Influenza virus-like particles as antigen carrier platform for conserved influenza viral epitopes

Background.

Various virus-like particles (VLPs) have been shown to strongly stimulate the innate and adaptive immune system including cytotoxic T-cell immune responses. This makes VLPs promising candidates for antigen-carrier platforms for various epitopes. Influenza A VLPs produced in insect cells have undergone detailed analysis as vaccine candidates against influenza disease; currently they are tested in clinical trials phase II.

This proposal deals with the establishment of insect cell produced influenza A VLPs as modular tool for antigen presentation in regards to future vaccine design. Epitopes derived from the conserved HA2 domain of influenza hemagglutinin, which due to steric hindrance are normally not recognized by the immune system will be displayed in different ways on the surface of influenza VLPs. The composition and molecular structure of these VLPs will be characterized by mass spectrometry and electron microscopic techniques. Furthermore, detailed analysis of immune responses; the immunmodulatory influence of engineered VLPs on the immune response against displayed epitopes and antigens will be elucidated in the mouse model. Taken together, detailed structural and immunological data will provide a solid starting point for the further development of VLP vaccines carrying conserved influenza antigens. Basic knowledge on the characteristics, possibilities and limitations of influenza A VLPs as a new surface display platform will greatly contribute to existing efforts and strategies for recombinant vaccine design in general.

Aims and methods.

Conserved epitopes from influenza A virus will be inserted into the antigenic sites A and/or B of influenza HA by inverse PCR (Muster et al., 1994; Ernst et al., 1998; Krammer et al., 2010). For this purpose an existing baculovirus transfer vector which harbours the sequence of the HA gene from the H1 strain A/New Caledonia/99 will be used. The baculovirus expression system facilitates the co-expression of two and more proteins and is therefore, suitable for the expression of large protein complexes like VLPs (Roy et al., 2003; Noad et al., 2003; Grgagic et al., 2006; Krammer and Grabherr, 2010). The first step is to generate recombinant baculovirus by co-transfection of baculovirus transfer vectors harbouring the gene of interest with linearized baculovirus DNA into insect cells. This procedure is highly effective and convenient procedure and recombinant baculovirus can be obtained within a few days. Yield will be determined by quantitation of M1 and HA proteins and by testing the HA activity of the VLPs. Thereby, a correlation of inserted number of amino acids and HA activity can be determined. VLPs will be characterised in terms of yield, stability, shape and baculovirus background.

Strategies for separation of virus with polymethacrylate monoliths so-called CIM-disks and CIM tubes will be applied. The viruses can be purified to homogeneity with a high yield of infectious virus as measured by plague assays. Purity is assed by chemical and biophysical methods such as electrophoresis Western blots, TEM and Nanocyte. This work will be done in collaboration with Prof. Alois Jungbauer, Department of Biotechnology, BOKU. Immunological studies will include the detailed characterization of humoral and cellular immune responses against VLPs and displayed epitopes after parenteral and mucosal (nasal) immunisation.

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