**2. Adjuvants**

## **2.1 Toll-like receptors and pathogen-associated molecular patterns**

The word *adjuvant* is derived from the Latin root of *adjuvare*, which means to help*.* Thus, an adjuvant can be defined as any product which increases or modulates the specific humoral or cellular immune response against an antigen. The interaction between the innate and the adaptive immune responses is paramount in generating an antigen-specific immune response. The initiation of innate immune responses begins with the interaction of pathogen-associated molecular patterns (PAMPs) on the pathogen side with patternrecognizing receptors (PRR) such as Toll-like receptors (TLRs) on the host cells involved in the innate immunity (e.g., dendritic cells). A major functional criterion commonly used for the evaluation of various new adjuvants involves their ability to stimulate the innate immunity cells. This would include engaging and other PRRs and the co-receptors and intracellular adaptor signalling proteins with which they are associated. PAMPs and their derivatives are utilized by adjuvant developers to harness the power of innate immunity to channel the immune response in a desired direction.

Based on the identification of several TLRs and PAMPs recognized by them, various PAMP agonists were tested as adjuvans. Examples of TLR-PAMP specific interaction include bacterial or viral unmethylated immunostimulating CpG oligonucleotides interacting with TLR9, liposaccharide and its component monophosphoryl lipid A (MPLA) interacting with TRL4. These two types of adjuvants are in advanced stage of testing in clinical trials and some already licensed vaccines contain MPLA in liposomal form. Further liposomal or lipidbased particle formulations of both CpG and MPLA are under development and testing.

## **2.2 Muramyl dipeptide and other muropeptides**

Very specific group of PAMPs is represented by peptidoglycans (PGN). Both Gram-positive and Gram-negative bacteria contain PGN which consists of numerous glycan chains that are cross-linked by oligopeptides. These glycan chains are composed of alternating Nacetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with the amino acids coupled to the muramic acid. Muropeptides are breakdown products of PGN that bear at least the MurNAc moity and one amino acid (Traub et al., 2006). One of the prominent muropeptides is muramyl dipeptide (MDP), which is known since the 1970s.

Recently, the molecular bases for MDP recognition and subsequent stimulation of the host immune system have been uncovered. Myeloid immune cells (monocytes, granulocytes,

currently impossible to culture. Of course, this approach is limited to the identification of protein or glycoprotein antigens, omitting such important vaccine components as polysaccharides and glycolipids. Nevertheless, reverse vaccinology can enable scientists to systemically classify the potential protective antigens, thereby helping to improve the existing vaccines and to develop efficient preparations against virtually any pathogen that

As regards the process of activation of the immune system to produce an adaptive immune response, it is generally observed that the antigen by itself may not be adequate as a stimulating agent. Many potential antigens have no apparent immunizing activity at all when tested alone. In general, seamy side of pure recombinant protein antigens and synthetic peptide antigens is their poor immunogenicity. Therefore, potent adjuvants are

The word *adjuvant* is derived from the Latin root of *adjuvare*, which means to help*.* Thus, an adjuvant can be defined as any product which increases or modulates the specific humoral or cellular immune response against an antigen. The interaction between the innate and the adaptive immune responses is paramount in generating an antigen-specific immune response. The initiation of innate immune responses begins with the interaction of pathogen-associated molecular patterns (PAMPs) on the pathogen side with patternrecognizing receptors (PRR) such as Toll-like receptors (TLRs) on the host cells involved in the innate immunity (e.g., dendritic cells). A major functional criterion commonly used for the evaluation of various new adjuvants involves their ability to stimulate the innate immunity cells. This would include engaging and other PRRs and the co-receptors and intracellular adaptor signalling proteins with which they are associated. PAMPs and their derivatives are utilized by adjuvant developers to harness the power of innate immunity to

Based on the identification of several TLRs and PAMPs recognized by them, various PAMP agonists were tested as adjuvans. Examples of TLR-PAMP specific interaction include bacterial or viral unmethylated immunostimulating CpG oligonucleotides interacting with TLR9, liposaccharide and its component monophosphoryl lipid A (MPLA) interacting with TRL4. These two types of adjuvants are in advanced stage of testing in clinical trials and some already licensed vaccines contain MPLA in liposomal form. Further liposomal or lipidbased particle formulations of both CpG and MPLA are under development and testing.

Very specific group of PAMPs is represented by peptidoglycans (PGN). Both Gram-positive and Gram-negative bacteria contain PGN which consists of numerous glycan chains that are cross-linked by oligopeptides. These glycan chains are composed of alternating Nacetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with the amino acids coupled to the muramic acid. Muropeptides are breakdown products of PGN that bear at least the MurNAc moity and one amino acid (Traub et al., 2006). One of the prominent

Recently, the molecular bases for MDP recognition and subsequent stimulation of the host immune system have been uncovered. Myeloid immune cells (monocytes, granulocytes,

muropeptides is muramyl dipeptide (MDP), which is known since the 1970s.

has had its genome sequence determined.

**2. Adjuvants** 

required for highly purified antigen-based vaccines to be effective.

channel the immune response in a desired direction.

**2.2 Muramyl dipeptide and other muropeptides** 

**2.1 Toll-like receptors and pathogen-associated molecular patterns** 

neutrophils, and also DCs) possess two types of intracellular receptor for MDP, namely NOD2 and Cryopyrin (inflammasome-NALP-3 complex) (Agostini et al., 2004; Girardin and Philpott, 2004; McDonald et al., 2005). These two receptors recognize MDP/MDP analogues minimal recognition motifs for bacterial cell wall peptidoglycans (Girardin & Philpott, 2004; McDonald et al., 2005). NOD2 is also expressed in specialised epithelial cells, Paneth cells, localised in crypts of Lieberkün, which are producers of antimicrobial peptides having direct antimicrobial activity together with signalling functions within the immune system. Induction of an innate immune response against *Cryptosporidium parvum* infection by the liposomal preparation of lipophilic norAbuGMDP was demonstrated by us in newborn goats (Turanek et al., 2005) and this data is supportive of the present view of the role of MDP recognition in inducing both specific and innate immune responses.

Here GMDP abbreviates N-acetylglucosaminylmuramyl dipeptide. Another recently reported sensor of MDP is Cryopyrin (also known as CIAS1 and NALP3), which is a member of the NOD-LRR family (Agostini et al., 2004). Cryopyrin is a part of the inflammasome complex that is responsible for processing caspase-1 to its active form. Caspase-1 cleaves the precursors of interleukin IL-1β and IL-18, thereby activating these proinflammatory cytokines and promoting their secretion. IL-1β is known to be a strong endogenous pyrogen induced by MDP. We showed that norAbu-MDP analogues were not pyrogenic even at a high concentration, much higher than the concentrations used for vaccination. We supposed that the modification introduced into the structure of MDP to get norAbuMDP analogues had not changed their affinity to NOD2 but had substantially decreased the affinity to cryopyrin. This hypothesis is in accordance with our data on pyrogenicity and is being currently tested in appropriate *in vitro* models. In addition, murabutide, another nonpyrogenic derivative of MDP was shown not to be able to induce detectable level of IL-1ß in sera of treated volunteers (Darcissac et al., 2001).

The expression of NOD2 in dendritic cells is of importance with respect to the application of MDP analogues as adjuvants. Nanoparticles like liposomes are able to provide a direct codelivery of a danger signal (e.g., MDP) together with the recombinant antigen and therefore to induce an immune response instead of an immune tolerance. This is especially important for weak recombinant antigens or peptide antigens. Clearly, the recognition of MDP by DCs is crucial for the application of MDP analogues as adjuvants. Although the immunostimulatory effects of MDPs have been described for over three decades, the process of molecular recognition and binding of MDP/MDP analogues to NOD2 and cryopyrin receptors remains unclear. Within the cell, MDP/MDP analogues trigger intracellular signalling cascades that culminate in the transcriptional activation of inflammatory mediators such as the nuclear transcription factor NF-κB. The biological effects of muramyl peptides have been described for over three decades. The mechanism underlying their internalization of MDP to the cytosol, where it is sensed by NOD2 and cryopyrin, remains unclear. Liposomes probably play the role of efficient carriers for MDP and its analogues on the pathway from extracellular milieu into the cytosol, where they trigger intracellular signalling cascades that culminate in transcriptional activation of inflammatory mediators such as the nuclear transcription factor NF-κB pathway. In case of liposomal formulation of various MDP analogues, the relevant intracellular pharmacokinetics, molecular recognition and binding affinity towards NOD2 and Cryopyrin remain to be determined. Such differences found for various MDP analogues are responsible for their various biological activities (e.g., pyrogenicity, ability to induce the innate immune response etc.) and, therefore, could be utilised for a precise tuning of the intensity and type of immune response. Since the discovery and first synthesis of MDP, about one thousand various derivatives of MDP have been designed, synthesised, and tested to develop an appropriate drug for an immunotherapeutic application that would be free of the side effect exerted by MDP. The main side effects of MDP are pyrogenicity, rigor, headache, flue-like symptoms, hypertension etc. Only several preparations reached the stage of clinical testing and only Mifamurtide (Fig. 1) was approved for the treatment of osteosarcoma.
