Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

Purification of a lectin from Cratylia mollis crude extract seed by a single step PEG/phosphate aqueous two-phase system.

The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500-8000 g/mol), PEG concentration (12.5-17.5% w/w), phosphate (10-15% w/w) concentration, and pH (6-8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box-Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 °C and lost hemagglutinating activity at 80 °C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.

An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro.

Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.

Isolation of a Hemagglutinin with Potent Antiproliferative Activity and a Large Antifungal Defensin from Phaseolus vulgaris cv. Hokkaido Large Pinto Beans.

Lectins (hemagglutinins) are defined as sugar-binding proteins or glycoproteins with various biological activities. A 60 kDa dimeric hemagglutinin with a blocked N-terminus was isolated in large yield (190 mg/60 g) from the common edible bean Phaseolus vulgaris cv. Hokkaido large pinto bean. Its hemagglutinating, antifungal, and antitumor activities as well as the effects of carbohydrate and metal ions on its hemagglutinating activity were examined. It inhibited the proliferation of nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7), and hepatoma (HepG2) cells. The IC50 values toward HepG2, MCF7, and CNE2 cells after treatment for 48 h were 8.1, 6.07, and 7.49 µM, respectively, which were relatively low among lectins of different P. vulgaris cultivars. From the pinto beans, a 10888 Da antifungal peptide with similarity to plant defensins as revealed by mass spectroscopic analysis was also isolated with a yield of 3.2 mg of proteins from 60 g of beans. The large defensin was capable of inhibiting mycelial growth in Mycosphaerella arachidicola, Setosphaeria turcica, Bipolaris maydis, and Fusarium oxysporum but not in Valsa mali.

Purification and Characterization of a Novel Hemagglutinin with Inhibitory Activity toward Osteocarcinoma Cells from Northeast China Black Beans.

In the present study, we isolated a novel hemagglutinin from an edible legume and explored its growth-inhibitory effect on osteocarcinoma and liver cancer cells. The protein was purified by liquid chromatography techniques which entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono Q, and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion dependent and stable over a wide range of temperature and pH values. Antiproliferative activity was observed in the tumor cell lines MG-63 and HepG2 but not in the normal cell line WRL 68. Osteocarcinoma cells treated with the hemagglutinin underwent obvious cell shrinkage, chromatin condensation, mitochondrial membrane depolarization, and apoptosis. The mRNA expression level of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) were found to be up-regulated to different extents after treatment of this hemagglutinin.

Purification and characterization of a novel lectin from Chinese leek seeds.

A novel lectin, CLSL, was purified from Chinese leek seeds by ion exchange chromatography on SP Sephadex C-25 and gel filtration chromatography on Sephadex G50. The lectin had a molecular weight of 23.6 kDa and was composed of two identical subunits linked by disulfide bonds, a conclusion based on SDS-PAGE under reducing and nonreducing conditions. CLSL was a glycoprotein with a carbohydrate content of 3.6%. It exerted potent agglutinating activity against rat red blood cells at a concentration of 8.9 µg/mL. Hemagglutination of rat erythrocytes was inhibited by d-fructose, mannitol, and sorbose at the concentration of 20 mM. The hemagglutinating activity of CLSL was maintained at 100 °C for 60 min and under acidic pH conditions but was lost at neutral and alkaline pH conditions. The hemagglutinating activity was stimulated by Ca(2+), Fe(2+), and Cu(2+) but inactivated by Ba(2+) at a concentration of 10 mM. Ba(2+)-mediated inactivation of CLSL was caused by CLSL conformational change induced by barium ions, according to the results of circular dichroism and fluorescence spectroscopy. Deconvolution of the CLSL circular dichroism indicated that it was an α-helical lectin with α-helix and ß-fold contents of 35.8% and 8.6%, respectively. CLSL could also selectively inhibit cell proliferation.

Surface modifications of influenza proteins upon virus inactivation by ß-propiolactone.

Inactivation of intact influenza viruses using formaldehyde or ß-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution FT-ICR MS-based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full-length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids' side chain length and polarity.

Purification, partial characterization and immobilization of a mannose-specific lectin from seeds of Dioclea lasiophylla mart.

Lectin from the seeds of Dioclea lasiophylla (DlyL) was purified in a single step by affinity chromatography on a Sephadex® G-50 column. DlyL strongly agglutinated rabbit erythrocytes and was inhibited by monosaccharides (D-mannose and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Similar to other Diocleinae lectins, DlyL has three chains, α, ß and γ, with mass of 25,569 ± 2, 12,998 ± 1 and 12,588 ± 1 Da, respectively, and has no disulfide bonds. The hemagglutinating activity of DlyL was optimal in pH 8.0, stable at a temperature of 70 °C and decreased in EDTA solution, indicating that lectin activity is dependent on divalent metals. DlyL exhibited low toxicity on Artemia sp. nauplii, but this effect was dependent on the concentration of lectin in solution. DlyL immobilized on cyanogen bromide-activated Sepharose® 4B bound 0.917 mg of ovalbumin per cycle, showing the ability to become a tool for glycoproteomics studies.

Serologic assays for influenza surveillance, diagnosis and vaccine evaluation.

Serological techniques play a critical role in various aspects of influenza surveillance, vaccine development and evaluation, and sometimes in diagnosis, particularly for novel influenza virus infections of humans. Because individuals are repeatedly exposed to antigenically and genetically diverse influenza viruses over a lifetime, the gold standard for detection of a recent influenza virus infection or response to current vaccination is the demonstration of a seroconversion, a fourfold or greater rise in antibody titer relative to a baseline sample, to a circulating influenza strain or vaccine component. The hemagglutination-inhibition assay remains the most widely used assay to detect strain-specific serum antibodies to influenza. The hemagglutination-inhibition assay is also used to monitor antigenic changes among influenza viruses which are constantly evolving; such antigenic data is essential for consideration of changes in influenza vaccine composition. The use of the hemagglutinin-specific microneutralization assay has increased, in part, owing to its sensitivity for detection of human antibodies to novel influenza viruses of animal origin. Neutralization assays using replication-incompetent pseudotyped particles may be advantageous in some laboratory settings for detection of antibodies to influenza viruses with heightened biocontainment requirements. The use of standardized protocols and antibody standards are important steps to improve reproducibility and interlaboratory comparability of results of serologic assays for influenza viruses.

Screening from the world's largest TCM database against H1N1 virus.

The swine influenza virus (H1N1) 2009 pandemic highlights the importance of having effective anti-viral strategies. Recently, oseltamivir (Tamiflu) resistant influenza viruses are identified; which further emphasizes the urgency in developing new antiviral agents. In influenza virus replication cycle, viral surface glycoprotein, hemagglutinin, is responsible for viral entry into host cells. Hence, a potentially effective antiviral strategy is to inhibit viral entry mechanism. To develop novel antiviral agent that inhibits viral entry, we analyzed 20,000 traditional Chinese medicine (TCM) ingredients in hemagglutinin subtype H1 sialic acid binding site found on H1N1 virus. We then performed molecular dynamics simulations to investigate receptor-ligand interaction of the candidates obtained from docking. Here, we report three TCM derivatives that have high binding affinities to H1 sialic acid binding site residues based on structure-based calculations. The top three derivatives, xylopine_2, rosmaricine_14 and rosmaricine_15, all have an amine group that interact with Glu83 and a pyridinium group that interact with Asp103. Molecular dynamics simulations show that these derivatives form strong hydrogen bonding with Glu83 but interact transiently with Asp103. We therefore suggest that an enhanced hemagglutinin inhibitor, based on our scaffold, should be designed to bind both Glu83 and Asp103 with high affinity.

Biological activities of aqueous and organic extracts from tropical marine sponges.

We report on screening tests of 66 extracts obtained from 35 marine sponge species from the Caribbean Sea (Curaçao) and from eight species from the Great Barrier Reef (Lizard Island). Extracts were prepared in aqueous and organic solvents and were tested for hemolytic, hemagglutinating, antibacterial and anti-acetylcholinesterase (AChE) activities, as well as their ability to inhibit or activate cell protein phosphatase 1 (PP1). The most interesting activities were obtained from extracts of Ircinia felix, Pandaros acanthifolium, Topsentia ophiraphidites, Verongula rigida and Neofibularia nolitangere. Aqueous and organic extracts of I. felix and V. rigida showed strong antibacterial activity. Topsentia aqueous and some organic extracts were strongly hemolytic, as were all organic extracts from I. felix. The strongest hemolytic activity was observed in aqueous extracts from P. acanthifolium. Organic extracts of N. nolitangere and I. felix inhibited PP1. The aqueous extract from Myrmekioderma styx possessed the strongest hemagglutinating activity, whilst AChE inhibiting activity was found only in a few sponges and was generally weak, except in the methanolic extract of T. ophiraphidites.

Prepandemic H5N1 influenza vaccine adjuvanted with AS03: a review of the pre-clinical and clinical data.

BACKGROUND: Universal and timely administration of a prepandemic vaccine is considered to be one of the most effective measures to reduce the incidence of pandemic influenza infection and consequently its morbidity and mortality. OBJECTIVES: To provide the reader with basic insights into influenza virus infections, the threat of a pandemic and the challenges it poses for vaccine development. METHODS: This review summarizes the reported preclinical and clinical data obtained with the prepandemic H5N1 vaccine adjuvanted with AS03. RESULTS: The AS03-adjuvanted prepandemic H5N1 influenza vaccine allows for antigen sparing, has a good safety and acceptable reactogenicity profile, induces an immune response that not only meets all European Committee for Medicinal Products (CHMP) and FDA requirements for the vaccine strain but also generates neutralizing antibodies that broadly cross-react against H5N1 drift strains, and finally conveys protection in a ferret model against lethal challenges with homologous and heterologous H5N1 viruses.

Studies on protein characteristics and toxic constituents of Simarouba glauca oilseed meal.

In order to exploit the protein rich (47.7 g/100g) simarouba meal in food/feed, studies were conducted on its chemical composition with emphasis on protein characteristics and toxic constituents. Simarouba meal contained high calcium (143 mg/100g) and sodium (79 mg/100g). Saponins with triterpenoid aglycone (3.7 g/100g), alkaloids (1.01 g/100g), phenolics (0.95 g/100g) and phytic acid (0.73 g/100g) were the major toxic constituents identified in simarouba meal. TLC and HPLC results indicated that among different fractions of simarouba saponins, one dominant fraction accounted for about 28%. Proteins of simarouba recorded high in vitro digestibility (88%). SDS-PAGE revealed four major protein bands in molecular weight ranges of 20-24, 36-45 and 55-66 kDa. Apart from, glutamic acid (23.43 g/100g protein) and arginine (10.75 g/100g protein), simarouba protein contained high essential amino acids like leucine (7.76 g/100g protein), lysine (5.62 g/100g protein) and valine (6.12 g/100g protein). Among nutritional indices, simarouba meal recorded a good EAA Index (75.02), C-PER (1.90) and PDCAAS (1.0-Adult group).

Purification and characterization of human phosphatidylserine synthases 1 and 2.

PS (phosphatidylserine) in mammalian cells is synthesized by two distinct base-exchange enzymes, PSS1 (PS synthase 1) and PSS2, which are responsible for the conversion of PC (phosphatidylcholine) and PE (phosphatidylethanolamine) respectively into PS in intact cells. The PS synthesis in cultured mammalian cells is inhibited by exogenous PS, and this feedback control occurs through inhibition of PSSs by PS. In the present study, we purified epitope-tagged forms of human PSS1 and PSS2. The purified PSS2 was shown to catalyse the conversion of PE, but not PC, into PS, this being consistent with the substrate specificity observed in intact cells. On the other hand, the purified PSS1 was shown to catalyse the conversion of both PC and PE into PS, although PSS1 in intact cells had been shown not to contribute to the conversion of PE into PS to a significant extent. Furthermore, we found that the purified PSS2, but not the purified PSS1, was inhibited on the addition of PS to the enzyme assay mixture, raising the possibility that there was some difference between the mechanisms of the inhibitory actions of PS towards PSS1 and PSS2.

Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities.

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.

T-helper 1 and 2 serum cytokine assay in chronic opioid addicts.

There are a few studies with conflicting results on the effects of opioids on the functioning of immune system. This study was performed to investigate the in vitro production of interferon-gamma and interleukin-10 after antigenic stimulation of cells using whole blood from opioid addicts. Blood samples were taken from 20 chronically opioid-addicted persons, who voluntarily enrolled for detoxification (10 opium and 10 heroin addicts). Blood samples were also taken from 10 healthy individuals with no history of drug abuse as the control. Cell culture was performed in a whole blood culture assay. Diluted blood samples were stimulated with phytohemagglutinin or with lipopolysaccharide and the supernatants were collected to measure cytokine production. The results demonstrated a significant decrease in interferon-gamma production and an increase in interleukin-10 secretion in heroin addicts, relative to the control group (35.9+/-26.3 versus 110.2+/-60.3 pg/mL, p<0.01 and 71.8+/-28.4 versus 17.1+/-13.5 pg/mL, p<0.01, respectively), however the changes in these values in opium addicts were not significant compared to healthy individuals. The results could suggest that opioid addiction leads to a shift in the Th1/Th2 cytokine balance of peripheral CD4+ cells towards the Th2 response, and opioid addicts demonstrate reduced mitogenic responsiveness of lymphocytes relative to healthy individuals.

Correctors of protein trafficking defects identified by a novel high-throughput screening assay.

High-throughput small-molecule screens hold great promise for identifying compounds with potential therapeutic value in the treatment of protein-trafficking diseases such as cystic fibrosis (CF) and nephrogenic diabetes insipidus (NDI). The approach usually involves expressing the mutant form of the gene in cells and assaying function in a multiwell format when cells are exposed to libraries of compounds. Although such functional assays are useful, they do not directly test the ability of a compound to correct defective trafficking of the protein. To address this we have developed a novel corrector-screening assay for CF, in which the appearance of the mutant protein at the cell surface is measured. We used this assay to screen a library of 2000 compounds and have isolated several classes of trafficking correctors that had not previously been identified. This novel screening approach to protein-trafficking diseases is robust and general, and could enable the selection of molecules that could be translated rapidly to a clinical setting.

The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling.

Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.

Molecular characterization of a new lectin from the marine alga Ulva pertusa.

A new lectin, named UPL1, was purified from a green alga Ulva pertusa by an affinity chromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectin was about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinating activity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. The lectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and had higher activity within pH 6-8. The N-terminal amino acid sequence of the purified lectin was determined (P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned by rapid amplification of cDNA ends (RACE) method (AY433960). Sequence analysis of upl1 indicated it was 1084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the mature UPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 amino acids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not show amino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigm of a novel lectin family.

Soamsan, a traditional Korean medicine, enhances antigen-specific immune responses in low-responder mice via the combined activity of glycoproteins and endotoxins.

Soamsan is a traditional anti-cancer treatment in oriental medicine. It is thought that this material modulates immune responses. To determine whether Soamsan treatment has any effect on the induction of antigen-specific immune responses, C57BL/6 mice, which are low-responders to hen egg-white lysozyme (HEL), were injected with HEL, and their specific immune responses were measured while they were fed Soamsan. Oral administration of Soamsan enhanced the anti-HEL antibody response as well as the T-cell proliferative response to the antigen. Analyses of the HEL-specific antibody isotypes showed that Soamsan treatment resulted in increased levels of HEL-specific antibodies, irrespective of isotype. In particular, however, HEL-specific antibodies of the IgG2b, IgG3, and IgM isotypes, which are associated with direct stimulation of B cells, were significantly increased by the Soamsan treatment. Stimulation of C57BL/6 splenocytes in vitro showed that the presence of Soamsan significantly augmented the proliferative activity induced by both B and T cell mitogens. This augmentation was associated with glycoprotein(s) with a molecular weight mass of about 100 kDa, as well as with endotoxin-like compounds. These results suggest that Soamsan modulates and enhances antigen-specific immune responses.