatic repulsion between fragments, could be responsible for DODAB BFs aggregation and/or fusion. DLS data also showed that diameters increase upon addition of Na2HPO4 concentrations above 0.5mM (Figure 4A), and this diameter increase up to 400 nm is related to a decrease in the zeta-potential of the fragments (Figure 4D). The charge screening of DODAB charged heads by Na2HPO4 explains the decrease in zeta-potential (Figure 4D) as well as the tighter bilayer packing represented by higher mean phase transition temperature for the bilayer [54]. Addition of dAMP leads to a decrease in diameter and zeta-potential of the assemblies (Figures 4B and E). It was shown that DODAB bilayer fragments are able to order dAMP molecules on their surface, causing the dAMP bases to stack [68]. In this case, bulky moieties of dAMP would be exposed at the fragments surface representing a steric hindrance to fragments aggregation and/or fusion which would also contribute to colloid stabilization in dispersion. For polynucleotides such as poly(dA), charge neutralization leads to flocculation whereas charge overcompensation upon increasing poly(dA) concentration leads to colloidal restabilization due to electrostatic repulsion (Figures 4C and F) [54]. This behavior has been often described for polyelectrolytes interacting with particles of opposite charge [67,68]. Beyond the neutralization point, the system regains stability due to charge overcompensation. This phenomenon was observed only for the electrolyte poly (dA) and not for the electrolytes Na2HPO4 and dAMP, which are unable to completely neutralize the bilayer [54], as shown in Figure 4. Colloid instability induced by oligonucleotide or salt could be associated with bilayer fusion [54]. In contrast, mononucleo‐ tide neither reduced colloid stability over the low range of concentrations tested nor caused BF fusion [54].

**Figure 3.** Quantitative analysis of cytokines secreted by lymph node cells from DODAB/18/14-Tcra and Al(OH)3/18/14-Tcra immunized mice (on the left column, A-D) or from 18kDa-hsp, DODAB/18kDa-hsp, PSS/DODAB/ 18kDa-hsp, SiO2/DODAB/18 kDa-hsp and Al(OH)3/18kDa-hsp immunized mice (on the right column, E-F). Cells from lymph nodes of BALB/c mice previously immunized with 10 μg of 18/14-*Tcra* administered alone, in DODAB BF or in Al(OH)3 were *in vitro* stimulated with medium, 160 μg/ml of 18/14-*Tcra* or 2.5 µg/mL of ConA for 48 hours and the supernatants collected for cytokine analysis by sandwich kit enzyme-linked immunosorbent assay (ELISA). The results were expressed as mean of the cytokine concentration of two distinct assays ± standard deviation. Limits of detection are shown as horizontal dashed lines. Similarly, cells from lymph nodes of BALB-c mice previously immunized with 15 μg of 18 kDa-hsp from *M. leprae* administered alone or in DODAB BF, PSS/DODAB, silica/DODAB or Al (OH)3 were *in vitro* stimulated with medium, 250 μg/ml of 18 kDa-hsp or 2.5 µg/mL of ConA for 48 hours before following cytokines analysis as above (reproduced with permission from reference [27]). Reprinted from Vaccine, 27/42, Nilton Lincopan, Noelí M. Espíndola, Adelaide J. Vaz,Maria Helena B. da Costa, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity *in vivo*, 5760-5771.Copyright

2009, with permission from Elsevier.

10 Immune Response Activation

**Figure 4.** Effect of [Na2HPO4], [dAMP] or [poly (dA)] concentrations on the zeta-average diameter (A, B and C) and zeta-potential of DODAB BF at 0.5 mM DODAB (D, E and F).Reprinted from reference [54].Reprinted from Biochimica et Biophysica Acta (BBA)-Biomembranes, 1808/3, Julio H.K. Rozenfeld,Tiago R. Oliveira,M. Teresa Lamy,Ana M. Carmo‐ na-Ribeiro, Interaction of cationic bilayer fragments with a model oligonucleotide, 649-655.Copyright 2011, with per‐ mission from Elsevier.

Particles are finding a large variety of biomedical and pharmaceutical applications since their size scale can be similar to that of biomacromolecules (e.g., proteins, DNA) and structures (e.g., bacteria and viruses). Their utility for imaging, gene and drug delivery, and vaccine design is undeniable [69,70].Particulate systems are naturally targeted to antigen presenting cells (APC) so that particles deliver antigens to APC more efficiently than soluble antigen [71,72].Positively charged particles with diameters of 500 nm and below were shown to be optimal for dendritic cells uptake [42].DODAB bilayers electrostatically combine with a vast variety of negatively charged biomolecules or biological structures [14]. Silica [17], latex [21,73,74] or hydrophobic drug particles [51,75] have been coated with DODAB with optimal bilayer deposition on particles achieved by coalescence of bilayer fragments at an adequate ionic strength [17,76]. Figure 5 shows how DODAB can cover oppositely charged polystyrene nanoparticles modi‐ fying their charge as shown in reference [19]. These cationic nanoparticles contrast with alum regarding their small size and very low polydispersity as shown in Table 1 taken from reference [19]. The optimal bilayer coverage of polystyrene sulfate (PSS) nanoparticles with a DODAB bilayer produces homodisperse particles that successfully present a mixture of purified 18/14 *Taenia crassiceps* proteins (18/14-Tcra) to the immunological system [19]. presenting cells (APC) so that particles deliver antigens to APC more efficiently than soluble antigen [71,72].Positively charged particles with diameters of 500 nm and below were shown to be optimal for dendritic cells uptake [42].DODAB bilayers electrostatically combine with a vast variety of negatively charged biomolecules or biological structures [14]. Silica [17], latex [21,73,74] or hydrophobic drug particles [51,75] have been coated with DODAB with optimal bilayer deposition on particles achieved by coalescence of bilayer fragments at an adequate ionic strength [17,76]. Figure 5 shows how DODAB can cover oppositely charged polystyrene nanoparticles modifying their charge as shown in reference [19]. These cationic nanoparticles contrast with alum regarding their small size and very low polydispersity as shown in Table 1 taken from reference [19]. The optimal bilayer coverage of polystyrene sulfate (PSS) nanoparticles with a DODAB bilayer produces homodisperse particles that successfully present a mixture of purified 18/14 *Taenia crassiceps* proteins (18/14-Tcra) to the immunological system [19].

log [DODAB]

Mean diameter (nm)

Figure 5.Effect of [DODAB] (in mM) on mean z-average diameter (A) and zeta-potential (B) of PSS particles at 5 x 109 particles/mL, 25 C, in 1 mM NaCl. Bare particle diameter is 301 ± 2 nm. Regions I, II and III define particle charge, which is negative, zero and positive, respectively, from reference [19]. Reprinted from International Journal of Pharmaceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid bilayers for antigen presentation, 216-222.Copyright 2007, with permission from Elsevier. **Figure 5.** Effect of [DODAB] (in mM) on mean z-average diameter (A) and zeta-potential (B) of PSS particles at 5 x 109 particles/mL, 25 °C, in 1 mM NaCl. Bare particle diameter is 301 ± 2 nm. Regions I, II and III define particle charge, which is negative, zero and positive, respectively, from reference [19]. Reprinted from International Journal of Pharma‐ ceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid bilayers for antigen presentation, 216-222.Copyright 2007, with permission from Elsevier.

PSS - - 301 ± 2 -60 ± 1 0.064 ± 0.020 DODAB 2.00 - 81 ± 1 45 ± 2 0.230 ± 0.006 PSS/DODAB 0.01 - 309 ± 2 48 ± 2 0.040 ± 0.010 18/14-*Tcra* - 25 310 ± 5 -52 ± 1 0.214 ± 0.030 DODAB/18/14-*Tcra* 0.01 25 295 ± 3 6 ± 6 0.167 ± 0.023 PSS/DODAB/18/14-*Tcra* 0.01 25 328 ± 3 11 ± 8 0.060 ± 0.020 Al(OH)3 - - 883 ± 29 28 ± 3 0.381 ± 0.013 Al(OH)3/18/14-*Tcra* - 25 9574 ± 2361 -23 ± 1 0.525 ± 0.030

[Ag] (μg/mL)

Table 1.Physical properties of particles, DODAB dispersion, DODAB-covered particles, proteins and proteins/DODAB-

electron microscopy is 301±2 nm.The Al(OH)3 and PSS were tested at final concentration of 0.05 and 0.075 mg/mL, respectively,from reference [19]. Reprinted from International Journal of Pharmaceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid bilayers for antigen presentation, 216-222.

particles /mL; PSS particle diameter from transmission

Zeta-Potential

(mV) Polydispersity

**Dispersion [DODAB]**

Copyright 2007, with permission from Elsevier.

**(mM)**

**[Ag] (μg/mL)** **Mean diameter**

**Zeta-Potential**

**(mV) Polydispersity**

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 13

[80]. System‐

**(nm)**

**Table 1.** Physical properties of particles, DODAB dispersion, DODAB-covered particles, proteins and proteins/DODABcovered particles at 1 mM NaCl.Particles concentration is 5 x 109 particles /mL; PSS particle diameter from transmission electron microscopy is 301±2 nm.The Al(OH)3 and PSS were tested at final concentration of 0.05 and 0.075 mg/mL, respectively,from reference [19]. Reprinted from International Journal of Pharmaceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid bilayers for antigen presentation, 216-222.

Several cationic agents have been employed for DNA compaction such as cationic peptides and proteins [77], cationic lipids [78], cationic polyelectrolytes, cationic surfactants, or iron (III) [79].DNA compaction has also been used to model chromatin structure and its influence on gene expression. The self-assembled complex of basic histone proteins wrapped by approxi‐ mately two turns of DNA is a nucleosome, which is the building block in the chromatin structure where DNA of lengths on the order of meters suffers compaction into an∼10 μm diameter cell nucleous. Phage DNA, for example, is remarkable for its density of packing. In solution the 40 kbp T7 genome with its contour length of 13.6 μm might span a space several micrometers across and in an infected bacterium, ∼1 μm across. Thus, confinement to a 55 nm

atic studies on the physical chemistry of the association between cationic nanoparticles and DNA yield rather complex phase diagrams as a function of particle size and concentration [81,82].The way in which positively charged nanoparticles tie up DNA is not obvious, and mechanisms change dramatically with particle size [83]. Only the smallest (10 nm) particles allowed transcription to occur at intermediate loading densities. Larger particles shut tran‐ scription down rather abruptly [81]. Cationic nanoparticles have found many uses such as efficient cell transfection agents *in vitro* [84-86] and complexation with long-chained DNA as a simple model of chromatin for transcription studies [81, 87].The compaction of long duplex DNA by cationic nanoparticles (NP) used as a primary model of histone core particles has been systematically studied regarding the effect of salt concentration, particle size, and particle charge by means of single-molecule observations from fluorescence and transmission electron microscopy [87]. DNA compaction proceeds through the formation of beads-on-a-string structures of various morphologies with DNA adsorbed amount per particle depending weakly on NP concentration but increasing with particle size and being optimal at an inter‐

capsid represents a compaction marked by a density increase by a factor of ∼10<sup>4</sup>

PSS - - 301 ± 2 -60 ± 1 0.064 ± 0.020 DODAB 2.00 - 81 ± 1 45 ± 2 0.230 ± 0.006 PSS/DODAB 0.01 - 309 ± 2 48 ± 2 0.040 ± 0.010 18/14-*Tcra* - 25 310 ± 5 -52 ± 1 0.214 ± 0.030 DODAB/18/14-*Tcra* 0.01 25 295 ± 3 6 ± 6 0.167 ± 0.023 PSS/DODAB/18/14-*Tcra* 0.01 25 328 ± 3 11 ± 8 0.060 ± 0.020 Al(OH)3 - - 883 ± 29 28 ± 3 0.381 ± 0.013 Al(OH)3/18/14-*Tcra* - 25 9574 ± 2361 -23 ± 1 0.525 ± 0.030

covered particles at 1 mM NaCl.Particles concentration is 5 x 10<sup>9</sup>

(mM)

Copyright 2007, with permission from Elsevier.

Dispersion [DODAB]


Particles are finding a large variety of biomedical and pharmaceutical applications since their size scale can be similar to that of biomacromolecules (e.g., proteins, DNA) and structures (e.g., bacteria and viruses). Their utility for imaging, gene and drug delivery, and vaccine design is undeniable [69,70].Particulate systems are naturally targeted to antigen presenting cells (APC) so that particles deliver antigens to APC more efficiently than soluble antigen [71,72].Positively charged particles with diameters of 500 nm and below were shown to be optimal for dendritic cells uptake [42].DODAB bilayers electrostatically combine with a vast variety of negatively charged biomolecules or biological structures [14]. Silica [17], latex [21,73,74] or hydrophobic drug particles [51,75] have been coated with DODAB with optimal bilayer deposition on particles achieved by coalescence of bilayer fragments at an adequate ionic strength [17,76]. Figure 5 shows how DODAB can cover oppositely charged polystyrene nanoparticles modi‐ fying their charge as shown in reference [19]. These cationic nanoparticles contrast with alum regarding their small size and very low polydispersity as shown in Table 1 taken from reference [19]. The optimal bilayer coverage of polystyrene sulfate (PSS) nanoparticles with a DODAB bilayer produces homodisperse particles that successfully present a mixture of purified 18/14

presenting cells (APC) so that particles deliver antigens to APC more efficiently than soluble antigen [71,72].Positively charged particles with diameters of 500 nm and below were shown to be optimal for dendritic cells uptake [42].DODAB bilayers electrostatically combine with a vast variety of negatively charged biomolecules or biological structures [14]. Silica [17], latex [21,73,74] or hydrophobic drug particles [51,75] have been coated with DODAB with optimal bilayer deposition on particles achieved by coalescence of bilayer fragments at an adequate ionic strength [17,76]. Figure 5 shows how DODAB can cover oppositely charged polystyrene nanoparticles modifying their charge as shown in reference [19]. These cationic nanoparticles contrast with alum regarding their small size and very low polydispersity as shown in Table 1 taken from reference [19]. The optimal bilayer coverage of polystyrene sulfate (PSS) nanoparticles with a DODAB bilayer produces homodisperse particles that successfully present a mixture of purified 18/14 *Taenia* 

I II III

**+** + **+ +** + **+** + + + **+ + +**

Figure 5.Effect of [DODAB] (in mM) on mean z-average diameter (A) and zeta-potential (B) of PSS particles at 5 x 109 particles/mL, 25 C, in 1 mM NaCl. Bare particle diameter is 301 ± 2 nm. Regions I, II and III define particle charge, which is negative, zero and positive, respectively, from reference [19]. Reprinted from International Journal of Pharmaceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid

**Figure 5.** Effect of [DODAB] (in mM) on mean z-average diameter (A) and zeta-potential (B) of PSS particles at 5 x 109 particles/mL, 25 °C, in 1 mM NaCl. Bare particle diameter is 301 ± 2 nm. Regions I, II and III define particle charge, which is negative, zero and positive, respectively, from reference [19]. Reprinted from International Journal of Pharma‐ ceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid bilayers for


log (DDA mM)

log [DODAB]

PSS - - 301 ± 2 -60 ± 1 0.064 ± 0.020 DODAB 2.00 - 81 ± 1 45 ± 2 0.230 ± 0.006 PSS/DODAB 0.01 - 309 ± 2 48 ± 2 0.040 ± 0.010 18/14-*Tcra* - 25 310 ± 5 -52 ± 1 0.214 ± 0.030 DODAB/18/14-*Tcra* 0.01 25 295 ± 3 6 ± 6 0.167 ± 0.023 PSS/DODAB/18/14-*Tcra* 0.01 25 328 ± 3 11 ± 8 0.060 ± 0.020 Al(OH)3 - - 883 ± 29 28 ± 3 0.381 ± 0.013 Al(OH)3/18/14-*Tcra* - 25 9574 ± 2361 -23 ± 1 0.525 ± 0.030

Mean diameter (nm)

Table 1.Physical properties of particles, DODAB dispersion, DODAB-covered particles, proteins and proteins/DODAB-

electron microscopy is 301±2 nm.The Al(OH)3 and PSS were tested at final concentration of 0.05 and 0.075 mg/mL, respectively,from reference [19]. Reprinted from International Journal of Pharmaceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid bilayers for antigen presentation, 216-222.

particles /mL; PSS particle diameter from transmission

Zeta-Potential

(mV) Polydispersity

bilayers for antigen presentation, 216-222.Copyright 2007, with permission from Elsevier.

antigen presentation, 216-222.Copyright 2007, with permission from Elsevier.


0

Zeta Potential (mV)

40

300

350

1000

Mean Diameter (nm)

1100

1200

B


[Ag] (μg/mL)

(mM)

covered particles at 1 mM NaCl.Particles concentration is 5 x 10<sup>9</sup>

Copyright 2007, with permission from Elsevier.

Dispersion [DODAB]

*Taenia crassiceps* proteins (18/14-Tcra) to the immunological system [19].

A

*crassiceps* proteins (18/14-Tcra) to the immunological system [19].

12 Immune Response Activation

**Table 1.** Physical properties of particles, DODAB dispersion, DODAB-covered particles, proteins and proteins/DODABcovered particles at 1 mM NaCl.Particles concentration is 5 x 109 particles /mL; PSS particle diameter from transmission electron microscopy is 301±2 nm.The Al(OH)3 and PSS were tested at final concentration of 0.05 and 0.075 mg/mL, respectively,from reference [19]. Reprinted from International Journal of Pharmaceutics, 340 / 1–2, N. Lincopan, N.M. Espíndola, A.J. Vaz, A.M. Carmona-Ribeiro, Cationic supported lipid bilayers for antigen presentation, 216-222. Copyright 2007, with permission from Elsevier.

Several cationic agents have been employed for DNA compaction such as cationic peptides and proteins [77], cationic lipids [78], cationic polyelectrolytes, cationic surfactants, or iron (III) [79].DNA compaction has also been used to model chromatin structure and its influence on gene expression. The self-assembled complex of basic histone proteins wrapped by approxi‐ mately two turns of DNA is a nucleosome, which is the building block in the chromatin structure where DNA of lengths on the order of meters suffers compaction into an∼10 μm diameter cell nucleous. Phage DNA, for example, is remarkable for its density of packing. In solution the 40 kbp T7 genome with its contour length of 13.6 μm might span a space several micrometers across and in an infected bacterium, ∼1 μm across. Thus, confinement to a 55 nm capsid represents a compaction marked by a density increase by a factor of ∼10<sup>4</sup> [80]. System‐ atic studies on the physical chemistry of the association between cationic nanoparticles and DNA yield rather complex phase diagrams as a function of particle size and concentration [81,82].The way in which positively charged nanoparticles tie up DNA is not obvious, and mechanisms change dramatically with particle size [83]. Only the smallest (10 nm) particles allowed transcription to occur at intermediate loading densities. Larger particles shut tran‐ scription down rather abruptly [81]. Cationic nanoparticles have found many uses such as efficient cell transfection agents *in vitro* [84-86] and complexation with long-chained DNA as a simple model of chromatin for transcription studies [81, 87].The compaction of long duplex DNA by cationic nanoparticles (NP) used as a primary model of histone core particles has been systematically studied regarding the effect of salt concentration, particle size, and particle charge by means of single-molecule observations from fluorescence and transmission electron microscopy [87]. DNA compaction proceeds through the formation of beads-on-a-string structures of various morphologies with DNA adsorbed amount per particle depending weakly on NP concentration but increasing with particle size and being optimal at an inter‐ mediate salt concentration [87]. Three different complexation mechanisms were proposed: free DNA adsorption onto NP surface, DNA wrapping around NP, and NP collection on DNA chain [87]. On the other hand, particle size has been recognized as an important parameter that determines the mechanism of particle entry into cells. Particles with a diameter of 200 nm or less enter cells almost exclusively via the clathrin-coated pathway whereas particles with a larger diameter penetrate cells via caveolae-mediated endocytosis [88,89].Cationic biomimetic particles produced from adsorption of dioctadecyldimethylammonium bromide (DODAB) bilayers onto polystyrene sulfate (PSS) microspheres have been described by our group since 1992 [18,73,76,90].These cationic bilayer-covered particles exhibit a narrow size distribution and can be produced at any desired size ranging from 70-500 nm of mean hydrodynamic diameter [90]. Polystyrene sulfate (PSS) particles with different sizes were covered by a dioctadecyldimethylammonium bromide (DODAB) bilayer yielding the so-called cationic biomimetic particles (PSS/DODAB). These cationic particles are highly organized, present a narrow size distribution and were obtained over a range of particle sizes [53,90].Thereafter, upon adding λ, T5 or T2-DNA to PSS/DODAB particles, supramolecular assemblies PSS/ DODAB/DNA were obtained and characterized over a range of DNA concentrations and particle sizes (80-700 nm). Over the low DNA concentration range, PSS/DODAB/DNA assemblies were cationic, colloidally stable with moderate polydispersity and high cytotoxicity against *E. coli*. From the DNA concentration corresponding to charge neutralization, neutral or anionic supramolecular assemblies PSS/DODAB/DNA exhibited low colloid stability, high polydispersity and moderate cytotoxicity [53]. Some nucleosome mimetic assemblies were observed by atomic force microscopy (AFM) at charge neutralization (zeta-potential equal to zero) [55].Figure 6 shows how cationic nanoparticles can induce DNA compaction [53].

DNA sequences containing unmethylated CpG dinucleotide are recognized as danger signals by the immune system since they are typical of bacteria and viruses but rare in vertebrates [91, 92]. Natural or synthetic sequences containing unmethylated CpG motifs activate cells that express Toll-like receptor 9 to induce an innate immune response characterized by the production of Th1 and proinflammatory cytokines [92]. Hence, CpG has been extensively used in the induction of cellular immune responses against cancer [63, 93], intracellular infections by pathogens [94, 95] and allergies [62,96]. Both CpG [92] and DODAB BF [27] were reported to improve Th1 responses against antigens when used separately. Recently, DODAB BF and CpG were combined in a single assembly aiming at the comparison between the small, stable and cationic DODAB BF carrying ovalbumin (OVA) and the small, stable and anionic DODAB BF/OVA/CpG assemblies of very similar sizes but opposite charges [97]. Both adjuvants produced similar enhanced Th1 immune responses despite their opposite charges emphasiz‐ ing the novel concept that particle charge does not matter. In comparison with the traditional alum, the size minimization for the cationic assemblies elicited different responses: alum drove the Th2 whereas DODAB BF/OVA/CpG and DODAB BF/OVA drove the Th1 response. The effects of DODAB BF or DODAB BF/CpG adjuvants with opposite charges but very similar sizes showed that the charge is not important but the size is [97]. Table 2 shows the physical properties of the assemblies. A comparison between mean hydrodynamic diameter (Dz), zetapotentials and polydispersities for DODAB BF/OVA/CpG and Al(OH)3/OVA/CpG as repro‐ duced from reference [97]. At 20 μM CpG and 0.1 mg/mL Al(OH)3, the dispersion was characterized by a low colloidal stability (large Dz, low zeta-potential (ζ) and high polydis‐ persity). OVA stabilized both Al(OH)3 or Al(OH)3/CpG to a certain extent (Table 2). However, sizes were still larger than those determined for assemblies based on DODAB BF (Table 2). In particular, polydispersity for DODAB BF/OVA/CpG was notably low (0.150) and well below the one obtained for DODAB BF only (0.251), suggesting that OVA/CpG induced stabilization

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 15

**Dispersion Dz** ± **δ (nm) ζ** ± **δ (mV) Polydispersity** ± **δ** DODAB BF 67 ± 0 47 ± 1 0.251 ± 0.006 DODAB BF/ OVA 274 ± 2 21 ± 0 0.291 ± 0.008 DODAB BF/ OVA/ CpG 245 ± 1 -26 ± 1 0.150 ± 0.020

Al(OH)3 3147 ± 197 16 ± 2 0.415 ± 0.018 Al(OH)3/ OVA 916 ± 17 -29 ± 1 0.231 ± 0.020 Al(OH)3/ CpG 3584 ± 74 9 ± 1 0.407 ± 0.026 Al(OH)3/ OVA/ CpG 570 ± 19 -35 ± 2 0.210 ± 0.020

**Table 2.** Physical properties of alum or DODAB BF dispersions combined with ovalbumin (OVA) and/or CpG

oligonucleotide. Concentrations are 0.1mg/mL OVA, 0.1mM DODAB BF, 0.1mg/mL Al(OH)3 and 20μM CpG. Reprinted from reference [97]. Reprinted from Journal of Controlled Release, 160/2, Julio H.K. Rozenfeld, Sandriana R. Silva, Priscila A. Ranéia, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in vivo*, 367-373.Copyright 2012, with permission from

of the DODAB BF dispersion (Table 2).

Elsevier.

**Figure 6.** DNA compaction by biomimetic cationic particles of polystyrene microspheres covered by a DODAB cationic bilayer from reference [53].Adapted with permission from Rosa H, Petri DF, Carmona-Ribeiro AM.Interactions be‐ tween bacteriophage DNA and cationic biomimetic particles. J Phys Chem B. 2008; 112(51):16422-30.Copyright (2008) American Chemical Society.

DNA sequences containing unmethylated CpG dinucleotide are recognized as danger signals by the immune system since they are typical of bacteria and viruses but rare in vertebrates [91, 92]. Natural or synthetic sequences containing unmethylated CpG motifs activate cells that express Toll-like receptor 9 to induce an innate immune response characterized by the production of Th1 and proinflammatory cytokines [92]. Hence, CpG has been extensively used in the induction of cellular immune responses against cancer [63, 93], intracellular infections by pathogens [94, 95] and allergies [62,96]. Both CpG [92] and DODAB BF [27] were reported to improve Th1 responses against antigens when used separately. Recently, DODAB BF and CpG were combined in a single assembly aiming at the comparison between the small, stable and cationic DODAB BF carrying ovalbumin (OVA) and the small, stable and anionic DODAB BF/OVA/CpG assemblies of very similar sizes but opposite charges [97]. Both adjuvants produced similar enhanced Th1 immune responses despite their opposite charges emphasiz‐ ing the novel concept that particle charge does not matter. In comparison with the traditional alum, the size minimization for the cationic assemblies elicited different responses: alum drove the Th2 whereas DODAB BF/OVA/CpG and DODAB BF/OVA drove the Th1 response. The effects of DODAB BF or DODAB BF/CpG adjuvants with opposite charges but very similar sizes showed that the charge is not important but the size is [97]. Table 2 shows the physical properties of the assemblies. A comparison between mean hydrodynamic diameter (Dz), zetapotentials and polydispersities for DODAB BF/OVA/CpG and Al(OH)3/OVA/CpG as repro‐ duced from reference [97]. At 20 μM CpG and 0.1 mg/mL Al(OH)3, the dispersion was characterized by a low colloidal stability (large Dz, low zeta-potential (ζ) and high polydis‐ persity). OVA stabilized both Al(OH)3 or Al(OH)3/CpG to a certain extent (Table 2). However, sizes were still larger than those determined for assemblies based on DODAB BF (Table 2). In particular, polydispersity for DODAB BF/OVA/CpG was notably low (0.150) and well below the one obtained for DODAB BF only (0.251), suggesting that OVA/CpG induced stabilization of the DODAB BF dispersion (Table 2).

mediate salt concentration [87]. Three different complexation mechanisms were proposed: free DNA adsorption onto NP surface, DNA wrapping around NP, and NP collection on DNA chain [87]. On the other hand, particle size has been recognized as an important parameter that determines the mechanism of particle entry into cells. Particles with a diameter of 200 nm or less enter cells almost exclusively via the clathrin-coated pathway whereas particles with a larger diameter penetrate cells via caveolae-mediated endocytosis [88,89].Cationic biomimetic particles produced from adsorption of dioctadecyldimethylammonium bromide (DODAB) bilayers onto polystyrene sulfate (PSS) microspheres have been described by our group since 1992 [18,73,76,90].These cationic bilayer-covered particles exhibit a narrow size distribution and can be produced at any desired size ranging from 70-500 nm of mean hydrodynamic diameter [90]. Polystyrene sulfate (PSS) particles with different sizes were covered by a dioctadecyldimethylammonium bromide (DODAB) bilayer yielding the so-called cationic biomimetic particles (PSS/DODAB). These cationic particles are highly organized, present a narrow size distribution and were obtained over a range of particle sizes [53,90].Thereafter, upon adding λ, T5 or T2-DNA to PSS/DODAB particles, supramolecular assemblies PSS/ DODAB/DNA were obtained and characterized over a range of DNA concentrations and particle sizes (80-700 nm). Over the low DNA concentration range, PSS/DODAB/DNA assemblies were cationic, colloidally stable with moderate polydispersity and high cytotoxicity against *E. coli*. From the DNA concentration corresponding to charge neutralization, neutral or anionic supramolecular assemblies PSS/DODAB/DNA exhibited low colloid stability, high polydispersity and moderate cytotoxicity [53]. Some nucleosome mimetic assemblies were observed by atomic force microscopy (AFM) at charge neutralization (zeta-potential equal to zero) [55].Figure 6 shows how cationic nanoparticles can induce DNA compaction [53].

**Figure 6.** DNA compaction by biomimetic cationic particles of polystyrene microspheres covered by a DODAB cationic bilayer from reference [53].Adapted with permission from Rosa H, Petri DF, Carmona-Ribeiro AM.Interactions be‐ tween bacteriophage DNA and cationic biomimetic particles. J Phys Chem B. 2008; 112(51):16422-30.Copyright

(2008) American Chemical Society.

14 Immune Response Activation


**Table 2.** Physical properties of alum or DODAB BF dispersions combined with ovalbumin (OVA) and/or CpG oligonucleotide. Concentrations are 0.1mg/mL OVA, 0.1mM DODAB BF, 0.1mg/mL Al(OH)3 and 20μM CpG. Reprinted from reference [97]. Reprinted from Journal of Controlled Release, 160/2, Julio H.K. Rozenfeld, Sandriana R. Silva, Priscila A. Ranéia, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in vivo*, 367-373.Copyright 2012, with permission from Elsevier.

At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] established 0.1 mM DODAB as suitable for obtaining stable and cationic DODAB BF/ OVA assemblies [97]. At 0.1 mM DODAB, 0.1 mg/mL OVA and 0.006 mM CpG, the zetapotential is zero showing charge neutralization [97]. At [CpG]> 0.006 mM, good colloidal stability for the anionic assemblies due to charge overcompensation was observed whereas at 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies turned out to be highly effective *in vivo* generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA delayed-type hypersensitivity (DTH) reaction and the secretion of IFN-gamma and IL-12 resulted 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively [97].Figure 7 A and B illustrate the colloidal stability of the assemblies over a range of DODAB concentrations.

the secretion of IFN-γ and IL-12 increased by 33 and 49 %, respectively when compared to DODAB BF/OVA-immunized mice group and 52% and 35% when compared to OVA/CpGgroup. In contrast, the highest secretion of IL-10 and IL-13 was observed in cultures of cells from mice that were immunized with Al(OH)3 /OVA whereas all other assemblies resulted in poor production of these cytokines [97]. Immune responses were similar for anionic DODAB BF/OVA/CpG and cationic DODAB BF/OVA of similar sizes showing that the charge is not important but the size is. The adsorption of antigen on the surface of DODAB large vesicles was shown to stimulate active antigen capture and presentation by dendritic cells (DCs) [41]. Administration of antigen adsorbed on DODAB large vesicles (LV) resulted in formation of an antigen depot at the site of injection which hampered the rapid clearance of antigen that takes place in absence of a carrier [100]. In contrast to DODAB LV, the small DODAB BF/ antigen assemblies did not result in any observable depot effect [27, 97]. Furthermore, since the depot was absent for DODAB BF/CpG/OVA the immunostimulatory effect must have occurred via direct effect of the assemblies on the lymphonode antigen presenting cells [97]. Only nanoparticles can specifically target lymph node-resident cells [101]. CpG combined with DODAB BF yielded improved cellular Th1 response [97] possibly due to the appropriate targeting of DODAB BF/CpG/antigen to DCs in charge of expressing endosomal toll like receptor 9 (TLR9) in the lymph nodes [102]. The enhanced Th1 response by anionic DODAB BF/OVA/CpG relative to OVA alone was evidenced by the 6 times increase in DTH reaction, the 42 times increase in IFN-γ secretion by lymph node cells in culture and the 9 times increase

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 17

in IL-12 secretion also by lymph node cells in culture (Figure 7) [97].

optimal physical properties for efficient antigen presentation [106].

deacetylated to yield chitosan as shown in Figure 8.

**polymers**

Other delivery systems based on anionic [103] or cationic lipid bilayer [41,44,100,104,105] have also been successful for improving Th1 response against important antigens such as those of influenza [103], hepatitis A and B [103,104], and fungal infections [105]. In general, cationic lipids are known for the production of a large inflammatory response [41, 44].However, for small cationic bilayer fragments as immunoadjuvants, this adverse reaction is absent [27, 97]. The small and anionic DODAB BF/OVA/CpG assemblies [97] also did not elicit adverse reactions similarly to other anionic assemblies [103]. There was no depot effect for these small assemblies [97] and their net negative charge ensured the absence of the adverse reactions observed previously for the large cationic liposomes and vesicles [100]. Recently, based on cross reactivity with *Neisseria lactamica* outer membrane vesicles (OMV) antigens, DODAB BF were combined with OMV to develop a vaccine against *Neisseria meningitidis* in young children [106]. Complexes of 25 μg of OMV in 0.1 mM of DODAB BF were colloidally stable, exhibiting

**3. Cationic nanostructures based on chitosan and other biocompatible**

Chitin is a long-chain polymer of N-acetylglucosamine, a derivative of glucose which can be

The delayed-type hypersensitivity reaction (DTH) is an important *in vivo* response mediated by cells that can be quantified from the footpad sweelling test [44]. Mice immunized with OVA alone or with DODAB BF/CpG or with OVA / Al(OH)3 exhibited a footpad swelling equal to the one observed for naive mice [97]. The largest increase in footpad swelling was observed for DODAB BF/OVA/CpG mice immunization, which was about 6 times larger than the one observed for naive mice and 1.3 times larger than the one observed for DODAB BF/OVA [97]. Figure 7 C shows the improved DTH response in mice induced by the assemblies as adapted from reference [97].

Since OVA isoelectric point is 4.5 [98], this protein is negatively charged at 6.3, the pH of water. Thus, OVA adsorption onto DODAB BF is initially electrostatically driven.The OVA titration with DODAB BF determined ranges of DODAB concentration for occurrence of stable DODAB BF/OVA assemblies as illustrated in Figure 7 A and B. Similar results had been previously described also for DODAB BF/ bovin serum albumin or DODAB BF/ purified antigens from *Taenia crassiceps* [27].When the net charge of the assemblies is zero (at charge neutralization), maximal aggregation was observed for the assemblies. Further increasing DODAB concen‐ tration, stabilized them to a certain extent (Figure 7 B). However, sizes and polydispersities were still higher than those of DODAB BF in absence of OVA. Optimal colloidal stability was only achieved upon CpG addition to the system yielding high and negative zeta-potentials plus remarkably small polydispersity [98]. Oligonucleotides with less than 20 nucleotide residues usually behave as rigid charged rods in solution [99]. CpG would also adsorb as rigid charged rods on vacant positive sites of DODAB BF/ OVA assemblies inducing charge overcompensation and recovery of colloidal stability as previously described for a model oligonucleotide above charge neutralization [56].

Figure 7 also illustrates the improvement in the cellular OVA-specific response from the analysis of cytokines secreted by lymph node cells of mice as adapted from reference [97]. IFNγ and IL-12 production is associated to the Th1 response whereas IL-10 and IL-13 production reflects the Th2 response. Levels of IFN-γ and IL-12 secretion observed in cell cultures of mice immunized with OVA or Al(OH)3/ OVA were low and close to the detection limit for these cytokine assays (Figure 7). For mice immunized with OVA/CpG or DODAB BF/OVA assem‐ blies, the secretion of IFN-γ and IL-12 substantially increased in comparison to secretion from cultured cells of OVA-immunized mice [97]. For the DODAB BF/OVA/CpG immunized mice, the secretion of IFN-γ and IL-12 increased by 33 and 49 %, respectively when compared to DODAB BF/OVA-immunized mice group and 52% and 35% when compared to OVA/CpGgroup. In contrast, the highest secretion of IL-10 and IL-13 was observed in cultures of cells from mice that were immunized with Al(OH)3 /OVA whereas all other assemblies resulted in poor production of these cytokines [97]. Immune responses were similar for anionic DODAB BF/OVA/CpG and cationic DODAB BF/OVA of similar sizes showing that the charge is not important but the size is. The adsorption of antigen on the surface of DODAB large vesicles was shown to stimulate active antigen capture and presentation by dendritic cells (DCs) [41]. Administration of antigen adsorbed on DODAB large vesicles (LV) resulted in formation of an antigen depot at the site of injection which hampered the rapid clearance of antigen that takes place in absence of a carrier [100]. In contrast to DODAB LV, the small DODAB BF/ antigen assemblies did not result in any observable depot effect [27, 97]. Furthermore, since the depot was absent for DODAB BF/CpG/OVA the immunostimulatory effect must have occurred via direct effect of the assemblies on the lymphonode antigen presenting cells [97]. Only nanoparticles can specifically target lymph node-resident cells [101]. CpG combined with DODAB BF yielded improved cellular Th1 response [97] possibly due to the appropriate targeting of DODAB BF/CpG/antigen to DCs in charge of expressing endosomal toll like receptor 9 (TLR9) in the lymph nodes [102]. The enhanced Th1 response by anionic DODAB BF/OVA/CpG relative to OVA alone was evidenced by the 6 times increase in DTH reaction, the 42 times increase in IFN-γ secretion by lymph node cells in culture and the 9 times increase in IL-12 secretion also by lymph node cells in culture (Figure 7) [97].

At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] established 0.1 mM DODAB as suitable for obtaining stable and cationic DODAB BF/ OVA assemblies [97]. At 0.1 mM DODAB, 0.1 mg/mL OVA and 0.006 mM CpG, the zetapotential is zero showing charge neutralization [97]. At [CpG]> 0.006 mM, good colloidal stability for the anionic assemblies due to charge overcompensation was observed whereas at 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies turned out to be highly effective *in vivo* generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA delayed-type hypersensitivity (DTH) reaction and the secretion of IFN-gamma and IL-12 resulted 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively [97].Figure 7 A and B illustrate

The delayed-type hypersensitivity reaction (DTH) is an important *in vivo* response mediated by cells that can be quantified from the footpad sweelling test [44]. Mice immunized with OVA alone or with DODAB BF/CpG or with OVA / Al(OH)3 exhibited a footpad swelling equal to the one observed for naive mice [97]. The largest increase in footpad swelling was observed for DODAB BF/OVA/CpG mice immunization, which was about 6 times larger than the one observed for naive mice and 1.3 times larger than the one observed for DODAB BF/OVA [97]. Figure 7 C shows the improved DTH response in mice induced by the assemblies as adapted

Since OVA isoelectric point is 4.5 [98], this protein is negatively charged at 6.3, the pH of water. Thus, OVA adsorption onto DODAB BF is initially electrostatically driven.The OVA titration with DODAB BF determined ranges of DODAB concentration for occurrence of stable DODAB BF/OVA assemblies as illustrated in Figure 7 A and B. Similar results had been previously described also for DODAB BF/ bovin serum albumin or DODAB BF/ purified antigens from *Taenia crassiceps* [27].When the net charge of the assemblies is zero (at charge neutralization), maximal aggregation was observed for the assemblies. Further increasing DODAB concen‐ tration, stabilized them to a certain extent (Figure 7 B). However, sizes and polydispersities were still higher than those of DODAB BF in absence of OVA. Optimal colloidal stability was only achieved upon CpG addition to the system yielding high and negative zeta-potentials plus remarkably small polydispersity [98]. Oligonucleotides with less than 20 nucleotide residues usually behave as rigid charged rods in solution [99]. CpG would also adsorb as rigid charged rods on vacant positive sites of DODAB BF/ OVA assemblies inducing charge overcompensation and recovery of colloidal stability as previously described for a model

Figure 7 also illustrates the improvement in the cellular OVA-specific response from the analysis of cytokines secreted by lymph node cells of mice as adapted from reference [97]. IFNγ and IL-12 production is associated to the Th1 response whereas IL-10 and IL-13 production reflects the Th2 response. Levels of IFN-γ and IL-12 secretion observed in cell cultures of mice immunized with OVA or Al(OH)3/ OVA were low and close to the detection limit for these cytokine assays (Figure 7). For mice immunized with OVA/CpG or DODAB BF/OVA assem‐ blies, the secretion of IFN-γ and IL-12 substantially increased in comparison to secretion from cultured cells of OVA-immunized mice [97]. For the DODAB BF/OVA/CpG immunized mice,

the colloidal stability of the assemblies over a range of DODAB concentrations.

from reference [97].

16 Immune Response Activation

oligonucleotide above charge neutralization [56].

Other delivery systems based on anionic [103] or cationic lipid bilayer [41,44,100,104,105] have also been successful for improving Th1 response against important antigens such as those of influenza [103], hepatitis A and B [103,104], and fungal infections [105]. In general, cationic lipids are known for the production of a large inflammatory response [41, 44].However, for small cationic bilayer fragments as immunoadjuvants, this adverse reaction is absent [27, 97]. The small and anionic DODAB BF/OVA/CpG assemblies [97] also did not elicit adverse reactions similarly to other anionic assemblies [103]. There was no depot effect for these small assemblies [97] and their net negative charge ensured the absence of the adverse reactions observed previously for the large cationic liposomes and vesicles [100]. Recently, based on cross reactivity with *Neisseria lactamica* outer membrane vesicles (OMV) antigens, DODAB BF were combined with OMV to develop a vaccine against *Neisseria meningitidis* in young children [106]. Complexes of 25 μg of OMV in 0.1 mM of DODAB BF were colloidally stable, exhibiting optimal physical properties for efficient antigen presentation [106].

### **3. Cationic nanostructures based on chitosan and other biocompatible polymers**

Chitin is a long-chain polymer of N-acetylglucosamine, a derivative of glucose which can be deacetylated to yield chitosan as shown in Figure 8.

node cells in culture (Figure 7) [97].

above charge neutralization [56].

chitosan molecules, a new water-soluble compound, glycated chitosan (GC), was synthesized [109,110]. GC is a non-toxic biodegradable product used in laser immunotherapy (LIT) which combines local laser irradiation and local administration of GC at primary or metastatic cancers where the tumoral antigens of the irradiated tumor cells combined with GC elicit a potent immune response against the cancer [111,112]. After the first step involving tumor irradiation with a laser beam that causes swelling and disruption of tumor cells by thermal effect, the local injection of GC as immunoadjuvant would lead to the capture of the tumoral antigens by dendritic cells and migration to the lymph nodes where the antigens would be presented to T cells, thus activating cytotoxic T-lymphocytes [113-115]. LIT using GC induced regression of primary and secondary tumours in rats and caused resistance to repeated challenges with tumours of the same type [116]. Furthermore, rats developed immunity could be adoptively

Since they are biocompatible, biodegradable by deacetylases, mucoadhesive, and nontoxic, with antimicrobial, antiviral, and adjuvant properties, chitin, chitosan and their derivatives have been widely applied in medicine, pharmacy and vaccine design [107, 108]. Chitosan is soluble in diluted acids but is insoluble in water due to deprotonation of its amino moiety [109]. The poor solubility of chitosan at the pH of water represents a serious limitation for its applications as an immunoadjuvant in the clinics [107]. Several chitosan derivatives have been obtained to circumvent this limitation.For example, by attaching galactose moieties to the chitosan molecules, a new water-soluble compound, glycated chitosan (GC), was synthesized [109,110]. GC is a non-toxic biodegradable product used in laser immunotherapy (LIT) which combines local laser irradiation and local administration of GC at primary or metastatic cancers where the tumoral antigens of the irradiated tumor cells combined with GC elicit a potent immune response against the cancer [111,112]. After the first step involving tumor irradiation with a laser beam that causes swelling and disruption of tumor cells by thermal effect, the local injection of GC as immunoadjuvant would lead to the capture of the tumoral antigens by dendritic cells and migration to the lymph nodes where the antigens would be presented to T cells, thus activating cytotoxic Tlymphocytes [113-115]. LIT using GC induced regression of primary and secondary tumours in rats and caused resistance to repeated challenges with tumours of the same type [116]. Furthermore, rats developed immunity could be

seen from the kinetics obtained after adding DODAB BF at a final concentration of 0.005 (); 0.01 (); 0.02 (); 0.05 (); 0.1 (); 0.2 (); 0.5 () and 1mM DODAB () to 0.1mg/mL OVA. In (B), kinetical data are detailed for the larger DODAB BF concentrations.Assemblies were prepared in 1mM NaCl.In (C), delayed-type hypersensitivity response for BALB/c mice immunized with OVA in different adjuvant formulations determined from the footpad swelling (nm) standard error of the mean. Final concentrations are 0.1mM DODAB BF, 20M CpG and 0.1mg/mL Al(OH)3 and 0.1mg/mL OVA. p< 0.05 compared to naive (), p< 0.05 compared to OVA/Al(OH)3 (#) and p< 0.05 compared to OVA/DODAB/CpG () from reference [97].Adapted from Journal of Controlled Release, 160/2, Julio H.K. Rozenfeld, Sandriana R. Silva, Priscila A. Ranéia, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in vivo*, 367-373 .Copyright 2012,

Other delivery systems based on anionic [103] or cationic lipid bilayer [41,44,100,104,105] have also been successful for improving Th1 response against important antigens such as those of influenza [103], hepatitis A and B [103,104], and fungal infections [105]. In general, cationic lipids are known for the production of a large inflammatory response [41, 44].However, for small cationic bilayer fragments as immunoadjuvants, this adverse reaction is absent [27, 97]. The small and anionic DODAB BF/OVA/CpG assemblies [97] also did not elicit adverse reactions similarly to other anionic assemblies [103]. There was no depot effect for these small assemblies [97] and their net negative charge ensured the absence of the adverse reactions observed previously for the large cationic liposomes and vesicles [100]. Recently, based on cross reactivity with *Neisseria lactamica* outer membrane vesicles (OMV) antigens, DODAB BF were combined with OMV to develop a vaccine against *Neisseria meningitidis* in young children [106]. Complexes of 25 µg of OMV in 0.1 mM of DODAB BF were colloidally stable, exhibiting optimal physical properties for efficient antigen presentation [106].

Chitin is a long-chain polymer of N-acetylglucosamine, a derivative of glucose which can be deacetylated to yield

deacetylation

**3. Cationic nanostructures based on chitosan and other biocompatible polymers.**

Chitosan nanoparticles have been obtained by ionotropic gelation, complex coacervation, emulsion and microemulsion techniques, and self-assembly of hydrophobically modified chitosan [117]. Ionotropic gelation consists of the ionic crosslinking of chitosan with multiva‐ lent counter-ions such as sodium tripolyphosphate (TPP) by adding a dilute chitosan acid solution to a solution of TPP or vice versa, with stirring [118]. Chitosan particles of nanometric size were obtained for chitosan concentrations up to 2.8 g L−1 and TPP concentrations from 0.21 to 0.43 g L−1. The size and surface charge of particles can be modified by varying the ratio of chitosan and stabilizer. The main problematic aspects of the technique are the poor colloidal stability of the dispersion which may require the addition of stabilizers, and the need of using very dilute solutions which may be inconvenient when large amounts of nanoparticles are required [117]. Complex coacervation is achieved by mixing two oppositely charged polye‐ lectrolytes. The polyelectrolyte or coacervate complex is structured as nanoparticles. Chitosan– poly (acrylic acid) (PAA) nanoparticles carrying a positive charge with sizes from 50 to 400 nm were obtained by the dropwise addition of dilute chitosan solutions [119]. Carboxyme‐ thylcellulose and alginate have also been complexed with chitosan to prepare nanoparticles. Chitosan–carboxymethylcellulose nanoparticles were subsequently coated with plasmid DNA (pDNA) [120]. Chitosan–alginate nanoparticles were loaded with insulin [121]. Nanoparticles have also been obtained in which the polyanion is the active principle itself as for example heparin or DNA or even siRNA. Chitosan–heparin nanoparticles crosslinked with TPP were described [122]. Figure 9 shows on the left a scanning electron micrograph of chitosan– heparin nanoparticles adapted from reference [117] and, on the right, an atomic force micrograph of

particles can be modified by varying the ratio of chitosan and stabilizer. The main problematic aspects of the technique are the poor colloidal stability of the dispersion which may require the addition of stabilizers, and the need of using very dilute solutions which may be inconvenient when large amounts of nanoparticles are required [117]. Complex coacervation is achieved by mixing two oppositely charged polyelectrolytes. The polyelectrolyte or coacervate complex is structured as nanoparticles. Chitosan–poly (acrylic acid) (PAA) nanoparticles carrying a positive charge with sizes from

and TPP concentrations from 0.21 to 0.43 g L<sup>−</sup><sup>1</sup>

. The size and surface charge of

Cationic Nanostructures for Vaccines http://dx.doi.org/10.5772/57543 19

Chitosan nanoparticles have been obtained by ionotropic gelation, complex coacervation, emulsion and microemulsion techniques, and self-assembly of hydrophobically modified chitosan [117]. Ionotropic gelation consists of the ionic crosslinking of chitosan with multivalent counter-ions such as sodium tripolyphosphate (TPP) by adding a dilute chitosan acid solution to a solution of TPP or vice versa, with stirring [118]. Chitosan particles of nanometric size were obtained for

transferred [116].

adoptively transferred [116].

chitosan concentrations up to 2.8 g L<sup>−</sup><sup>1</sup>

with permission from Elsevier.

chitosan as shown in Figure 8.

Figure 8.Chitin yielding chitosan by deacetylation.

**Figure 8.** Chitin yielding chitosan by deacetylation.

inducing charge overcompensation and recovery of colloidal stability as previously described for a model oligonucleotide

Figure 7 also illustrates the improvement in the cellular OVA-specific response from the analysis of cytokines secreted by lymph node cells of mice as adapted from reference [97]. IFN-γ and IL-12 production is associated to the Th1 response whereas IL-10 and IL-13 production reflects the Th2 response. Levels of IFN- γ and IL-12 secretion observed in cell cultures of mice immunized with OVA or Al(OH)3/ OVA were low and close to the detection limit for these cytokine assays (Figure 7). For mice immunized with OVA/CpG or DODAB BF/OVA assemblies, the secretion of IFN- γ and IL-12 substantially increased in comparison to secretion from cultured cells of OVA-immunized mice [97]. For the DODAB BF/OVA/CpG immunized mice, the secretion of IFN- γ and IL-12 increased by 33 and 49 %, respectively when compared to DODAB BF/OVA-immunized mice group and 52% and 35% when compared to OVA/CpG-group. In contrast, the highest secretion of IL-10 and IL-13 was observed in cultures of cells from mice that were immunized with Al(OH)3 /OVA whereas all other assemblies resulted in poor production of these cytokines [97]. Immune responses were similar for anionic DODAB BF/OVA/CpG and cationic DODAB BF/OVA of similar sizes showing that the charge is not important but the size is. The adsorption of antigen on the surface of DODAB large vesicles was shown to stimulate active antigen capture and presentation by dendritic cells (DCs) [41]. Administration of antigen adsorbed on DODAB large vesicles (LV) resulted in formation of an antigen depot at the site of injection which hampered the rapid clearance of antigen that takes place in absence of a carrier [100]. In contrast to DODAB LV, the small DODAB BF/antigen assemblies did not result in any observable depot effect [27, 97]. Furthermore, since the depot was absent for DODAB BF/CpG/OVA the immunostimulatory effect must have occurred via direct effect of the assemblies on the lymphonode antigen presenting cells [97]. Only nanoparticles can specifically target lymph node -resident cells [101]. CpG combined with DODAB BF

anionic DODAB BF/OVA/CpG relative to OVA alone was evidenced by the 6 times increase in DTH reaction, the 42 times increase in IFN-γ secretion by lymph node cells in culture and the 9 times increase in IL-12 secretion also by lymph

*vivo*. In (A), the effect of time and DODAB concentration on zeta-average diameter of DODAB BF/OVA assemblies is **Figure 7.** Nanostructures of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant ac‐ tivity *in vivo*. In (A), the effect of time and DODAB concentration on zeta-average diameter of DODAB BF/OVA assem‐ blies is seen from the kinetics obtained after adding DODAB BF at a final concentration of 0.005 (∎); 0.01 (○); 0.02 (▲); 0.05 (▽); 0.1 (◆); 0.2 (□); 0.5 (●) and 1mM DODAB (△) to 0.1mg/mL OVA. In (B), kinetical data are detailed for the larger DODAB BF concentrations.Assemblies were prepared in 1mM NaCl.In (C), delayed-type hypersensitivity re‐ sponse for BALB/c mice immunized with OVA in different adjuvant formulations determined from the footpad swel‐ ling (nm) ± standard error of the mean. Final concentrations are 0.1mM DODAB BF, 20μM CpG and 0.1mg/mL Al(OH)<sup>3</sup> and 0.1mg/mL OVA. p< 0.05 compared to naive (○), p< 0.05 compared to OVA/Al(OH)3 (#) and p< 0.05 compared to OVA/DODAB/CpG (△) from reference [97].Adapted from Journal of Controlled Release, 160/2, Julio H.K. Rozenfeld, Sandriana R. Silva, Priscila A. Ranéia, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Stable assemblies of cationic bi‐ layer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in vivo*, 367-373.Copyright 2012, with permission from Elsevier.

Figure 7.Nanostructures of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in* 

Since they are biocompatible, biodegradable by deacetylases, mucoadhesive, and nontoxic, with antimicrobial, antiviral, and adjuvant properties, chitin, chitosan and their derivatives have been widely applied in medicine, pharmacy and vaccine design [107, 108]. Chitosan is soluble in diluted acids but is insoluble in water due to deprotonation of its amino moiety [109]. The poor solubility of chitosan at the pH of water represents a serious limitation for its applications as an immunoadjuvant in the clinics [107]. Several chitosan derivatives have been obtained to circumvent this limitation.For example, by attaching galactose moieties to the

Since they are biocompatible, biodegradable by deacetylases, mucoadhesive, and nontoxic, with antimicrobial, antiviral,

Chitin is a long-chain polymer of N-acetylglucosamine, a derivative of glucose which can be deacetylated to yield

seen from the kinetics obtained after adding DODAB BF at a final concentration of 0.005 (); 0.01 (); 0.02 (); 0.05 (); 0.1 (); 0.2 (); 0.5 () and 1mM DODAB () to 0.1mg/mL OVA. In (B), kinetical data are detailed for the larger DODAB BF concentrations.Assemblies were prepared in 1mM NaCl.In (C), delayed-type hypersensitivity response for BALB/c mice immunized with OVA in different adjuvant formulations determined from the footpad swelling (nm) standard error of the mean. Final concentrations are 0.1mM DODAB BF, 20M CpG and 0.1mg/mL Al(OH)3 and 0.1mg/mL OVA. p< 0.05 compared to naive (), p< 0.05 compared to OVA/Al(OH)3 (#) and p< 0.05 compared to OVA/DODAB/CpG () from reference [97].Adapted from Journal of Controlled Release, 160/2, Julio H.K. Rozenfeld, Sandriana R. Silva, Priscila A. Ranéia, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in vivo*, 367-373 .Copyright 2012,

Other delivery systems based on anionic [103] or cationic lipid bilayer [41,44,100,104,105] have also been successful for improving Th1 response against important antigens such as those of influenza [103], hepatitis A and B [103,104], and fungal infections [105]. In general, cationic lipids are known for the production of a large inflammatory response [41, 44].However, for small cationic bilayer fragments as immunoadjuvants, this adverse reaction is absent [27, 97]. The small and anionic DODAB BF/OVA/CpG assemblies [97] also did not elicit adverse reactions similarly to other anionic assemblies [103]. There was no depot effect for these small assemblies [97] and their net negative charge ensured the absence of the adverse reactions observed previously for the large cationic liposomes and vesicles [100]. Recently, based on cross reactivity with *Neisseria lactamica* outer membrane vesicles (OMV) antigens, DODAB BF were combined with OMV to develop a vaccine against *Neisseria meningitidis* in young children [106]. Complexes of 25 µg of OMV in 0.1 mM of DODAB BF were colloidally stable, exhibiting optimal physical properties for efficient antigen presentation [106].

Figure 8.Chitin yielding chitosan by deacetylation. **Figure 8.** Chitin yielding chitosan by deacetylation.

with permission from Elsevier.

chitosan as shown in Figure 8.

Since they are biocompatible, biodegradable by deacetylases, mucoadhesive, and nontoxic, with antimicrobial, antiviral, and adjuvant properties, chitin, chitosan and their derivatives have been widely applied in medicine, pharmacy and vaccine design [107, 108]. Chitosan is soluble in diluted acids but is insoluble in water due to deprotonation of its amino moiety [109]. The poor solubility of chitosan at the pH of water represents a serious limitation for its applications as an immunoadjuvant in the clinics [107]. Several chitosan derivatives have been obtained to circumvent this limitation.For example, by attaching galactose moieties to the

Figure 7.Nanostructures of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in vivo*. In (A), the effect of time and DODAB concentration on zeta-average diameter of DODAB BF/OVA assemblies is

**Figure 7.** Nanostructures of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant ac‐ tivity *in vivo*. In (A), the effect of time and DODAB concentration on zeta-average diameter of DODAB BF/OVA assem‐ blies is seen from the kinetics obtained after adding DODAB BF at a final concentration of 0.005 (∎); 0.01 (○); 0.02 (▲); 0.05 (▽); 0.1 (◆); 0.2 (□); 0.5 (●) and 1mM DODAB (△) to 0.1mg/mL OVA. In (B), kinetical data are detailed for the larger DODAB BF concentrations.Assemblies were prepared in 1mM NaCl.In (C), delayed-type hypersensitivity re‐ sponse for BALB/c mice immunized with OVA in different adjuvant formulations determined from the footpad swel‐ ling (nm) ± standard error of the mean. Final concentrations are 0.1mM DODAB BF, 20μM CpG and 0.1mg/mL Al(OH)<sup>3</sup> and 0.1mg/mL OVA. p< 0.05 compared to naive (○), p< 0.05 compared to OVA/Al(OH)3 (#) and p< 0.05 compared to OVA/DODAB/CpG (△) from reference [97].Adapted from Journal of Controlled Release, 160/2, Julio H.K. Rozenfeld, Sandriana R. Silva, Priscila A. Ranéia, Eliana Faquim-Mauro, Ana M. Carmona-Ribeiro, Stable assemblies of cationic bi‐ layer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity *in vivo*, 367-373.Copyright 2012,

0.0 0.2 0.4 0.6 0.8

 

 

OVA

OVA/CpG

DODAB/OVA

DODAB/OVA/CpG

Al(OH)3 /OVA

Al(OH)3 /OVA/CpG

OVA

OVA/CpG

DODAB/OVA

DODAB/OVA/CpG

Al(OH)3 /OVA

Al(OH)3 /OVA/CpG

01234

012

012

 

 

 

Cytokine concentration (ng/mL)

 

IL-10

 ConA OVA Medium

IFN-

IL-13

IL-12

inducing charge overcompensation and recovery of colloidal stability as previously described for a model oligonucleotide

Figure 7 also illustrates the improvement in the cellular OVA-specific response from the analysis of cytokines secreted by lymph node cells of mice as adapted from reference [97]. IFN-γ and IL-12 production is associated to the Th1 response whereas IL-10 and IL-13 production reflects the Th2 response. Levels of IFN- γ and IL-12 secretion observed in cell cultures of mice immunized with OVA or Al(OH)3/ OVA were low and close to the detection limit for these cytokine assays (Figure 7). For mice immunized with OVA/CpG or DODAB BF/OVA assemblies, the secretion of IFN- γ and IL-12 substantially increased in comparison to secretion from cultured cells of OVA-immunized mice [97]. For the DODAB BF/OVA/CpG immunized mice, the secretion of IFN- γ and IL-12 increased by 33 and 49 %, respectively when compared to DODAB BF/OVA-immunized mice group and 52% and 35% when compared to OVA/CpG-group. In contrast, the highest secretion of IL-10 and IL-13 was observed in cultures of cells from mice that were immunized with Al(OH)3 /OVA whereas all other assemblies resulted in poor production of these cytokines [97]. Immune responses were similar for anionic DODAB BF/OVA/CpG and cationic DODAB BF/OVA of similar sizes showing that the charge is not important but the size is. The adsorption of antigen on the surface of DODAB large vesicles was shown to stimulate active antigen capture and presentation by dendritic cells (DCs) [41]. Administration of antigen adsorbed on DODAB large vesicles (LV) resulted in formation of an antigen depot at the site of injection which hampered the rapid clearance of antigen that takes place in absence of a carrier [100]. In contrast to DODAB LV, the small DODAB BF/antigen assemblies did not result in any observable depot effect [27, 97]. Furthermore, since the depot was absent for DODAB BF/CpG/OVA the immunostimulatory effect must have occurred via direct effect of the assemblies on the lymphonode antigen presenting cells [97]. Only nanoparticles can specifically target lymph node -resident cells [101]. CpG combined with DODAB BF yielded improved cellular Th1 response [97] possibly due to the appropriate targeting of DODAB BF/CpG/antigen to DCs in charge of expressing endosomal toll like receptor 9 (TLR9) in the lymph nodes [102]. The enhanced Th1 response by anionic DODAB BF/OVA/CpG relative to OVA alone was evidenced by the 6 times increase in DTH reaction, the 42 times increase in IFN-γ secretion by lymph node cells in culture and the 9 times increase in IL-12 secretion also by lymph

above charge neutralization [56].

node cells in culture (Figure 7) [97].

0 10 20 30 40 50 60

OVA

with permission from Elsevier.

t (min)
