Chimeric hepatitis B surface antigen virus-like particles expressing the strep A epitope p*17 elicit a humoral immune response in mice.

To optimize immunogenicity, bacterial epitopes in putative vaccine constructs can be presented to immune cells as multiple repeated structures on a defined nanoparticle. Virus-like particles (VLPs) are viral capsid proteins that self-assemble to form compact and highly ordered nanoparticles that are within the optimal size range for uptake by dendritic cells. VLPs mimic the live virus in size and form but contain no viral genetic material, are therefore noninfective and are the basis of safe and effective vaccines against hepatitis B virus (HBV) and human papillomavirus (HPV). Due to their particulate nature, molecular stability, and expression of high density and repetitive antigen displays, recombinant cell culture-derived VLPs are ideal platforms for the delivery of small molecules, including bacterial epitopes. We developed a putative vaccine by expressing a minimal epitope from the bacterium Streptococcus pyogenes (Strep A) on the surface of a recombinant VLP comprising multiple copies of HBV small envelope protein (HBsAg-S). Strep A is responsible for a wide spectrum of human infections and postinfectious diseases that disproportionately affect children and young adults living in resource-poor communities. No vaccine is currently available to offer sufficiently broad protection from the numerous and diverse strains of Strep A endemic in these at-risk populations. The Strep A antigen targeted by our vaccine construct is p*17, a cryptic epitope from a highly conserved region of the Strep A M-protein with demonstrated enhanced immunogenicity and broad protective potential against Strep A. To ensure surface expression and optimal immunogenicity, we expressed p*17 within the immunodominant "a" determinant of HBsAg-S. The recombinant VLPs (VLP-p*17) expressed in HEK293T cells spontaneously formed 22 nm particles and induced the production of high titers of p*17-specific IgG in BALB/c mice immunized with three 0.5 µg doses of VLP-p*17 formulated with adjuvant.

Correlation between Fatty Acid Profile and Anti-Inflammatory Activity in Common Australian Seafood by-Products.

Marine organisms are a rich source of biologically active lipids with anti-inflammatory activities. These lipids may be enriched in visceral organs that are waste products from common seafood. Gas chromatography-mass spectrometry and fatty acid methyl ester (FAME) analyses were performed to compare the fatty acid compositions of lipid extracts from some common seafood organisms, including octopus (Octopus tetricus), squid (Sepioteuthis australis), Australian sardine (Sardinops sagax), salmon (Salmo salar) and school prawns (Penaeus plebejus). The lipid extracts were tested for anti-inflammatory activity by assessing their inhibition of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse cells. The lipid extract from both the flesh and waste tissue all contained high amounts of polyunsaturated fatty acids (PUFAs) and significantly inhibited NO and TNFα production. Lipid extracts from the cephalopod mollusks S. australis and O. tetricus demonstrated the highest total PUFA content, the highest level of omega 3 (ω-3) PUFAs, and the highest anti-inflammatory activity. However, multivariate analysis indicates the complex mixture of saturated, monounsaturated, and polyunsaturated fatty acids may all influence the anti-inflammatory activity of marine lipid extracts. This study confirms that discarded parts of commonly consumed seafood species provide promising sources for the development of new potential anti-inflammatory nutraceuticals.

Brominated indoles from a marine mollusc inhibit inflammation in a murine model of acute lung injury.

New drug leads for the treatment of inflammation are urgently needed. Marine molluscs are widely used as traditional medicines for the treatment of inflammation. Here we report the positive effects of a hypobranchial gland (HBG) extract and the dominant bioactive compound 6-bromoisatin from the Muricidae mollusc Dicathais orbita, for reducing lipopolysaccharide (LPS) induced acute lung inflammation in a mouse model. Both 6-bromoisatin and the HBG extract suppressed the inflammatory response in mice that were pre-treated by oral gavage at 48, 24 and 1 h prior to LPS infusion. The inflammatory antagonists were tested at concentrations of 0.5 mg/g and 0.1 mg/g HBG extract and 0.1 mg/g and 0.05 mg/g 6-bromoisatin in carrier oil and all treatments reduced inflammation as indicated by a significant suppression of inflammatory markers present in bronchoalveolar lavage fluid (BALF), in comparison to LPS induced positive control mice administered the carrier oil alone (p < 0.0001). Tumour necrosis factor-alpha (TNFα) and interleukin-1 beta (IL-1ß) levels, in addition to total protein concentration were all significantly reduced in BALF from mice treated with the extract or 6-bromoisatin. Furthermore, all treatment groups showed significant reductions in neutrophil sequestration and preservation of the lung tissue architecture compared to the positive control (p < 0.0001). The combined results from this study and our previous in vitro studies indicate that 6-bromoisatin in the HGB extracts inhibit the activation of inflammatory signalling pathway. The results from this study further confirm that the HBG extract from Muricidae molluscs and 6-bromoisatin are bioavailable and effective in vivo, thus have potential for development as natural therapeutic agents for inflammation.

Anti-Inflammatory Activity and Structure-Activity Relationships of Brominated Indoles from a Marine Mollusc.

Marine molluscs are rich in biologically active natural products that provide new potential sources of anti-inflammatory agents. Here we used bioassay guided fractionation of extracts from the muricid Dicathais orbita to identify brominated indoles with anti-inflammatory activity, based on the inhibition of nitric oxide (NO) and tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages and prostaglandin E2 (PGE2) in calcium ionophore-stimulated 3T3 ccl-92 fibroblasts. Muricid brominated indoles were then compared to a range of synthetic indoles to determine structure-activity relationships. Both hypobranchial gland and egg extracts inhibited the production of NO significantly with IC50 of 30.8 and 40 µg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα and PGE2 with IC50 of 43.03 µg/mL and 34.24 µg/mL, respectively. The purified mono-brominated indole and isatin compounds showed significant inhibitory activity against NO, TNFα, and PGE2, and were more active than dimer indoles and non-brominated isatin. The position of the bromine atom on the isatin benzene ring significantly affected the activity, with 5Br > 6Br > 7Br. The mode of action for the active hypobranchial gland extract, 6-bromoindole, and 6-bromoisatin was further tested by the assessment of the translocation of nuclear factor kappa B (NFκB) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NFκB in the LPS-stimulated RAW264.7 macrophages by 48.2%, whereas 40 µg/mL of 6-bromoindole and 6-bromoistain caused a 60.7% and 63.7% reduction in NFκB, respectively. These results identify simple brominated indoles as useful anti-inflammatory drug leads and support the development of extracts from the Australian muricid D. orbita, as a new potential natural remedy for the treatment of inflammation.

Anti-inflammatory activity of hyperimmune plasma in a lipopolysaccharide-mediated rat air pouch model of inflammation.

Tumour necrosis factor-α (TNFα) and polymorphonuclear neutrophils play key and interrelated roles in the inflammatory response against infectious agents. However, these entities can mediate significant tissue damage if their biological activity becomes deregulated. We have previously shown that canine hyperimmune frozen plasma (HFP) contains anti-TNFα activity that is attributable to elevated levels of soluble TNFα receptor 1 (sTNFR1). The aim of this study was to determine the effect of HFP on TNFα levels and neutrophil infiltration in a lipopolysaccharide (LPS)-mediated rat air pouch model of inflammation. Rats were administered either HFP, HFP which had been pre-incubated with anti-sTNFR1 antibody (5 ng/ml), fresh frozen plasma (FFP), physiological saline (PS) at 2 ml/day or Carprofen at 5 mg/kg for 3 days prior to LPS challenge. Pouch fluid was withdrawn at 1, 6, 12, 24 and 48 h post-LPS challenge and assayed for TNFα by ELISA, and for total leukocytes and neutrophils by microscopic examination. At 6 h post-LPS challenge, both TNFα levels and neutrophil counts were significantly lower in HFP-treated rats than was found in FFP, PS or Carprofen treated animals (p<0.05). In a sTNFR1 blocking experiment, incubation of HFP with anti-sTNFR1 antibody resulted in significant increases in neutrophil numbers and TNFα levels, which suggests that the anti-TNFα activity observed in HFP may be due to elevated levels of sTNFR1. The data also revealed a significant inverse correlation between total leukocyte counts and sTNFR1 levels present in pouch fluid (r= -0.73, p<0.0001). Our observations suggest that HFP warrants further investigation as a possible means for modulating acute inflammatory processes where TNFα is a key mediator.

Immunological response to parenteral vaccination with recombinant hepatitis B virus surface antigen virus-like particles expressing Helicobacter pylori KatA epitopes in a murine H. pylori challenge model.

Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S-KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 10(3)) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 10(2)). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 10(4) and 2.6 × 10(4), respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.

Caelestines A-D, brominated quinolinecarboxylic acids from the Australian ascidian Aplidium caelestis.

Four new brominated natural products, caelestines A-D (1-4), have been isolated from the Australian ascidian Aplidium caelestis. The structures of 1-4 were determined by analysis of their NMR and MS data. This is the first report of brominated quinolinecarboxylic acids from nature. Compound 1 has been previously synthesized but not spectroscopically characterized. Compounds 1-4 were tested against three mammalian cell lines (MCF-7, NFF, and MM96L) and a panel of microbial strains and showed only minor cytotoxicity.

Detection of anti-TNFalpha activity in canine hyperimmune serum using a TNFalpha inhibition assay.

BACKGROUND: Increased serum tumor necrosis factor-alpha (TNFalpha) activity has been associated with onset of serious inflammatory diseases in dogs. Development of treatment with TNFalpha-antagonists has been limited by the unavailability of suitable reagents and potency assays for TNFalpha. OBJECTIVES: The objectives of this study were to optimize a cell-based assay to measure anti-TNFalpha activity in serum and plasma from hyperimmune (vaccinated with an Escherichia coli J5 bacterin) and unvaccinated canine donors; to use the assay to determine whether hyperimmune serum inhibits TNFalpha activity in vivo; and to determine whether soluble TNF receptor-1 (sTNFR1, a naturally occurring TNFalpha antagonist) contributes to anti-TNFalpha activity. METHODS: Commercial plasma and serum from hyperimmune-frozen plasma (HFP) donors and unvaccinated fresh-frozen plasma (FFP) donors were used in the study. An L929-cell TNFalpha-inhibition assay (LTIA) was optimized to measure anti-TNFalpha activity. Using a rat subcutaneous pouch model of inflammation, the effects of HFP, FFP, a synthetic TNFalpha antagonist (Etanercept), and carprofen on TNFalpha activity were compared in vivo. Immunofluorescence was used to measure soluble sTNFR1 concentration. RESULTS: Using the optimized LTIA, HFP serum but not FFP serum decreased canine TNFalpha activity (P<.01). HFP plasma and Etanercept (but not FFP plasma or carprofen) significantly decreased TNFalpha activity in pouch exudates (P<.05). A significantly higher concentration of sTNFR1 was found in HFP than FFP serum. CONCLUSIONS: Using the LTIA, anti-TNFalpha activity is readily measured in canine serum and inflammatory exudates. sTNFR1 appears to contribute to anti-TNFalpha activity in HFP serum. These results suggest HFP should be investigated further as a potential immunotherapeutic agent for controlling canine diseases in which TNFalpha is implicated.