Immunostimulatory sequence

In parallel to these initial descriptions, observations in the antisense field of study revealed that some antisense ODNs displayed sequence-specific immunostimulatory properties (Branda et al., 1993 Krieg et al., 1989, 1993 McIntyre et al., 1993 Pisetsky and Reich 1994 Tanaka et al., 1992). Exhaustive study revealed that immunostimulation by these ODNs could be attributed to the presence of CpG dinucleotides, in particular to base contexts (most importantly a hexamer) that were not restricted to a palindrome (Krieg et al., 1995) (Figure 22.1). Further, methylation of the cytosine in the CpG dinucleotide completely abrogated any immunostimulatory properties, confirming that the immunostimulatory properties of these ODNs were dependent on the presence of the unmethylated CpG dinucleotides (Krieg et al., 1995). Such immunostimulatory sequences based on unmethylated CpG dinucleotides are now known as CpG motifs. 

Pyles, R.B., D. Higgins, C. Chalk, A. Zalar, J. Eiden, C. Brown, G. VanNest, and L.R. Stanberry. 2002. Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection. J Virol 76 11387. 

Immunotoxicity of Oligonucleotide Therapeutics The pro-inflammatory activity of the ASOs produces a constellation of effects, which includes splenomegaly, lymphoid hyperplasia, and multi-organ lymphohistiocytic cell infiltrate driven by the production of pro-inflammatory cytokines [57,63,75]. Incorporation of immunostimulatory sequences like unmethylated CG dinucleotides mimic bacterial DNA [76], Unmethylated CG dinucleotides interact with receptors of the innate immune system such as TLR9 to produce a shift toward T-helper type 1 immunity [77,78], Oligonucleotides with these motifs modulate the immune system. We have recently reported on a direct evaluation for effects on immune function. 

Roman, M. et al., Immunostimulatory DNA sequences function as T helper-1-promoting... 

A number of modified nucleotides have been tested and described in siRNA design. These are mostly modifications of the 2 OH group of the ribose. By the incorporation of chemically modified nucleotides into siRNAs, the on-target efficiency of the siRNAs can be increased (42 4). On the other hand, different types of siRNA off-target effects can be reduced by the use of chemically modified nucleotides immunostimulatory effects (reviewed in (20)) as well as sequence-dependent miRNA-like off-target effects (26, 44,45) and passenger strand incorporation (46, 47). In addition, chemical modifications can be used to improve the cellular delivery of siRNAs in living animals and are important tools to enhance the serum stability of siRNAs (48). 

Roman, M., Martin-Orozco, E., Goodman, J.S., Nguyen, M.D., Sato, Y., Ronaghy, A. etal. (1997) Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat. Med., 3, 849-854. 

Some of the peptides derived from milk proteins have more than one functional role, e.g., peptides from the sequence 60-70 of (3-cascin show immunostimulatory, opioid and ACE-inhibitory activities. This sequence has been defined as a strategic zone (Migliore-Samour and Jolles, 1988). The sequence is protected from proteolysis because of its high hydrophobicity and the presence of proline residues. 

Newer classes of immunostimulatory oligonucleotides that are composed of RNA and bind different receptors are now being identified. Single- and double-stranded RNA have the potential to be proinflammatory, but the receptor and sequence-dependence are distinct from CpG oligonucleotides. These RNA oligonucleotides tend to bind to TLR3, 7, and 8 as well as the... 

Chimeric peptides are unnatural constructs consisting of bioactive compounds from at least two different peptide(s) and/or protein(s) or two sequences from different parts of the same protein. Such multifunctional peptide combinations are prepared to enhance the biological activity or selectivity of their components. New biological effects can also be achieved with the chimera. In this chapter the synthesis of three different types of chimeric peptides will be described. In a linear chimera, two peptide epitopes from different parts of glycoprotein D (gD) of herpes simplex virus (HSV) are combined. A branched chimera, built from linear peptides, consists of tuftsin oligomers with immunostimulatory activity and an epitope peptide of HSV gD. The third compound is a cyclic chimeric molecule, where a-cono-toxin GI as a host peptide is modified by the incorporation of a core epitope from HSV gD as a guest sequence. 

Sequence-dependent toxicity Palindromic sequences and dinucleotide motifs containing CpG sequences in particular, have been shown to posses potent immunostimulatory properties in rodents. Similar effects do not seem to occur in primates. 

Antisense ODNs, small ODNs (15-30 mers) that are complementary to the sequence of pre- or mature mRNA sequences, have been shown to inhibit specifically the expression of numerous genes [128], Abdou et al. reported the use of -CyD bearing galactose moiety for antisense phosphodiester-ODN delivery into cells [129]. In addition, Zhao et al. found inhibitory effects of 6 and 5 on immune stimulation by immunostimulatory phosphorothioate-ODN containing the CpG motif in splenocytes isolated from GDI mice and in vivo [130]. 

Sato, Y. et al, Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science, 273,352,1996. 

Yu D, Kandimalla ER, Cong YP et al (2002) Design, synthesis, and immunostimulatory properties of CpG DNAs containing alkyl-linker substitutions role of nucleosides in the flanking sequences. J Med Chem 45 4540 548... 

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