Modulation of immunogenicity and allergenicity by controlling the number of immunostimulatory oligonucleotides linked to Amb a 1.

BACKGROUND: Immunostimulatory DNA sequences (ISS) are potent immunomodulators that can drive T(H)1 responses to antigens or allergens. This effect can be dramatically enhanced by direct linkage of ISS to the protein. OBJECTIVE: Evaluate the effects of the number of ISS bound to the major ragweed allergen Amb a 1 on immunogenicity and allergenicity. METHODS: Immunogenicity in mice and allergenicity using PBMC or sera from subjects with ragweed allergy were assayed. RESULTS: Both antibody induction in vivo and antibody recognition in vitro were highly sensitive to the number of ISSs linked. IgE recognition of Amb a 1 in competitive ELISA or histamine release assays was inhibited by ISS linkage and showed an inverse relationship to the number of ISSs bound. Type and magnitude of antibody induction in mice was also highly dependent on the number of ISS bound. At the highest ISS to protein ratios, antibody induction was very low. Moderate ISS to protein ratios induced high antibody responses in which IgG(2a) generally predominated. Low ISS to protein ratios produced the highest overall antibody responses in which IgG(1) predominated. In contrast, varied ISS to protein ratios did not affect T-cell responses. In both in vivo mouse studies and in vitro human PBMC studies, all ISS to protein ratios evaluated induced similar responses represented by high levels of IFN-gamma and low levels of T(H)2 cytokines. CONCLUSION: Controlling the number of ISS bound to a protein allows manipulation of antibody recognition and induction while retaining the potent T(H)1 properties of an ISS-linked protein. CLINICAL IMPLICATIONS: Immunostimulatory DNA sequence-linked Amb a 1 conjugate represents a safe, novel therapeutic approach for treating ragweed allergy.

Induction of interferon-gamma from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha.

Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon-gamma (IFN-gamma) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS-induced IFN-gamma from NK cells is primarily dependent upon IFN-alpha release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC-released soluble factor(s) may contribute to IFN-gamma induction. Indeed, tumour necrosis factor-alpha (TNF-alpha) was identified as a significant contributor to ISS-mediated activation of NK cells and was observed to act in an additive fashion with IFN-alpha in the induction of IFN-gamma from NK cells and to up-regulate CD69 expression on NK cells. This activity of TNF-alpha, however, was dependent upon the presence of PDC-derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF-alpha.

Superior activity of the type C class of ISS in vitro and in vivo across multiple species.

CpG-C are a novel class of CpG motif-containing immunostimulatory sequences (ISS) that includes both a 5'-TCG element and a CpG-containing palindrome. CpG-C drive all known ISS activities and, in particular, are potent enhancers of IFN-alpha from plasmacytoid dendritic cells (PDCs). In our examination of CpG-C sequence requirements, we determined that optimal IFN-alpha-inducing activity could be achieved with longer palindromes. Longer palindromes also correlated with maintenance of the double-stranded (ds) form despite concentration and pH changes, indicating a preference for ds oligodeoxynucleotides (ODNs) by the ISS-induced signaling mechanism for IFN-alpha synthesis. This correlation did not hold for all arms of the ISS-induced immune response, since we did not observe increased B cell activity with the longer palindrome CpG-C ODNs. We further demonstrated that CpG-C retained activity in an in vitro primate system and induced the expression of several cytokines and IFN-alpha-inducible genes when CpG-C were administered in vivo to mice and primates. In conclusion, we have shown CpG-C to exert several types of immune functions across multiple species, and this novel class is thus an attractive candidate for ISS-based therapeutic strategies.

Polymyxin B enhances ISS-mediated immune responses across multiple species.

The immunostimulatory effects of bacterial DNA on mammalian cells have been localized to unmethylated CpG motifs, and synthetic CpG-containing oligodeoxynucleotides that mimic these effects are known as immunostimulatory sequences (ISS). We have found that the polycationic antibiotic, polymyxin B (PMXB), associates with ISS and serum albumin in vitro and forms microparticles that greatly increase the activity of ISS on plasmacytoid dendritic cells (PDCs). Specifically, ISS/PMXB greatly enhanced IFN-alpha production from PDCs and other activities downstream of IFN-alpha, including IFN-gamma secretion, NK lytic activity, and the expression of genes dependent upon IFN-alpha/IFN-gamma. This amplification was specific for the IFN-alpha pathway since other ISS activities, including B cell proliferation, B cell IL-6 secretion, and PDC maturation, were not affected by PMXB. Both the polycationic peptide and lipophilic fatty acid side chain domains of PMXB, as well as the presence of a third party stabilizing agent such as albumin or Tween 85, were required for particle formation and enhanced ISS activity. The ISS-enhancing activity of PMXB was observed across multiple species (human, primate, and mouse) and in vivo (primate, mouse). These data illustrate the usefulness of formulating ISS with a cationic lipopeptide such as PMXB, which focuses and greatly amplifies the ISS-induced pathway of IFN-alpha-mediated responses.

A minimal human immunostimulatory CpG motif that potently induces IFN-gamma and IFN-alpha production.

Recent reports have shown that immunostimulatory sequences (ISS) containing CpG motifs have minimal length requirements (>/=12 bases) for the exertion of immune-enhancing function upon mammalian cells. Herein we demonstrate that short ISS (5-7 bases), which exhibit no activity on their own, induce IFN-gamma and IFN-alpha secretion from human peripheral blood mononuclear cells when adsorbed to the surface of cationic poly(D,L-lactide-co-glycolide) microparticles (cPLGA). Utilizing this technique, we discovered a minimal ISS sequence for induction of IFN-gamma and IFN-alpha from human cells: 5'-TCGXX-3'. These short ISS/cPLGA formulations targeted PDC in similar fashion to longer ISS ODN, the activity of which does not require (but is enhanced by) cPLGA. PDC stimulated with short ISS/cPLGA responded with enhanced uptake of ISS and elevated production of cytokines, including IFN-alpha. However, ISS-responsive B cells did not respond to short ISS/cPLGA, underlining the plasmacytoid dendritic cell selectivity of this formulation. These results describe a novel technique for formulating active, but very short, ISS oligodeoxynucleotide that allows for the dissection and characterization of minimal immunostimulatory CpG motifs.

Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions.

Recent reports have identified two major classes of CpG motif-containing oligodeoxynucleotide immunostimulatory sequences (ISS): uniformly modified phosphorothioate (PS) oligodeoxyribonucleotides (ODNs), which initiate B cell functions but poorly activate dendritic cells (DCs) to make interferon (IFN)-alpha, and chimeric PS/phosphodiester (PO) ODNs containing runs of six contiguous guanosines, which induce very high levels of plasmacytoid DC (PDC)-derived IFN-alpha but poorly stimulate B cells. We have generated the first reported ISS, C274, which exhibits very potent effects on all human immune cells known to recognize ISS. C274 is a potent inducer of IFN-gamma/IFN-alpha from peripheral blood mononuclear cells and exhibits accelerated kinetics of activity compared with standard ISS. This ODN also effectively stimulates B cells to proliferate, secrete cytokines, and express costimulatory antigens. In addition, C274 specifically activates PDCs to undergo maturation and secrete cytokines, including very high levels of IFN-alpha. Sequence variation studies based on C274 were used to identify the general motif requirements for this novel and distinct class of ISS. In contrast, chimeric PO/PS CpG-containing ODNs with polyguanosine sequences exert a differential pattern of ISS activity compared with C274, perhaps in part as a result of their greatly different structural nature. This pattern is composed of high IFN-alpha/IFN-gamma induction and low DC maturation in the absence of B cell stimulation. In conclusion, we have generated a novel class of ISS that transcends the limitations ascribed to classes described previously in that it provides excellent stimulation of B cells and simultaneously activates PDCs to differentiate and secrete large amounts of type I IFN.