Výzkumný záměr Ú.Imunologie / Research Activity of D.of Immunology

The project is planned for years 2007 – 2011.

2007

• Synthesis of galactosylated cationic lipids.
• In vitro testing of transfection efficacy of galactosylated DNA/lipoplexes with various N/P and electron microscope DNA/lipoplexes analyses.
• Preparation of cationic non-galactosilated DNA/lipoplexes and in vitro testing of optimal N/P. Design and cloning of the fusion Osp cDNA constructs into prokaryote and eukaryote vectors.
• Isolation of the cDNA for OX40L and IFN- and cloning into vaccination plasmids.
• Testing of humoral and cellular immune response of the mice to the mucosal vaccination with recombinant hsp90 C. albicans and gp120HIV-1 proteins with mucosal adjuvants.
• Assessment of available primers in a representative group of bacterial strains, optimizing performance of the most appropriate primer system.
• Collecting material of human and animal origin for studying properties of microorganisms with relation to their diagnostics, pathogenicity factors and resistance to external impacts.
• Collecting material of human and animal origin for studying development and spread of bacterial resistance.
• Continuously solving problems related to nosocomial infections.
• Development and optimization of reduction methods for preparing and modifying silver nanoparticles as a new type of substance with antibacterial effects, studying the impact of production conditions on antibacterial activity of silver nanoparticles.
• Optimizing methods for testing antibacterial activity of silver nanoparticles.

2008

• Cloning of hsp90, hsp60, gp120, and fusion Osp cDNA 5§ fused with J-domain cDNA into DNA vaccination vectors.
• Optimization of the conditions for small (<100 nm) galactosylated DNA/lipoplex preparation and in vitro testing of hepatocyte cells specificity and transfection efficacy. Electron microscope analyses.
• In vivo testing of liver transfectability, liver specificity, and systemic toxicity of galactosylated DNA/lipoplex after systemic application to the mice (reporter DNA).
• Prokaryote expression of selected recombinant fusion Osp polypeptides B. afzelii, B. burgdorferi, and B. garinii and optimization of purification conditions. Testing of immunogenicity of particular polypeptides and their epitopes in mice.
• In vivo testing of mucosal tissue transfectablity and adverse effects of cationic non-galactosylated DNA/lipoplexes after local application to the mice (reporter DNA).
• Testing of the optimal doses and time schedule for two DNA - antigen and OX40L or IFN- expressing - vaccines immunization.
• Determination of the humoral and cellular mucosal immune response of the mice to the optimized mucosal vaccination with recombinant hsp90 C. albicans protein and mucosal adjuvants and performing of vaginal challenge with live Candida.
• Collecting material of human and animal origin for studying properties of microorganisms with relation to their diagnostics, pathogenicity factors and resistance to external impacts.
• Collecting material of human and animal origin for studying development and spread of bacterial resistance.
• Elaborating and introducing new methods for early detection of bacterial resistance on phenotype and genotype levels.
• Continuously solving problems related to nosocomial infections.
• Studying the impact of modifiers on stability and subsequently antibacterial activity of silver nanoparticles.
• Development and optimization of methods for studying antifungal activity of silver nanoparticles.
• Initializing testing the effects of substances based on silver nanoparticles on bacterial biofilm.

2009

• Testing of the humoral and cellular immune response of mice immunized systematically with gp120+MBL or hsp90 C. albicans DNA targeted to the liver by complexing into optimized galactosylated DNA/lipoplexes – identification optimal conditions (DNA dose).
• Testing of the humoral and cellular immune response of the mice mucosaly immunized with cationic DNA/lipoplexes expressing hsp90 and gp120 – optimization of the conditions (DNA dose).
• Vaccination of mice with DNA vaccines expressing gp120, hsp90, hsp60, Osp antigens N’-terminally fused with J-domain.
• Formulation of optimal schedule for particular antigen and modulation molecule during DNA vaccination.
• Formulation of optimal conditions for systemic and mucosal DNA vaccinations with particular DNA vaccines.
• Collecting material of human and animal origin for studying properties of microorganisms with relation to their diagnostics, pathogenicity factors and resistance to external impacts.
• Collecting material of human and animal origin for studying development and spread of bacterial resistance.
• Elaborating schemes for rapid and reproducible laboratory diagnostics of mycobacterioses.
• Continuously solving problems related to nosocomial infections.
• Optimizing preparation of antibacterial substances containing silver nanoparticles with respect to their potential use in elimination of bacteria, yeasts and fungi.
• Testing the effects of substances containing silver nanoparticles on bacterial biofilm.

2010

• Determination of the immune response of mice vaccinated by DNA priming and DNA or protein boosting by optimized application approaches for particular antigens.
• Testing of the protectivity of selected combined vaccination schedules by challenge experiments in mice (borreliosis, candidosis) and guinea pigs (trichophytosis).
• Determination of the cytokine response of the antigen-specific T cells after immunization with DNA vaccines expressing Lol-p-I, Phl-p-V a Bet-v-I allergen together with immunization with DNA vaccines expressing immunomodulation molecules (OX40L, IFN-, eventually IL-2, IL-12).
• Collecting material of human and animal origin for studying properties of microorganisms with relation to their diagnostics, pathogenicity factors and resistance to external impacts.
• Collecting material of human and animal origin for studying development and spread of bacterial resistance.
• Elaborating new schemes for rapid and reproducible laboratory diagnostics of fungal and bacterial diseases based on molecular genetic methods.
• Continuously solving problems related to nosocomial infections.
• Studying optimization of small-scale preparation of antibacterial substances containing silver nanoparticles preserving their high biological effect on bacterial, yeast and fungal strains.
• Testing the effects of substances containing silver nanoparticles on bacterial biofilm.
• Studying possibilities of developing resistance of bacterial strains to systems containing silver nanoparticles.

2011

• Continuation in determination of the immune response of mice vaccinated by DNA priming and DNA or protein boosting by optimized application approaches for particular antigens – formulation of the optimal immunization schedules.
• Continuation in the testing of the protectivity of selected combined vaccination schedules by challenge experiments in mice (borreliosis, candidosis) and guinea pigs (trichophytosis) - formulation of the optimal immunization and challenge experiments.
• Continuation in determination of the cytokine response of the antigen-specific T cells after immunization with DNA vaccines expressing Lol-p-I, Phl-p-V a Bet-v-I allergen together with immunization with DNA vaccines expressing immunomodulation molecules (OX40L, IFN-, eventually IL-2, IL-12). Formulation of potentially protecting immunization schedules.
• Defining sources and ways of transmission of multiresistant bacteria in human and animal setting using molecular biology methods.
• Final analysis of the factors of development and spread of bacterial resistance including assessing the impact of selection pressure of antibiotics.
• Final analysis of the prevalence of multiresistant bacterial strains in animal population and defining basic presumption for protecting the human food chain from contamination with multiresistant bacteria.
• Final analysis of antibacterial and antifungal effect of substances with silver nanoparticles, assessing the effect on bacterial biofilm and their potential use in practical applications.