The Cholera Toxin as a Biotechnological Tool

It was as early as 1886 when Robert Koch proposed that the symptoms caused by Vibrio cholerae were initiated by a "poison" produced by the pathogen. However, it was not until 1959 that this postulate could be demonstrated by reproducing the disease in an animal model [De, 1959]. Today, cholera toxin (CT) is known to exhibit toxic effects in human cells and produces dehydrating diarrhea in humans. It is produced almost exclusively by few serogroups of V. cholera, however, sometimes may be naturally produced by other organisms, as the opportunistic pathogen V. mimicus [Nishibuchi and Seidler, 1983; Spira and Fedorka-Cray, 1984].

ribose unit from NAD + oxidizing agent to an arginine residue of Gs protein. This covalent modification leads to the loss of GTPase activity of the Gs protein, which remains attached to GTP, keeping the adenylate cyclase (AC) enzyme active that will produce increasing amounts of cAMP. Over 100 times the normal concentration of cAMP, the intestinal mucosa cells open a Clchannels in the cytoplasmic membrane, resulting in an influx of ions and water to the gut lumen that causes the characteristic acute diarrhea of cholera [Spangler, 1992]. As little as 5 µg of purified CT administered orally is sufficient to induce significant diarrhea in human volunteers while ingestion of 25 µg of CT elicits a full 20 litres cholera purge [Levine et al., 1983].

Immune properties
Adjuvants are substances that have the ability to enhance the immune response when coadministered with poor immunogenic molecules. CT is a bacterial immunogen with a great function as an adjuvant to a variety of antigens when given by systemic and mucosal route whether these are linked to or simply mixed with the toxin, generating a long-term immune response (Elson 1989;Vajdy and Lycke 1992).
These properties may be explained by three main characteristics of the molecule. First, CT is remarkably stable to proteases, bile salts and other compounds in the intestine. Secondly, its high affinity to GM1 ganglioside receptor, which is present on most mammalian cells including the M cells covering the Peyers patches, as well as all antigen-presenting cells (APC), facilitates the uptake and presentation of the toxin to the gut mucosal immune system. Finally, CT has strong inherent adjuvant and immunomodulating activities that depend both on its cell binding capability and its enzymatic ADP-ribosylating function (Sanchez and Holmgren 2008).
Pioneer studies carried out in 1972 showed that CT delivered by the intravenous route with a foreign antigen behaved as an adjuvant [Northrup and Fauci, 1972], a fact confirmed later by several groups using a number of unrelated antigens of little immunogenicity [Bianchi et al., 1990;Elson and Ealding, 1984]. Additional studies revealed that upon co-administration of CT and antigen through parenteral, mucosal, and transcutaneous routes resulted in substantial enhancement of mucosal immunoglobulin A (IgA) and serum IgG responses to the co-administered antigen [Chen and Strober, 1990;Drew et al., 1992;Reuman et al., 1991]. In addition to enhancing humoral immune responses, CT also augmented cellular immune responses to co-administered antigens enhancing induction of CD4 + T helper (Th) and class I-restricted cytolitic T lymphocyte responses [Nurkkala et al.;Simmons et al., 1999]. In most cases, CT induced a Th2 bias response [Lavelle et al., 2004;Okahashi et al., 1996]. However, other studies have reported Th1 [Sasaki et al., 2003;Taniguchi et al., 2008] or mixed Th1/Th2 responses following oral, sublingual and intranasal immunization with antigens in the presence of CT [Cuburu et al., 2007;Fecek et al., 2010]. More importantly, subsequent studies showed that CT elicited a long-term memory response and thus was detectable long after the initial immune response [Soenawan et al., 2004;Vajdy and Lycke, 1992].
CT also acts as mucosal adjuvant against a variety of pathogens. Examples include, tetanus toxoid [Jackson et al., 1993], Helicobacter felis [Jiang et al., 2003], Schistosoma japonicum , Helicobacter pylori [Raghavan et al., 2002], and Sendai virus [Liang et al., 1988]. There are many other examples where it was shown that CT has significant potential

Mechanism of adjuvant activity
The mechanism of adjuvanticity of CT is still unclear but is has been related to: (i) the induction of increased permeability of the intestinal epithelium leading to enhanced uptake of co-administered antigens; (ii) the induction of enhanced antigen presentation by various APC; (iii) the promotion of isotype differentiation in B cells leading to increased IgA formation; and (iv) exhibition of complex stimulatory as well as inhibitory effects on T cell proliferation and cytokine production. Among these many effects, those leading to enhanced antigen presentation by various APC are probably of the greatest importance [Sanchez and Holmgren, 2011].
As mentioned before, the polarity of the immune response generated by CT is a matter of debate. Some studies indicate that CT primes naïve T cells in vitro and drives them towards a Th2 phenotype, with production of interleukins IL-4 (a cytokine needed for B cell differentiation), IL-5, IL-6 and IL-10, but little IFN-(a cytokine needed to evoke Th1 responses) and suppression of IL-12 production by dendritic cells (DC) [Braun et al., 1999;Klimpel et al., 1995;Wilson et al., 1991]. Moreover, after immunization of animals with CT co-administered antigens, IL-4 levels were significantly elevated in gut-associated tissues and in spleen, while the levels of IFN-either decreased or remained static [Akhiani et al., 1997;Marinaro et al., 1995]. These results are supported by evidence of increased secretory IgA, serum IgA and IgE levels [Adel-Patient et al., 2005;Bourguin et al., 1991], and higher titers of IgG1 than IgG2a [Glenn et al., 1998;Lycke et al., 1990].
In contrast, others have reported that CT induces a mixed Th1/Th2 type of immune response with the production of IFN-and IL-4 [Fromantin et al., 2001;Imaoka et al., 1998]. In addition, it has been shown that CT induces strong Th17-type responses after intranasal delivery [Datta et al.;Lee et al., 2009]. Furthermore, CT markedly increased antigen-presentation by DC, macrophages, and B cells [Bromander et al., 1991;George-Chandy et al., 2001]. Also, CT upregulates the expression of MHC/HLA-DR molecules, CD80/B7.1 and CD86/B7.2 co-stimulatory molecules, as well as chemokine receptors CCR7 and CXCR4, on both murine and human DC, among other APC [Cong et al., 1997;Gagliardi et al., 2000]. Importantly, CT also induced the secretion of IL-1 from both DC and macrophages. IL-1 not only induces the maturation of DC, but also acts as an efficient mucosal adjuvant when co-administered with protein antigens and might mediate a significant part of the adjuvant activity of CT [Staats and Ennis, 1999]. Treatment with CT has been demonstrated to induce maturation and mobilization of DC [Lavelle et al., 2003]. Also, CT interferes with the differentiation of monocytes into DC, giving rise to a distinct population (Ma-DC), which displays an activated macrophage-like phenotype, induces a strong allogeneic and antigen specific response, and promotes the polarization of naïve CD4 + T lymphocytes toward a Th2 profile [Raghavan et al., 2010]. In additon, CT enhanced IL-6 secretion by peritoneal mast cell [Leal-Berumen et al., 1996] and production of IL-1 , IL-6, and IL-10 together with inhibition of IL-12, TNF-, and nitric oxid in macrophages [Cong et al., 2001], depleted the CD8 + intraepithelial lymphocyte population [Flach et al., 2005], and induced isotype differentiation of B cells acting synergistically with IL-4 [Salmond et al., 2002]. Recent studies show that CT enhances STAT3 gene expression www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free.
Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. Fig. 2. Proposed mechanism of action by CT as a mucosal adjuvant. CT induces increased permeability of the intestinal epithelium leading to 1) enhanced uptake of co-administered antigens and 2) enhanced antigen-presentation by various APC. 3) It causes the depletion of CD8 + lymphocyte population that may produce inhibitory cytokines, and 4) induces maturation and mobilization of DC. In addition, 5) CT promotes a strong Th2 dominant response to bystander antigens, and can either 6) induce or inhibit a Th1 response. Moreover, 7) CT induces strong Th17-type responses. Furthermore, 8) mucosal epithelial cells contribute to the adjuvant activity of CT by secreting a number of chemokines and acting on polymorphonuclear leukocytes, macrophages, eosinophils and T cells.

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in murine B cells, and may critically modulate immune responses in both a proinflammatory and anti-inflammatory direction, depending on the circumstances and the types of cells involved Sjoblom-Hallen et al., (2010).
It has been suggested that mucosal epithelial cells may also play a role in adjuvanticity. Human epithelial cells express and secrete high levels of the chemoattractant cytokines IL-8, GRO , GRO , GRO , and ENA-78 in response to stimulation with TNF-, IL-1 , or infection with enteroinvasive microorganisms. These chemokines attract and activate polymorphonuclear leukocytes. Activated epithelial cells also secret MCP-1, MIP-1 , MIP-1 , and RANTES, which variably act on monocytes/macrophages, eosinophils, and subpopulations of T-cells [Freytag and Clements, 2005]. One possibility is that CT interacts with epithelial cells triggering expression of one or more immunomodulatory factors that recruite APC and immune effector cells or activate those cells, or both [Lopes et al., 2000;Soriani et al., 2002].
A proposed mechanism of action of CT as adjuvant is shown in Fig. 2.

Genetic modifications of CT
The inherent enterotoxicity of CT has limited its widespread use as a vaccine component and adjuvant. In dogs, protection due to CT occurred only with doses that caused transient, sometimes severe, diarrhea [Pierce et al., 1982]. Moreover, murine models demonstrated that intranasal sensitization with CT as adjuvant led to increased lung inflammation with a massive recruitment of macrophages as well as accumulation in the olfactory nerves, epithelium and the olfactory bulbs of mice after binding to GM1 gangliosides [Fischer et al., 2005]. These limitations have led to mucosal strategies involving nontoxic mutants and purified B subunits.
Although early reports showed that mutants without the ADP-ribosyltransferase activity lack their adjuvant properties , later studies showed that non-toxic mutants retained their adjuvant and immunogenic properties [Douce et al., 1997;Yamamoto et al., 1997] without central nervous system (CNS) toxicity [Hagiwara et al., 2006]. This suggests that the ADP-ribosyltransferase activity is not essential for its immunogenic properties, though it contributes to the adjuvant effect.
In a different approach, the CTA1 fragment linked to a synthetic analogue of Staphylococcus aureus protein A, the D fragment with affinity for APC, [Agren et al., 1997], proved to be non-toxic [Eriksson et al., 2004]. The fusion protein CTA1-DD binds specifically to immunoglobulins on the surface of antigen-presenting B cells through the DD polypeptide, and induces the ADP ribosylation by CTA1. Although this produces a good immune response when administered intranasally, it has been shown not to work as well after oral administration. This limitation was overcome by fusing CTA1-DD with immunostimulating complexes, such as ISCOMs (lipophilic immune stimulating complexes), producing both Th1/Th2 responses at systemic and mucosal levels [Andersen et al., 2007]. A recent report showed that CTA1 potently enhances a GeneGun-delivered DNA prime for human and simian immunodeficiency viruses antigens boost in macaques and mice [Bagley et al., 2011].

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Immunological and adjuvant properties of CTB
Several studies using different conditions and routes of administration have described that CTB has several immunomodulatory properties opening many perspectives for future therapeutic and biotechnological applications. In this regard, intranasal immunization of women with CTB resulted in the production of long-lasting IgG and IgA anti-CTB in serum, nasal and vaginal secretions in a dose-dependent manner [Bergquist et al., 1997].
However, its capacity as mucosal adjuvant has proven to be much less than that of the toxin when given together with non-coupled antigens by the oral route [Sanchez and Holmgren, 2008]. Recombinant CTB has been successfully used as a mucosal adjuvant in vaccines for human use such as the cholera vaccine itself [Quiding et al., 1991], and the vaccine against enterotoxigenic E. coli that causes diarrhea [Peltola et al., 1991;Qadri et al., 2000]. Analogously, CTB proved to b e g o o d a d j u v a n t f o r a Streptococcus pneumoniae cellular vaccine [Malley et al., 2004] and a severe acute respiratory syndrome-associated coronavirus vaccine [Qu et al., 2005] when administered intranasally in mice.
CTB is a useful carrier protein for induction of mucosal IgA antibodies against chemically coupled antigens. In this regard, mice immunized intraduodenally with the horseradish peroxidase (HRP) covalently coupled to CTB showed a 33-120 fold higher level of IgA anti-HRP in intestinal washes as well as increased levels of serum IgG anti-HRP [McKenzie and Halsey, 1984]. In addition, CTB chemically conjugated to the protein I/II of Streptococcus mutans when administered in mice by oral [Russell and Wu, 1991], intranasal [Wu and Russell, 1998], and intragastric routes [Wu and Russell, 1993] results in the production of antistreptococcal IgG and IgA in serum and mucosa, as well as the presence of large numbers of antibody-secreting cells in salivary glands, mesenteric lymph nodes, and spleens. Similar results were found with CTB conjugated to human gamma globulin (HGG) and the recombinant Neisseria gonorrhoeae transferrin binding proteins, TbpA and TbpB. Vaginal and intranasal immunizations with CTB-HGG resulted in high levels of anti-HGG antibodies [Johansson et al., 1998], while rCTB-TbpA and rCTB-TbpB administered intranasally induced antibody responses in the serum and genital tract [Price et al., 2005]. Moreover, CTB was chemically conjugated to type III capsular polysaccharide from www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free.
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Streptococcus group B [Shen et al., 2000] or to protein-polysaccharide conjugates [Bergquist et al., 1995] and in both cases, after subcutaneous administration, high levels of specific antibodies were detected. In addition to generating humoral response, simian immunodeficiency virus (SIV) virus-like particles (VLP) chemically conjugated to CTB showed higher levels of cytokine IFN--producing splenocytes and cytotoxic-T-lymphocyte activities of immune cells than VLPs plus CTB, indicating a generation of a Th1 response in mice by CTB-VLP [Kang et al., 2003]. Finally, CTB chemically conjugated to the Plasmodium vivax ookinete surface protein, Pvs25, proved to be a potent transmission-blocking antigen in both intranasal and subcutaneal routes in mice , and to protect against pharyngeal colonization by group A streptococcus when conjugated to the widely shared C repeat region of M6 protein [Bessen and Fischetti, 1990]. Another way of using CTB as an adjuvant is in genetic constructions based on the toxin and heterologous antigens. In general, these hybrid molecules are composed of antigens fused to the amino [Laloi et al., 1996;Song et al., 2004] or carboxyl [Kim et al., 2004;Wang et al., 2010] terminus of CTB, being GM1-binding much more efficient in the latter case [Liljeqvist et al., 1997], but also protein epitopes have been introduced at internal positions in CTB www.intechopen.com

Antigen
Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. [Dertzbaugh and Elson, 1993]. Some examples of genetic incorporation of epitopes to CTB include triple glutamic acid decarboxylase [Gong et al., 2009], dodecapeptide repeat of the serine-rich Entamoeba histolytica protein [Zhang et al., 1995] and human insulin B-chain [Sadeghi et al., 2002]. There are many studies showing the induction of immune responses through immunization of mice with CTB fused to soluble antigens expressed both in bacteria [Larsson et al., 2004;Lee et al., 2003;Sun et al., 1999;Tsuji et al., 2003] and in transgenic plants [Jani et al., 2004;Matsumoto et al., 2009]. In all cases there was generation of IgG and IgA antigen-specific antibodies and, in some cases, protection. Some examples of the adjuvant action of CTB are shown in Table 1.
One of the strategies for using CTB as an adjuvant genetically fused to antigens has been described by Arêas et al. and is based on the expression vector called pAEctxB (Fig. 3.). In the generation of the vector, the gene ctxB was modified to ensure that the codons were those most frequently used by E. coli, L. casei and S. typhimurium [Areas et al., 2002]. The genetically engineered ORF was then cloned into the expression vector pAE [Ramos et al., 2004] and includes two consecutive restriction sites MluI and HindIII. The resulting vector allows expression, under the control of a T7 promoter, of proteins fused to the C-terminus of CTB with 6 histidine residues at the N terminus, which facilitate protein purification by immobilized metal ion affinity chromatography.
The pAE-ctxB plasmid was used to clone the pneumococcal surface adhesin A (PspA) [Areas et al., 2004], the Leptospira interrogans protein LipL32 [Habarta et al., 2010], the fatty-acid binding protein from Schistosoma mansoni S14 [Henrique Roman Ramos, 2010], and the Bordetella pertussis type III secretion system effector protein Bsp22 (Olivera et al., unpublished results). Intradermal immunization with CTB-PspA induced high titers of anti-PspA IgG and partially protected mice after challenge with S. pneumonia [Areas et al., 2005]. Moreover, intranasal immunization with CTB-PsaA protected mice against colonization with S. pneumoniae without alteration of the natural oral or nasopharyngeal microbiota of mice [Pimenta et al., 2006]. CTB-Sm14 itself was not able to reduce Schistosoma mansoni worm burden on intranasally immunized BALB/c mice, but reduced the hepatic granulomas around trapped eggs. CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32, supporting CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.

CTB for mucosal immunotherapy
Mucosal administration by the oral, sublingual or nasal routes of many antigens can induce peripheral tolerance. Mucosal-induced tolerance has been recognized for a long time as a promising approach to prevent or treat allergic or autoimmune disorders and is characterized by a decreased immune response to systemic immunization with the same antigen [Sun et al., 2009;Sun et al., 1994]. In this regard, promising results have been obtained with auto-antigen coupled to CTB in order to induce oral tolerance. Although not known the mechanism by which CTB conjugated to antigens has the ability to potentiate the induction of oral tolerance, it is believed that in addition to the processes already mentioned before for CT, it may result in selected DC subsets with increased ability to induce different types of TGF--expressing suppressor T cells including CD4 + CD25 + Tr cells [Holmgren et al., 2005] and a direct depletion of effector T cells since CTB induces CD4+ and CD8+ T cell apoptosis [Christelle Basset, 2010].

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Fig. 3. Cloning strategy into pAEctxB plasmid
Oral delivery of CTB conjugated to myelin basic protein protected mice [Sun et al., 1996;Yuki et al., 2001] and rats [Sun et al., 2000b] against the development of experimental autoimmune encephalomyelitis. It was proposed that the inhibitory effect was a result of both the induction of TGF--producing Tr cells and down-regulation of IFN , IL-12, TNF , MCP-1 and RANTES in the CNS [Wang et al., 2009].
Oral administration of a CTB-insulin conjugate prevented diabetes in non-obese diabetic (NOD) mice [Arakawa et al., 1998;Bergerot et al., 1997;Gong et al., 2007;Petersen et al., 2003;Ploix et al., 1999], which was associated with a reduction in IFN production and Tr cell migration into pancreatic islets [Aspord et al., 2002;Sobel et al., 1998]. On the other hand, oral administration of CTB-proinsulin fusion protein showed an increased expression of IL-4 and IL-10 in the pancreas of NOD-treated mice, suggesting that Th2 lymphocytemediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis [Ruhlman et al., 2007].
Oral delivery of CTB conjugated to a 60 kDa heat-shock protein derived peptide prevented mucosal induced uveitis in rats, an effect that was associated with enhanced IL-10 and TGF-, and reduced IL-12 and IFN-production [Phipps et al., 2003]. Furthermore, a I/II phase clinical trial of the same peptide conjugated to CTB administered orally to 8 patients allowed the withdrawal of all immunosuppressive drugs in 5 of the 8 patients without a relapse of uveitis [Stanford et al., 2004].
In addition, oral administration of CTB in mice inhibits the induction of trinitrobenzene sulfonic acid-induced colitis and reverses such colitis after it has been established. This inhibition is associated with suppression of IL-12 and IFN-production [Boirivant et al., 2001;Coccia et al., 2005]. In a recent clinical trial, 40% of patients with active Crohn's disease responded to treatment with CTB [Stal et al., 2010].

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CTB conjugates were also effective in the induction of tolerance to type II collagen, leading to a suppression of chondritis in a model of autoimmune ear disease [Kim et al., 2001]. Oral administration of allogeneic antigen linked to CTB induced immunological tolerance against allograft rejection [Sun et al., 2000a]. Finally, transconjunctival immunotherapy using CTB could suppress clinical effects for experimental allergic conjunctivitis in guinea pigs [Oikawa et al., 2011].

Conclusion
CT has been studied for over 40 years. Both CT and its non-toxic derivatives or its B subunit, have shown to be excellent mucosal adjuvants. The possibility to use them as biotechnological tools in the development of new vaccines is being intensively studied in the present. In recent years, the prospect to use CTB fused to different protein antigens became relevant because these proteins can be expressed in high levels in a soluble form and directly purified in their active form, requiring only one fermentation step. In addition, several reports have shown that CTB can generate oral tolerance to different conjugated antigens, opening ways for the treatment of autoimmune diseases. Hopefully, future studies will focus on the use of CTB in such important issues.

Acknowledgements
This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT 07-00642 and PICT 07-00028 (RMG).   www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here.