HPV16 E6/E7 -based mRNA vaccine is therapeutic in mice bearing aggressive HPV-positive lesions.

HPV (Human papillomavirus) affects 600,000 people worldwide each year. Almost all cervical cancers are associated with a past HPV infection. In particular, the positivity to the high-risk type HPV16 is detected in most of the invasive cervical cancers.

A novel antagonist of Toll-like receptors 7, 8 and 9 suppresses lupus disease-associated parameters in NZBW/F1 mice.

Systemic Lupus Erythematosus is an autoimmune disease characterized by production of autoantibodies against nucleic acid-associated antigens. Endogenous DNA and RNA associated with these antigens stimulate inflammatory responses through Toll-like receptors (TLRs) and exacerbate lupus disease pathology. We have evaluated an antagonist of TLR7, 8 and 9 as a therapeutic agent in lupus-prone NZBW/F1 mice.

Immune-Stimulatory Dinucleotide at the 5'-End of Oligodeoxynucleotides Is Critical for TLR9-Mediated Immune Responses.

Oligodeoxynucleotides (ODNs) containing a CpG or certain synthetic dinucleotides, referred to as immune-stimulatory dinucleotides, induce Toll-like receptor 9 (TLR9)-mediated immune responses. Chemical modifications such as 2'-O-methylribonucleotides incorporated adjacent to the immune-stimulatory dinucleotide on the 5'-side abrogate TLR9-mediated immune responses. In this study, we evaluated the effect of the location of immune-stimulatory dinucleotides in ODNs on TLR9-mediated immune responses.

Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9.

Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2'-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates.

A Toll-like receptor 7, 8, and 9 antagonist inhibits Th1 and Th17 responses and inflammasome activation in a model of IL-23-induced psoriasis.

Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1β. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice.

Design of synthetic oligoribonucleotide-based agonists of Toll-like receptor 3 and their immune response profiles in vitro and in vivo.

Double-stranded RNA of viral origin and enzymatically synthesized poly I:C act as agonists of TLR3 and induce immune responses. We have designed and synthesized double-stranded synthetic oligoribonucleotides (dsORNs) which act as agonists of TLR3. Each strand of dsORN contains two distinct segments, namely an alignment segment composed of a heteronucleotide sequence and an oligo inosine (I) or an oligo cytidine (C) segment.

Novel oligonucleotides containing two 3'-ends complementary to target mRNA show optimal gene-silencing activity.

Oligonucleotides are being employed for gene-silencing activity by a variety of mechanisms, including antisense, ribozyme, and siRNA. In the present studies, we designed novel oligonucleotides complementary to targeted mRNAs and studied the effect of 3'-end exposure and oligonucleotide length on gene-silencing activity. We synthesized both oligoribonucleotides (RNAs) and oligodeoxynucleotides (DNAs) with phosphorothioate backbones, consisting of two identical segments complementary to the targeted mRNA attached through their 5'-ends, thereby containing two accessible 3'-ends; these compounds are referred to as gene-silencing oligonucleotides (GSOs).

Impact of nature and length of linker incorporated in agonists on toll-like receptor 9-mediated immune responses.

Oligodeoxynucleotides containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). We previously reported a novel class of TLR9 agonists, referred to as immune-modulatory oligonucleotides (IMOs), in which two 11-mers of the same sequence are attached via their 3'-ends through a 1,2,3-propanetriol linker and contain a synthetic immune-stimulatory motif, Cp7-deaza-dG. In the present study, we have examined the impact of length, nature, and stereochemistry of the linker incorporated in agonists for TLR9 activation.

Peptide conjugation at the 5'-end of oligodeoxynucleotides abrogates toll-like receptor 9-mediated immune stimulatory activity.

Bacterial and synthetic DNA containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). Our earlier studies showed that 5'-accessibility of synthetic oligodeoxynucleotides containing CpG motif (ODN) is required for TLR9-mediated immune stimulatory activity. Blocking the 5'-end of ODN through conjugation to a variety of moieties reduces immune stimulatory activity (Bioconjugate Chem.

Modifications incorporated in CpG motifs of oligodeoxynucleotides lead to antagonist activity of toll-like receptors 7 and 9.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides act as agonists of TLR9 and induce Th1-type immune responses. In the present study, we synthesized CpG containing ODNs in which C or G was substituted with 2'-O-methylribonucleotides, 5-methyl-dC, or 2'-O-methyl-5-methyl-C and studied their immune stimulatory activity alone and in combination with TLR agonists. In mouse and human primary cell-based assays, modified ODNs did not stimulate immune responses but inhibited TLR9 agonist-induced immune stimulatory activity.

Synthetic oligoribonucleotides containing arabinonucleotides act as agonists of TLR7 and 8.

In continuation of our studies with stabilized immune modulatory RNA (SIMRA) compounds, we have synthesized novel SIMRA compounds incorporating arabinonucleotides to study their effects on TLR7 and TLR8 activation. The SIMRA compounds containing ara-G, ara-C, ara-U or ara-A substitutions activated TLR8 in HEK293 cells. Interestingly, the SIMRA compound containing ara-C also activated TLR7 and stimulated immune responses in vivo in mice.

Oligodeoxyribonucleotide-based antagonists for Toll-like receptors 7 and 9.

Oligodeoxyribonucleotides containing unmethylated CpG motifs act as TLR9 agonists. In this study, we evaluated oligonucleotides containing an unmethylated CpG motif in which two nucleotides adjacent to the CpG dinucleotide were substituted with 2'-O-methylribonucleotides, resulting in TLR7 and TLR9 antagonists. In mouse and human cell cultures, antagonists did not stimulate immune activation but inhibited TLR7 and TLR9 agonist-induced activity.

Impact of secondary structure of toll-like receptor 9 agonists on interferon alpha induction.

Oligodeoxynucleotides containing a CpG motif and double- or multistranded structure-forming sequences act as agonists of Toll-like receptor 9 (TLR9) and induce high levels of interferon alpha (IFN-alpha) in addition to other Th1-type cytokines. In the present study, we evaluated three highly effective IFN-alpha-inducing agonists of TLR9 to determine the type of duplex structures formed and the agonist's ability to induce immune responses, including IFN-alpha induction, in human cell-based assays and in vivo in mice and nonhuman primates. Thermal melting studies showed that two of the agonists evaluated had a single melting transition with similar hyperchromicity in both heating and cooling cycles, suggesting the formation of intermolecular duplexes.

Agonists of Toll-like receptor 9 containing synthetic dinucleotide motifs.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9). Our previous studies have shown that ODNs containing two 5'-ends are more immunostimulatory than those with one 5'-end. In the present study, to understand the role of functional groups in TLR9 recognition and subsequent immune response, we substituted C or G of a CpG dinucleotide with 5-OH-dC, 5-propyne-dC, furano-dT, 1-(2'-deoxy-beta- d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, dF, 4-thio-dU, N(3)-Me-dC, N (4)-Et-dC, Psi-iso-dC, and arabinoC or 7-deaza-dG, 7-deaza-8-aza-dG, 9-deaza-dG, N(1)-Me-dG, N(2)-Me-dG, 6-Thio-dG, dI, 8-OMe-dG, 8-O-allyl-dG, and arabinoG in ODN containing two 5'-ends.

Sodium arsenite induces heat shock protein 70 expression and protects against secretagogue-induced trypsinogen and NF-kappaB activation.

Heat shock proteins (HSPs), induced by a variety of stresses, are known to protect against cellular injury. Recent studies have demonstrated that prior beta-adrenergic stimulation as well as thermal or culture stress induces HSP70 expression and protects against cerulein-induced pancreatitis. The goal of our current studies was to determine whether or not a non-thermal, chemical stressor like sodium arsenite also upregulates HSP70 expression in the pancreas and prevents secretagogue-induced trypsinogen and NF-kappaB activation.

Stabilized immune modulatory RNA compounds as agonists of Toll-like receptors 7 and 8.

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptor (TLR)7 and TLR8. However, single-stranded RNA is rapidly degraded by ubiquitous RNases, and the studies reported to date have used RNA with lipid carriers. To overcome nuclease susceptibility of RNA, we have synthesized several RNAs incorporating a range of chemical modifications.

Protease-activated receptor-2 protects against pancreatitis by stimulating exocrine secretion.

Background: Protease-activated receptor-2 (PAR-2) is present in the pancreas, where it has been shown to play a protective role during pancreatitis. However, the mechanism by which it protects against pancreatitis still remains to be elucidated. Acute pancreatitis is associated with premature zymogen activation and a blockage in digestive enzyme secretion.

Novel oligodeoxynucleotide agonists of TLR9 containing N3-Me-dC or N1-Me-dG modifications.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs activate Toll-Like Receptor 9 (TLR9). Our previous studies have shown the role of hydrogen-bond donor and acceptor groups of cytosine and guanine in the CpG motif and identified synthetic immunostimulatory motifs. In the present study to elucidate the significance of N3-position of cytosine and N1-position of guanine in the CpG motif, we substituted C or G of a CpG dinucleotide with N3-Me-cytosine or N1-Me-guanine, respectively, in immunomodulatory oligodeoxynucleotides (IMOs).

Immunization with gp120-depleted whole killed HIV immunogen and a second-generation CpG DNA elicits strong HIV-specific responses in mice.

HIV-1 Immunogen is a gp120-depleted whole killed virus vaccine candidate formulated with Incomplete Freund's Adjuvant (HIV-IFA). We evaluated in a mouse model the immunogenicity of HIV-IFA by itself and when combined with HYB2055, an immunomodulatory oligonucleotide consisting of a novel DNA structure and synthetic CpR immunostimulatory motif, as an adjuvant. C57/BL6 mice were immunized with HIV-IFA alone or combined with HYB2055.

Immunomodulatory oligonucleotides containing a cytosine-phosphate-2'-deoxy-7-deazaguanosine motif as potent toll-like receptor 9 agonists.

Bacterial DNA and synthetic oligomers containing CpG dinucleotides activate the immune system through Toll-like receptor (TLR) 9. Here, we compare the immunostimulatory activity of three immunomers with different nucleotide sequences containing a synthetic cytosine-phosphate-2'-deoxy-7-deazaguanosine dinucleotide (CpR), called immunomodulatory oligonucleotides (IMOs), in mouse, human, and monkey systems. IMOs induced IL-12 and IFN-gamma secretion more than a control non-CpG IMO in mice.

Novel immunomodulatory oligonucleotides prevent development of allergic airway inflammation and airway hyperresponsiveness in asthma.

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG oligos) have been shown to prevent development of allergic airway inflammation and airway hyperresponsiveness (AHR) in mouse models of asthma. Recently, we reported immunomodulatory oligonucleotides (IMOs) containing novel structures (immunomers) and synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) motifs show potent stimulatory activity with distinct cytokine secretion profiles. Since type 2 T cells predominate in asthma and increase in type 1 cells can prevent the differentiation of naïve T lymphocytes to a type 2 phenotype, we hypothesized that IMOs can prevent the development of allergic airway inflammation and AHR in the ovalbumin (OVA)-sensitized and challenged mouse model.

A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif.

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3'-3-linked oligos, referred to as immunomers.

Pathophysiology of alcohol-induced pancreatic injury.

The evidence that ethanol abuse can lead to pancreatitis is overwhelming, but the mechanism(s) by which ethanol causes pancreatic injury and pancreatitis are not known. Many studies have focused on short-term effects of ethanol administration on exocrine pancreatic function, but the results reported have been variable and no clear picture has emerged. Attempts to induce pancreatitis by long-term ethanol administration have, for the most part, failed.

Self-stabilized CpG DNAs optimally activate human B cells and plasmacytoid dendritic cells.

We recently showed that 5'-terminal secondary structures in CpG DNA affect activity significantly more than those at the 3'-end [Biochem. Biophys. Res.

Secondary structures in CpG oligonucleotides affect immunostimulatory activity.

Oligodeoxynucleotides containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system. Our previous studies showed that the 5(')-end of a CpG oligonucleotide should be accessible for receptor recognition and subsequent immune stimulation. Activity is abrogated if this end is blocked by joining two CpG oligos through 5(')-5(') linkage.