Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.     

Enhanced immune response by vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 macrophages

Vacuoles are membrane vesicles in eukaryotic cells, the digestive system of cells that break down substances absorbed outside the cell and digest the useless components of the cell itself. Researches on anticancer and intractable diseases using vacuoles are being actively conducted. The practical application of the present study to animals requires the determination of the biocompatibility of vacuole. In the present study, we evaluated the effects of vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 cells. This showed a significant increase in the production of nitric oxide (NO) produced by macrophage activity. Using Reactive Oxygen Species (ROS) assay, we identified that ROS is increased in a manner dependent on vacuole concentration. Western blot analysis showed that vacuole concentration-dependently increased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2). Therefore, iNOS expression was stimulated to induce NO production. In addition, pro-inflammatory cytokines levels promoted, such as interleukin (IL) 6 (IL-6) and tumor necrosis factor (TNF) α (TNF-α). In summary, vacuoles activate the immune response of macrophages by promoting the production of immune-mediated transporters NO, ROS, and pro-inflammatory cytokines.     

Water-soluble polysaccharides from Salvia officinalis L. possessing immunomodulatory activity

A water-soluble polysaccharide complex (A) composed of galactose (17.9%), 3-O-methyl-galactose (3.0%), glucose (15.5%), mannose (8.3%), arabinose (30.4%), xylose (7.6%), fucose (2.6%), rhamnose (6.7%), and uronic acids (8.0%) has been isolated from the aerial parts of sage (Salvia officinalis L.) by cold water extraction. It showed a broad molecular-mass distribution pattern (Mw approximately 2000-93,000) with a predominance of polymers with Mw< 10,000. Ion-exchange chromatography of A afforded six polymeric fractions (A1-A6) in which arabinogalactans associated with galacturonan and/or rhamnogalacturonan backbones prevail. Sage polysaccharides were examined for their immunomodulatory activity in the comitogenic thymocyte test which is interpreted as being an in vitro correlate of adjuvant activity. The acidic polysaccharide fractions A2, A3 and A4 exhibited the highest mitogenic and comitogenic activities of all fractions tested, and relatively high SI(comit)/SI(mit) ratios approximately 3 indicate potential adjuvant properties of these polysaccharides.     

DNA的免疫激活作用——Ⅰ.作用机理研究进展

含有免疫激活序列的DNA或寡聚核苷酸对脊椎动物的免疫系统可以产生广泛的激活作用。本文综述DNA免疫激活作用的结构基础、细胞及分子作用机制等方面的研究进展。     

Immunostimulatory principles from Chlorella pyrenoidosa—Part 1: Isolation and biological assessment in vitro

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.     

Structural Reevaluation of Streptococcus pneumoniae Lipoteichoic Acid and New Insights into Its Immunostimulatory Potency

Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39Δcps and TIGR4Δcps, respectively. The data obtained allowed us to (re)define (i) the position and linkage of the repeating unit, (ii) the putative α-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i.e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Δlgt mutant of strain D39Δcps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113Δlgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity.     

Delivery of plant-made vaccines and therapeutics

As commercial approval of the first, purified, plant-based biopharmaceuticals for parenteral delivery to humans approaches, improved strategies for delivery of plant-made vaccines and therapeutics are required to ensure their further development and to fulfil the prospect of supplying a global solution for affordable medicines. To ensure that this occurs, research should investigate and characterise the host immune system in addition to the effects of adjuvants and carrier vehicles on consistency and efficacy of vaccination. In this review we explore the basic understandings of pharmaceutical delivery and its effect on immunogenicity in an effort to advance the plant-made pharmaceutical platform.     

Concise synthesis of clarhamnoside, a novel glycosphingolipid isolated from the marine sponge Agela clathrodes

The first total synthesis of a novel alpha-galactoglycosphingolipid clarhamnoside has been achieved through a straightforward strategy. A thiogalactosyl donor with a benzylidene group at C-4 and C-6 and nonparticipating p-methoxybenzyl group at C-2 was successfully employed in the stereocontrolled syntheses of alpha-GalGSLs. The N-Phth-protected trifluoroacetimidate donor for terminal disaccharide was successfully applied in constructing the [GalNAc beta-(1-->6)-Gal] glycosidic linkage.     

Encapsulation in liposomal nanoparticles enhances the immunostimulatory,adjuvant and anti-tumor activity of subcutaneously administered CpG ODN

Immunostimulatory oligodeoxynucleotides (ODN) containing cytosine-guanine (CpG) motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through triggering of the Toll-like receptor 9. We have previously shown that encapsulation in liposomal nanoparticles (LN) enhances the immunostimulatory activity of CpG ODN (LN-CpG ODN) (Mui et al. in J Pharmacol Exp Ther 298:1185, 2001). In this work we investigate the effect of encapsulation on the immunopotency of subcutaneously (s.c.) administered CpG ODN with regard to activation of innate immune cells as well as its ability to act as a vaccine adjuvant with tumor-associated antigens (TAAs) to induce antigen (Ag)-specific, adaptive responses and anti-tumor activity in murine models. It is shown that encapsulation specifically targets CpG ODN for uptake by immune cells. This may provide the basis, at least in part, for the significantly enhanced immunostimulatory activity of LN-CpG ODN, inducing potent innate (as judged by immune cell activation and plasma cytokine/chemokine levels) and adaptive, Ag-specific (as judged by MHC tetramer positive T lymphocytes, IFN-γ secretion and cytotoxicity) immune responses. Finally, in efficacy studies, it is shown that liposomal encapsulation enhances the ability of CpG ODN to adjuvanate adaptive immune responses against co-administered TAAs after s.c. immunization, inducing effective anti-tumor activity against both model and syngeneic tumor Ags in murine tumor models of thymoma and melanoma.     

DNA的免疫激活作用——Ⅱ应用基础研究进展

综述免疫激活序列 (ISS)应用基础方面的研究进展。ISS- DNA不仅有望成为新一代高效低毒的免疫佐剂 ,而且在抗肿瘤、抗感染、促进造血、治疗免疫缺陷以及防治哮喘等方面均有非常乐观的应用前景。