Domů

Specific goals

  1. Design, synthesis, and testing of the liposome carriers of DNA vaccine suitable for delivery of the DNA/lipoplex to liver ASGPr
    1. design of the structures for labeling of the cationic lipids with trimer galactose and testing them with DNA coding for reporter proteins - luciferase and b-galactosidase
      1. synthesis of particular structures
      2. in vitro testing of the transfection efficacy of the galactosylated DNA/lipoplex in relation to the N/P ratio
      3. testing of the conditions for preparation small galactosylated DNA/lipoplexes (<100 nm)
      4. electron microscope analysis of the galactosylated DNA/lipoplexes size distribution relative to conditions of the preparation
      5. in vivo testing of the liver targeting efficacy in experimental mice
      6. formulation of the relation between liver targeting efficacy and galactosylated DNA/lipoplexes size and N/P
      7. determination of the organ distribution of the galactosylated DNA/lipoplexes with optimized size and N/P
      8. determination of the side effects of optimized galactosylated DNA/lipoplexes systemic application
    2. determination of the humoral and cellular systemic immune response of mice immunized with optimized galactosylated DNA/lipoplexes expressing antigens
      1. gp120+MBL
      2. hsp60 T. mentagrophytes
      3. hsp90 C. albicans
    3. determination of the cellular and humoral systemic immune response to the immunization of the mice combining DNA priming and DNA or recombinant protein boosting in various time schedules

      4. Hypothesis: Targeting of the DNA vaccine to the liver is very efficacious approach inducing intensive expression of the antigen. The study of the immunization conditions for particular antigens will be necessary for determination of optimal conditions for each antigen and overall estimation of the DNA vaccination to the liver benefit.

  2. Preparation of the fusion Osp antigens derived from protective and non-autoimmunogenic epitopes of the OspA, OspB, and OspC B. burgdorferi, B. afzelii, and B. garinii.
    1. cloning of the fusion epitopes OspA, OspB and OspC and construction of the DNA vaccine.
    2. prokaryotic expression of the fusion Osp antigens.
      1. purification of the recombinant protein - fusion polypeptides.
      2. testing of the recombinant protein immunogenicity after systemic immunization of the mice.
        1. comparison of the immunogenicity between particular fusion proteins.
        2. comparison of particular epitopes recognition by specific serum antibodies.
      3. determination of the systemic humoral and cellular immune response to vaccination combining the DNA priming and DNA or recombinant protein boosting in various time schedules.

      Hypothesis: The immunization with fusion Osp DNA vaccine by targeting of the DNA to the liver or by combined vaccination is promising approach for elicitation of protective humoral immune response against B. afzelii and B. garinii infection.

  3. Testing of non-galactosylated cationic lipids for preparation of the DNA/lipoplexes for mucosal immunization
    1. optimization of the conditions for DNA/lipoplexes preparation from cationic lipids DOTAP, DOPE, DOTMA, DC-Chol and reporter gene-expressing DNA plasmids.
      1. in vitro determination of the optimal transfection efficacy in relation to the N/P.
      2. in vivo testing of the transfection efficacy in relation to the N/P after application
        1. by intranasal route
        2. by intravaginal route
        3. determination of the side effects in relation to the N/P.
      3. formulation of the optimal conditions for particular application routes.
    2. testing of optimized formulations for vaccination of mice with DNA expressing gp120 and hsp90
      1. determination of the mucosal homoral and cellular immune response to the hsp90 C. albicans.
      2. determination of the mucosal homoral and cellular immune response to the HIV-1 gp120
      3. determination of the immune effects of mucosal adjuvants during immunization with recombinant hsp90 C. albicans and HIV-1 gp120 by measurement of the specific mucosal humoral and cellular immune response of the immunized mice.
        1. testing of the effect of the Bacillus antracis oedema toxin.
        2. testing of the effect of the B subunite of the cholera toxin.
        3. testing of the effect of the heat labile toxin I from E. coli.

      Hypothesis: Identification of the optimal vaccination approach inducing intense mucosal immune response is at present most promising procedure for prevention of genital HIV-1 transmission and perhaps it can prevent vaginal candidosis.

  4. Construction and testing of the DNA vaccines expressing immunomodulation molecules.
    1. isolation of the OX40L and IFN- and cloning into DNA vaccination plasmids.
    2. fusion of hsp90, hsp60, gp120, and Osp cDNA with J-domain and cloning into DNA vaccination plasmids.
    3. determination of the specific humoral and cellular immune response of the mice vaccinated with
      1. two separate DNA vaccines – one expressing the antigen and second expressing the immunomodulation molecule.
        1. determination of the optimal doses of both DNA.
        2. determination of the optimal time schedule for particular vaccines.
      2. DNA vaccine expressing antigens N’-terminally fused with J-domain.
    4. formulation of the optimal vaccination schedule for particular DNA vaccine combination of the antigen an immunomodulation molecule.
    5. determination of the efficacy of the vaccination approaches combining the DNA priming and the DNA or recombinant protein boosting in induction of preferred immune response type
      1. antigen-specific systemic humoral response (hsp90 – systemic candidosis, gp120 - HIV-1 infection, fusion Osp - borreliosis)
      2. antigen-specific systemic Th1 response (hsp60 – trichophytosis)
      3. antigen-specific mucosal (vaginal) humoral response (hsp90 - vaginal candidosis, gp120 – HIV-1 infection genital transmission)
      4. antigen-specific mucosal (vaginal) Th1 response (hsp90 – comparative approach for vaginal canidosis)
    6. testing of the protectivity of selected vaccination approaches by observing the course of experimental infection - candidosis and borreliosis in mice or trichophytosis in guinea pigs.
    7. estimation of the bi-cistronic DNA vaccine applicability as the result of 4)d)i)(2) goal.
    8. determination of the cytokine response of the antigen-specific T cells from mice immunized by DNA vaccines expressing the allergens Lol-p-I, Phl-p-V, and Bet-v-I and DNA vaccines expressing immunomodulation molecules (OX40L, IFN-a and eventually IL-2, or IL-12).

      9. Hypothesis: Assessment of the vaccination schedules eliciting dominant humoral or cellular and systemic or mucosal immune response is necessary for optimal vaccine design not only for prevention of infection diseases but also for potential therapy of allergic or autoimmune diseases with known antigens.

  5. To propose and verify diagnostic applicability of new techniques of PCR-detection of bacterial pathogens (especially in combination with high-resolution melting analysis, PCR-HRMA).
    1. To develop and test primer systems for amplification of sequentially variable products.
    2. To assess applicability of these systems in evaluating sequential variability of the products based on analyzing data obtained by HRMA. To achieve rapid detection and identification of microorganisms from clinical sample it is necessary to develop systems with as broad spectrum of detection as possible (panfungal PCR in fungal pathogens and broad-range PCR in bacterial pathogens), to detect virulence and/or resistance markers, specific systems for most adequate genes must be developed; and to identify and type cultures, McRAPD approach has to be improved.
    3. To select the most suitable systems in individual areas and to verify their diagnostic applicability in model clinical situations:
      1. to detect presence of the Mycoplasma pneumoniae a Chlamydophila pneumoniae during various stages of COPD,
      2. to detect infectious agents in hematooncological patients,
      3. to monitor development of intestinal microbial colonization in gastrointestinal tract diseases when probiotics are administered,
      4. to assess nosocomial spread of selected pathogenic microorganisms based on their typing and evaluating clonal structure of the population,
      5. to demonstrate virulence factors (such as enterotoxins, Panton-Valentine leukocidin in Staphylococcus aureus in relation to MRSA, cytolysins and hydrolyzing enzymes in enterococci in relation to VRE and others) and to assess their prevalence in multiresistant strains,
      6. to evaluate correlation of results of PCR-HRMA analysis of selected genes with the capability of selected microorganisms to produce biofilm,
      7. to evaluate correlation of results of PCR-HRMA analysis of gene polymorphisms influencing sensitivity to antimicrobial substances with the results of testing sensitivity to these substances and with clinical response to treatment with these substances.
    4. To standardize and validate techniques for individual applications.

      Hypothesis: The proposed new techniques will facilitate earlier detection of the presence of the microbe in the analyzed sample including identification of particular species and characterization of its key properties which will enable highly rational therapeutic approach.

  6. To develop a new type of specific, rapid and inexpensive detection (diagnostics) of pathogenic microorganisms based on measuring changes in physico-chemical properties of ultra-thin organized layers of metal nanoparticles modified by substances specific against given types of pathogens.
    1. To develop new methods for detection of pathogenic microorganisms.
    2. To standardize and validate methods for individual applications.
    3. To develop diagnostic kits.

      4. Hypothesis: Developing new methods will expand the possibilities of early detection of the microbe present in the examined sample.

  7. To solve problems related to development and spread of bacterial resistance.
    1. To design primers for amplification of selected genes of pathogenic microorganisms key for determining resistance and to assess the possibilities of detection of their sequential polymorphisms or mutation by HRMA.
    2. To analyze factors of development and spread of bacterial resistance by genetic methods.
    3. To analyze selection pressure of antibiotics and to assess the rate of its specificity.
    4. To study phenotype and genotype properties in groups of sensitive and resistant bacteria with regard to occurrence of selected pathogenicity factors.

      5. Hypothesis: Based on the results obtained, procedures will be formulated which decrease the risk of development and spread of bacterial resistance and improve antibiotic therapy.

  8. To solve problems related to nosocomial infections with focus on patients with hematooncological diseases.
    1. To define sources and ways of transmission of multiresistant bacteria by contemporary and developed molecular biology methods.
    2. To update initial presumptions of adequate antibiotic treatment.

      3. Hypothesis: Based on the acquired results it will be possible to characterize the ways of transmission of multiresistant bacteria and to define approaches decreasing the frequency of nosocomial infections.

  9. To analyze bacterial resistance in animal setting including foods of animal origin and to determine the extent of threat to human population.
    1. To analyze prevalence of multiresistant bacterial strains in animal population using current and developed molecular biology methods.
    2. To define sources and ways of transmission of multiresistant bacteria.
    3. To acquire knowledge leading to protection of human food chain from contamination with multiresistant bacteria.

      4. Hypothesis: The obtained results will enable to specify main risks of threat to human population resulting from prevalence of multiresistant bacteria in animal setting and the food chain and to define steps leading to their limitation.

  10. To develop new substances with antibacterial and antifungal effects containing silver nanoparticles.
    1. To optimize preparation and modification of silver nanoparticles aiming at preparation of a substance with high antibacterial and antifungal effects.
    2. To optimize methods for quantitative testing of antibacterial effects.
    3. To study potential development of bacterial or fungal resistance to preparations based on silver nanoparticles.
    4. To optimize methods for quantitative testing of antifungal effects.
    5. To optimize methods for assessing effects on bacterial biofilm.
    6. To analyze possible use of developed substances in medicine.

      7. Hypothesis: Based on the results published in literature as well as on the results of the first biological tests performed at Palacký University in Olomouc we can assume potential development of a new type of antibacterial and antifungal preparation based on silver nanoparticles with its potential use in both human and veterinary medicine.