A recently described bangle lectin (PHL) from the bacterium Photorhabdus asymbiotica was identified as a mainly fucose-binding protein that could play an important role in the host-pathogen interaction and in the modulation of host immune response. Structural studies showed that PHL is a homo-dimer that contains up to seven L-fucose-specific binding sites per monomer. For these reasons, potential ligands of the PHL lectin: α-L-fucopyranosyl-containing mono-, di-, tetra-, hexa- and dodecavalent ligands were tested. Two types of polyvalent structures were investigated - calix[4]arenes and dendrimers. The shared feature of all these structures was a C-glycosidic bond instead of the more common but physiologically unstable O-glycosidic bond. The inhibition potential of the tested structures was assessed using different techniques - hemagglutination, surface plasmon resonance, isothermal titration calorimetry, and cell cross-linking. All the ligands proved to be better than free L-fucose. The most active hexavalent dendrimer exhibited affinity three orders of magnitude higher than that of standard L-fucose. To determine the binding mode of some ligands, crystal complex PHL/fucosides 2 - 4 were prepared and studied using X-ray crystallography. The electron density in complexes proved the presence of the compounds in 6 out of 7 fucose-binding sites.
- MeSH
- antibakteriální látky chemie farmakologie terapeutické užití MeSH
- bakteriální infekce farmakoterapie mikrobiologie MeSH
- bakteriální proteiny antagonisté a inhibitory chemie izolace a purifikace metabolismus MeSH
- dendrimery chemie farmakologie terapeutické užití MeSH
- erytrocyty MeSH
- fukosa analogy a deriváty farmakologie terapeutické užití MeSH
- hemaglutinace účinky léků MeSH
- interakce hostitele a patogenu účinky léků MeSH
- krystalografie rentgenová MeSH
- lektiny antagonisté a inhibitory chemie izolace a purifikace metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely MeSH
- Photorhabdus metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope.
- MeSH
- aglutinace účinky léků MeSH
- aglutinační testy * metody MeSH
- bakteriální proteiny farmakologie MeSH
- erytrocyty cytologie účinky léků MeSH
- hemaglutinace účinky léků MeSH
- hemolýza účinky léků MeSH
- kvasinky cytologie účinky léků MeSH
- lektiny farmakologie MeSH
- lidé MeSH
- mikroskopie MeSH
- povrchová plasmonová rezonance MeSH
- proteiny z Escherichia coli farmakologie MeSH
- Saccharomyces cerevisiae cytologie účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Preparedness for an H5N1 influenza pre-pandemic requires effective and well-tolerated emergency vaccination strategies that provide both pandemic strain-specific and heterologous protection. OBJECTIVES: This was a pivotal study for the regulatory approval process for a candidate MF59-adjuvanted H5N1 vaccine. Its goals were to identify the preferred primary 2-dose vaccination schedule in adults and to assess whether the vaccine met European Committee for Medicinal Products for Human Use (CHMP) licensure criteria. METHODS: Healthy volunteers aged 18 to 60 years received 1 of 4 randomized schedules in which the 2 doses of vaccine were separated by a 1-, 2-, 3-, or 6-week interval. Three blood samples (~20 mL(-1)) were obtained from each subject: the first sample, immediately before administration of the first dose of vaccine; the second, immediately before administration of the second dose; and the third, 21 days after administration of the second dose. Hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) were assayed after each dose. Immunogenicity was assessed based on the CHMP licensure criteria for annual influenza vaccines (number of seroconversions or significant increase in HI titer >40%; mean geometric increase >2.5; and proportion of subjects achieving an HI titer ≥40 or SRH titer >25 mm(2) should be >70% [seroprotection]). Subjects recorded all adverse events occurring within 7 days of vaccine administration; information on any serious adverse events was collected throughout the study (duration, 202 days). RESULTS: All study participants (N = 240) were white, with a mean age of 33 years and a mean body mass index of 24.6 kg/m(2). Equal numbers of men and women were assigned to each vaccination schedule. The CHMP criterion for seroprotection was achieved when the 2 doses of vaccine were separated by 2 (76%), 3 (72%), and 6 (79%) weeks; similar results were obtained on MN and SRH analysis. On the SRH analysis, the candidate vaccine showed a heterologous immune response to the H5N1/turkey/Turkey/1/05 (NIBRG-23; clade 2) influenza antigen. The vaccine met 2 of the 3 European licensure criteria, with seroconversion rates of 69% and 65% in the groups assigned to a 2- and 3-week interval between doses, respectively, and geometric mean ratios of 4.3 and 4.5. There were no serious adverse events related to vaccination. The most common adverse events reported within 7 days of the first and second doses of vaccine were mild to moderate injection-site pain (63%-73% and 34%-48%, respectively) and fatigue (25%-30% and 13%-24%). CONCLUSIONS: Two 7.5-μg doses of MF59-adjuvanted H5N1 influenza vaccine given 2, 3, or 6 weeks apart afforded H5N1-specific immunity and met the CHMP licensure criterion for seroprotection in these healthy volunteers. Clinically relevant levels of heterologous immunity were observed when the 2 doses of vaccine were administered either 2 or 3 weeks apart; however, the licensure criterion for seroprotection was not met in this case.
- MeSH
- adjuvancia imunologická aplikace a dávkování škodlivé účinky MeSH
- chřipka lidská prevence a kontrola virologie MeSH
- dospělí MeSH
- hemaglutinace účinky léků MeSH
- hemolýza účinky léků MeSH
- imunologická paměť účinky léků MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- polysorbáty aplikace a dávkování škodlivé účinky MeSH
- protilátky virové krev MeSH
- rozvrh dávkování léků MeSH
- sekundární imunizace MeSH
- skvalen aplikace a dávkování škodlivé účinky imunologie MeSH
- testy inhibice hemaglutinace MeSH
- vakcíny proti chřipce aplikace a dávkování škodlivé účinky imunologie MeSH
- virus chřipky A, podtyp H5N1 imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky, fáze III MeSH
- práce podpořená grantem MeSH
- randomizované kontrolované studie MeSH
- MeSH
- dítě MeSH
- Escherichia coli genetika patogenita MeSH
- hemaglutinace účinky léků MeSH
- infekce močového ústrojí mikrobiologie MeSH
- infekce vyvolané Escherichia coli mikrobiologie MeSH
- kojenec MeSH
- koliciny biosyntéza MeSH
- lidé MeSH
- mannosa farmakologie MeSH
- pyelonefritida mikrobiologie MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- MeSH
- antigeny MeSH
- biologie buňky MeSH
- buněčná adheze MeSH
- buňky produkující protilátky imunologie MeSH
- časové faktory MeSH
- fibroblasty MeSH
- hemaglutinace účinky léků MeSH
- izotopy kobaltu MeSH
- kultivační techniky MeSH
- L buňky (buněčná linie) MeSH
- lektiny farmakologie MeSH
- lymfocyty imunologie účinky léků účinky záření MeSH
- myši MeSH
- reakce antigenu s protilátkou MeSH
- Salmonella typhi imunologie MeSH
- slezina imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
