Nanostructured DNA Vaccines
Nanostructured Polymer Systems Forming Complexes with DNA Vaccines for their Targeted Delivery into Dendritic Сells
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Melnikov V G
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- INEOS (Organo-Element Compounds), Russia, Moscow
- Université Catholique de Louvain / Unité de Chimie des Matériaux Inorganiques et Organiques, Belgium, Louvain-la-Neuve\nUniversity of Turku / Finnish-Russian Joint Biotechnology Laboratory, Finland, Turku
DNA vaccine technology is extremely topical for the construction of safe and effective vaccines against emerging infections and bioterrorism when timely development of specific protection means against new or genetically modified pathogens is needed. At present, using methods of molecular genetics, the necessary plasmid constructs could be quickly produced without any protein purification since the antigens encoded by the DNA vaccines are expressed in vivo. Application of nano-technologies and selection of corresponding nanostructured systems for the targeted DNA delivery into immunocompetent cells of the organism determines one of the perspective directions of DNA vaccine perfection in order to reach the needed immune responses with the lowering of the side effects. Nanostructures loaded with DNA or antigen sized 50-500 nm are the optimal target for effective capture by macrophages and dendritic cells (DCs) playing a key role in immunogenicity definition and generation of the corresponding immune response.
The aim of the Project is to construct new self-assembly macromolecular nanosystems for effective delivery of DNA vaccines into the macrophages and live dendritic cells and to study the immunogenicity of nanostructured DNA vaccines.
Development of the immune response is in much determined by the ability of phagocytic cells to process and present antigen on their surfaces in a complex with molecules of Class I/II Major Histocompatibility Complex (MHC). Proteins encoded by DNA vaccines are expressed limited by the host cells and, therefore, are available for presentation by Class I/II MHC molecules to induce antigen-specific response of T lymphocytes. Initiation of Т-cell immune response is connected with two- signal activation system (the first signal is provided by the binding of antigen-specific receptor with I/II Class МНС antigen/molecule complex; the second signal is provided by the interaction of co-receptor CD28 or CTLA-4 with В7.1 or В7.2 ligand, correspondingly). The success of this process needed for the primary Т-cell activation is defined by undoubtedly paramount role of antigen-presenting cells. DCs are the most effective antigen-presenting cells of reticoendothelial system. This cell type consitutively expresses В7 co-stimulators, I and II Class МНС molecules, and ICAM-I, LFA-3 adhesins. That is why these cells do not need any initiation of the surface structures participating in Т-cell response formation. A presence of specific immunogen and non-specific co-stimulator on the one and the same antigen-presenting cell creates conditions for initiation of two-signal system of selection of specific Т-cells and cytokine secretion providing T-cell proliferation and differentiation. Strategic dislocation site of immature DCs in lungs is respiratory epithelium and distal alveoli, mucosal surfaces and skin, where they form a barrier in the pathway of antigen penetration. The usage of mucosal surfaces for the application of nanostructured DNA vaccines could turn out to be one of the perspective approaches to a development of non-injectionable immunization pathways.
In order to reach the set objective, we propose:
- to synthesize new functional cationic polymers for biomedicinal application;
- to study polymer self-assembly with plasmid DNA being an expression vector encoding yersinia V antigen (LcrV) gene under CMV promoter regulatory control. For this purpose, it is supposed to synthesize both copolymers forming complexes with DNA and at the same time being рН sensitive, and corresponding diblock and triblock copolymers forming micelles in aqueous media where the core being the complex of DNA molecule with cationic blocks,
- to determine morphology of the obtained polymer/DNAcomplexes;
- to study the stability of polymer/DNA complexes to nuclease influence,
- to study the interaction of polymer/DNA complexes with cellular membranes of macrophages and DCs (to study the efficacy of polymer/DNA complex penetration (transfection) into the phagocytes),
- to measure LcrV expression by the cells transfected by polymer/DNA complexes in vitro;
- to study LcrV presentation by DCs;
- to study immune response formation at polymer/DNA complex immunization in vivo;
- to perform a comparative analysis of immune response types induced by the nanostructured DNA vaccines based on polymer/DNA complexes carrying LcrV gene and on an adjuvant vaccine based on highly purified LcrV protein.
To obtain the polymers, original synthesis methods elaborated by the Project participants will be used. Physico-chemical parameters of the polymers and nanostructures will be evaluated using UV, IR, NMR- spectroscopy and other analysis techniques. Morphology and size of polymer/DNA complexes will be studied by methods of transmission electron microscopy (ТЕМ) and small-angle X-ray-scattering. The nanostructures of polymer/DNA in solutions will be studied using fluorescent label technique (ethidium bromide/DNA – fluorescence). Cytotoxicity studies of polymers will be performed using flow cytofluoremetry and staining of finite cell lines (J774, U937, VTEC macrophage-like cells). Stability of polymer/DNA complexes to nuclease influence will be studied using agarose gel electrophoresis. A construction of plasmid vector carrying LcrV gene under the control of CMV promoter will be performed using standard techniques of molecular genetics. LcrV gene expression in various eukaryotic cells will be evaluated on the production of LcrV recombinant protein using Western blot analysis and immunofluorescent techniques with monoclonal anti-LcrV antibodies. Phagocytosis of polymer/DNA complexes will be studied using fluorescent flow cytometry and fluorescent microscopy. DCs will be obtained from bone marrow cell of inbred mice and maintained in the culture in vitro in the presence of recombinant growth factors and cytokines (GM-CSF,TNF-α). For the evaluation of the basal parameters of immune response formed by nanostructured DNA vaccines and vaccines based on DCs transfected by DNA the following immunological methods will be used: a) definition of in vitro proliferation of antigen-specific Т-cells (Н3-thymidine insertion technique); b) and expression detection of cellular activation markers (flow cytofluorimetry technique). Detection of cytokines (TNF-α, IL-1, IL-10, IL-12, INF-γ) will be performed by immuno-enzyme technique and using ELISPOT technique. Antibodies specific to LcrV will be detected using ELISA. LcrV recombinant antigen will be obtained and purified using the methods developed at the Institute of Immunological Engineering.
During the present Project performance, a number of new hydrophilic cationic polymers will be synthesized and the interconnection of their chemical structure and the structure forming polymer/DNA complexes will be studied, as well as the efficacy of the latter to induce immune response. Russian scientists participating in the Project will have a possibility to discuss the scientific problems under their study with the leading scientists of the USA and Europe, and to attend the largest scientific laboratories and to present the results of their studies at international conferences. The Project will result in a construction of a new generation of commercial polymers for the targeted delivery of DNA vaccines thus contributing to the development of market economy principles in Russia.