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

In case of the first goal of the project - induction of specific systemic immune response - as prevention of systemic infections (borreliosis, HIV-1 infection) and superficial skin infections (trichophytosis) – they will be designed and synthesized various synthetic forms of trimeric galactose with variable spacer between terminal trimeric galactose and the lipid. Those syntheses will be made as a commercial part order in biochemistry laboratories. Particular preparation will be tested in vitro and subsequently in experimental animal. The main goal is to find optimal approach for preparation of DNA/lipoplex structures as spherical or cochleate forms of cationic galactosylated lipids and negatively charged DNA. Critical points are the diameter of the complexes (<100 nm) and final cationic charge (the ratio between total lipide nitrogen and DNA phosphate - N/P = 1 - 3.5). The maximal DNA/lipoplex diameter 100 nm is based on observed size of fenestrae in liver capillaries which separate Disse’s spaces where the galactosylated DNA/lipoplexes can finally react with hepatocyte surface (ASGPr). Furthermore, the reduction of the N/P is important for minimalization of the non-specific interaction of the DNA/lipoplexes with negatively charged vessel surface during the path from injection site to the Disse’s spaces in liver. The size of the complexes can be reduced during preparation by use of the high ion strength conditions in combination with carbon filters extruder. Each preparation will be tested in vitro by comparison of the transfection efficacy in cells expressing the ASGPr (HepG2) and cells withouth ASGPr (293T, CHO, RD, ...). The best working preparations will be tested subsequently in experimental mice using reporter DNA plasmids. Thereafter, particular antigens expressing DNA vaccines will be complexed with lipids and the efficacy of the systemic immunization will be estimated by determination of specific humoral and cellular immune response. In some cases the animal model infection will be used for estimation of the in vivo protection (systemic candidosis and borreliosis in mice).
In case of the second goal of the project - induction of specific mucosal immune response as prevention of mucosal localized infection (vaginal candidosis) and mucosa-transferred infection (HIV-1 infection – they will be prepared DNA/lipoplexeses with strong final cationic charge (N/P = 3 – 10), which will be tested in vitro and subsequently in vivo. In in vivo experiments the toxicity of intranasal and intravaginal application in relation to N/P will be compared with transfection efficacy using reporter plasmids. Optimized N/P will be used for preparation of complexes with antigen-coding DNA vaccines whose immunogenicity will be estimated by determination of the antigen-specific humoral and cellular mucosal immune response of immunized animals. Furthermore, the efficacy of combination of the DNA vaccination and vaccination with recombinant protein will be determined. The last part of experiments will be focused on mucosal immune response to combination of the systemic DNA priming and mucosal DNA or recombinant protein boosting. The last approach was efficacious by immunization with HIV-1 gp120 antigens as emerge even from our experiments with gp120+MBL DNA construct.
The third goal - testing fusion DNA vaccines for modulation of antigen-specific immune response for targeted induction of humoral or cellular response as prevention of infectious diseases, inflammatory and allergic conditions – is focused on testing of the immune modulative properties of the OX40L, IFN-, and J-domain molecules during immunization of the mice with the above mentioned antigens. The effect of immune modulative DNA vaccines will be tested in two main forms: A) immunization with DNA vaccine coding for particular antigen together with application of the DNA vaccine coding for immune modulative molecule and B) immunization with mono/ or bi/cistronic DNA vaccine coding for antigen and immune modulative molecule. The first approach is supposed for OX40L and IFN- molecules because it allows compare various ratios between antigen-coding DNA and mudulative molecule-coding DNA vaccines as well as various time schedules for application of both DNA vaccines. Contrary, the second approach is suitable for testing of the efficacy of the fusion DNA vaccines (antigen N’-terminally fused with J-domain). The outlined modulative molecules serve as the examples for recent DNA vaccination approaches their spectrum will be extended according to new knowledge about efficacious Th1 or Th2 modulation.
The determination of the humoral and cellular immune response of the immunized animals will be the principal validation approach for each immunization schedule. The cellular immune response will be characterized by multi-parametric FACS determination of the intracellular cytokines level (IL-2, IL-4, IL-15, IFN-) in particular population of the cells (CD4, CD8, CD27, CD28, CD45, CD127) after antigen stimulation in vitro. The humoral antigen specific immune response will be determined by ELISA using our purified recombinant antigens and results will be confirmed by Western-immunoblot.
Research goals in microbiology can be characterized by an approach towards developing, extending, improving and accelerating diagnostic procedures in medical bacteriology and mycology, in particular in molecular genetics, extending knowledge in epidemiology of systemic and local infections of bacterial and fungal origin including studying virulence factors of individual strains. Furthermore, possibilities of new methods of rapid detection pathogenic organisms will be verified which use physico-chemical methods based on interaction of organized layers of metal nanoparticles.
An integral part of the research is analysis of mechanisms of selection pressure of antibiotics resulting in multiresistance, especially in widespread pathogens such as for example Staphylococcus aureus, Pseudomonas aeruginosa, enterococci and enterobacteria. The obtained results will be used to formulate procedures leading to limiting development and spread of resistance in both human and animal setting. The above mentioned goal is also related to prevention of nosocomial infections caused by multiresistant bacteria, particularly in immunocompromised (hematooncological) patients.
The essential goal of research in new types of antibacterial and antifungal agents based on silver nanoparticles is to develop a system stable enough in biological setting which at the same time would exhibit high antibacterial and antifungal activity. Another research goal is to study potential use of these substances containing silver nanoparticles as prevention against development of bacterial biofilm.