FUNCIONES Y SUS ESTÁNDARES DE FUNCIONAMIENTO, CONTEXTO OPERACIONAL

The human immune system represents a collective of cells, tissues, and organs that defends the human body against foreign pathogenic invaders such as viruses, bacteria, parasites, and fungi. It can be divided into innate and adaptive or acquired components. The innate immune system is the first quick line of defense. But neither are pathogens specifically recognized, nor mechanisms of safety against repetitive future infections initiated. Important elements of the innate immune system are the humoral component i.e. the complement system and the cellular components i.e. phagocytotic cells such as macrophages, granulocytes, and natural killer (NK) cells. Even though the innate immune system is very quick, it mostly fails to abolish the pathogen completely due to its lack of specificity. But, it triggers the acquired immune system which consists of antibodies as humoral components and lymphocytes as cellular components. Characteristic for the acquired immune system are its high specificity against pathogens and its memory function. Via clonal selection lymphocytes specific for one pathogenic antigen are produced. Some of these lymphocytes are differentiated to memory cells. The lymphocytes can be subdivided in B cells and T cells. B cells produce antibodies, whereas T cells act either as T helper cells (TH cells), supporting other immune cells or cytotoxic T

lymphocytes (CTL) that are able to induce the death of infected cells. TH cells are

positive for the antigen CD4 (cluster of differentiation antigen 4), whereas CTLs are positive for CD8. Naïve CD4+ _{T-cells (T}

H0 cells) are activated via antigens that

are presented by antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). According to the co-administered cytokines, the naïve TH0

cells are further differentiated to either TH1 or TH2 cells. TH1 cells support cellular

immune responses, while TH2 cells support the activation of B cells to produce

antibodies, thus inducing a humoral immune response. Within the immune system, APCs and especially DCs play a major role because they connect innate and acquired immune response.

Activation of the innate immune system occurs by the exposure to pathogen- associated molecular patterns (PAMPs) that are expressed by diverse infectious microorganisms (Medzhitov & Janeway 1997b). These PAMPs are recognized by specific pattern recognition receptors (PRRs) that are mainly present in APCs. Although PAMPs represent a very heterogeneous group, all PAMPs have in common their specificity for microbial pathogens that cannot be found in the host organism. Furthermore, PAMPs are part of the respective pathogenicity and are indispensable for the pathogen’s survival (Medzhitov & Janeway 1997a). The PRRs that exist in vertebrates are lectin receptors (Stahl et al. 1998), scavenger receptors (Peiser et al. 2002), and toll-like receptors (TLRs).

Presently, there are 10 human TLRs described in literature, although some of their natural ligands have not been identified so far. Most of these ligands are of microbial origin. But, there are also supposed to be endogenous ligands that are released during inflammation reactions due to cellular stress or damage (Matzinger 2002). An overview of the known TLRs and their corresponding ligands is given in Table 1 (Kaisho & Akira 2002).

Table 1: TLRs and ligands

TLR PAMP Origin of ligand Reference

TLR1 cooperates with TLR2

TLR2 Lipoproteins Gram-positive bacteria

(Yoshimura et al. 1999; Underhill et al. 1999)

Zymosan Yeast (Underhill et al. 1999)

Lipoarabinomannan Mycoplasma (Means et al. 1999a; Means et al. 1999b)

MALP-2 Mycobacteria (Takeuchi et al. 2000; Takeuchi

et al. 2001)

TLR3 Double-stranded RNA Virus (Alexopoulou et al. 2001)

TLR4 LPS Gram-negative

bacteria

(Qureshi et al. 1999; Hoshino et al. 1999)

F protein Respiratory

syncytial virus

(Kurt-Jones et al. 2000)

HSP60 Host (Ohashi et al. 2000; Vabulas et

al. 2001)

TLR6 Cooperates with TLR2 (Takeuchi et al. 1999) TLR7 Guanosine analogs,

single-stranded RNA

(Hornung et al. 2004)

TLR8 Guanosine analogs (Jurk et al. 2002)

TLR9 CpG motifs Bacteria, viruses (Hemmi et al. 2000) TLR10 unknown

TLR1, 2, 4, 5, and 6 are specialized on the recognition of ligands that are unique in bacteria and cannot be produced by the infected host itself. TLR3, 7, 8, and 9 are responsible for the detection of viral pathogens and the recognition of nucleic acids that do not only exist in microbial organisms. In these cases, the differentiation between own and foreign DNA does not only rely on the type of ligand but also on the accessibility of the particular TLRs. Thus, these TLRs are located in subcellular compartments.

1.2

History and mechanism of action of CpG ODNs

As described in Table 1, bacterial or viral DNA is recognized by TLR9 via non- methylated cytosine-phosphate-guanosine dinucleotides in particular sequence contexts (CpG motifs) that are sensed as the key danger signals (Krieg et al. 1995). Concerning the immunogenicity of viral or bacterial DNA, first findings were already made more than 100 years ago. Back in 1890, William Coley introduced the deliberate use of bacterial extracts for the treatment of cancer (Wiemann & Starnes 1994). Even though temporary tumor-restitution and a surprising complete remission rate of 10% (Coley 1991) were achieved, this approach was not further established since it lacked reproducibility and was ruled out by other methods such as radiotherapy. It took until the 1970ies before a second approach was conducted using attenuated mycobacteria Bacillus Calmette Guerin (BCG) for tumor therapy which was finally established as standard therapy for human bladder cancer (Morales 1978). Other research groups explored later that the anti-tumoral effect of the vaccination with BCG was related to a stimulation of the immune system (Tokunaga et al. 1984). More precisely, the bacterial DNA was responsible for the induction of natural killer cell (NK cell) activity and the production of type 1 and type 2 interferons (Yamamoto et al. 1992). After further investigations, the researchers concluded that self-complementary palindromes in the nucleotide sequence containing at least one cytosine-phosphate-guanosine (CpG) dinucleotide

(Kuramoto et al. 1992) were responsible for this stimulation of the immune system. Another research group independently reported that purified bacterial DNA as well as poly-(dG,dC) nucleotides in contrast to vertebrate DNA, induced the proliferation of murine B cells and immunoglobulin secretion (Messina et al. 1991). Furthermore this group explored that the mitogenicity of poly-(dC,dG)-nucleotides was abolished by methylation of cytosine (Pisetsky & Reich, III 1998). So they suggested that methylation interferes the formation of unique higher order structures, which are responsible for the immunogenic effect.

During experiments with antisense ODNs in 1995, Arthur M. Krieg coincidentially observed immune stimulatory effects such as strong B cell proliferation and immunoglobulin secretion of certain ODNs. After further analysis of the ODNs, Krieg found out that CpG dinucleotides in particular sequence contexts (XCGY whereas X can be random but C and Y can be random but G) are decisive to induce a stimulation of the immune system. Inversion of the base sequence (GpC) as well as methylation of cytosine abolished the immune stimulation. Concomitantly, it could be disproved that the formation of higher order structures related to a palindrome sequence is necessary for action (Krieg et al. 1995).

Summarizing all these insights, it can be explained how TLR9 succeeds to differentiate between own and foreign DNA (Bird et al. 1987):

a) CpG dinucleotides are statistically underrepresented in vertebrate DNA (1:60), whereas nucleotide utilization in bacterial DNA is random (1:16). b) The majority of cytosines (70%) in CpG dinucleotides are methylated in

vertebrate, but not in microbial DNA.

With this knowledge various synthetic ODNs were developed which were highly effective concerning their immune stimulation in murine in vitro and in vivo

experiments. But it was noticed that optimized sequences and settings of the murine experiments could not be transferred to nonhuman primates and humans, as there are significant evolutionary based differences in the recognition of CpG motifs. Whereas GACGTT was determined as the optimized CpG motif to activate the immune system of mice (Rankin et al. 2001) it is GTCGTT for nonhuman primates and humans (Hartmann & Krieg 2000) (Table 2). Moreover, the cell-types that contain TLR9 differ remarkably. In mice, TLR9 is also present in cells of the

DCs) and B cells (Rothenfusser et al. 2002). Murine MDCs are often utilized in in vitro models, since these cells are conveniently derivable from bone marrow precursors being treated with GM-CSF. But TLR9 is not present in human MDCs, so the significance of the data produced with this in vitro model is limited. Caution seems advisable when extrapolating murine CpG ODN data to primates and humans.

1.3

Synthetic CpG ODN classes for humans and their effects on