Recombinant proteins from filoviruses and their use

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Filovirus subunit protein immunogens are produced using a recombinant expression system and combined with one or more adjuvants in immunogenic formulations. The subunit proteins include GP95, GP-FL, VP40, VP24, and NP derived from Ebola Virus and Marburg Virus. Adjuvants include saponins, emulsions, alum, and dipeptidyl peptidase inhibitors. The disclosed immunogenic formulations are effective in inducing strong antibody responses directed against individual Filovirus proteins and intact Filovirus particles as well as stimulating cell-mediated immune responses to the Filoviruses.

A sequence listing file in ST.25 format on CD-ROM is appended to this application and fully incorporated herein by reference. The sequence listing information recorded in computer readable form is identical to the written sequence listing herein (per WIPO ST.25 para. 39, the information recorded on the form is identical to the written sequence listing). With respect to the appended CD-ROM, the format is ISO 9660; the operating system compatibility is MS- Windows; the single file contained on the CD-ROM is named "EBO.ADJ.03. ST25.txt" and is a text file produced by Patentln 3.3 software; the file size in bytes is 63 KB; and the date of file creation is 02 October 2006.

The invention relates to protection against Filovirus induced hemorrhagic fevers using recombinant proteins. The invention, more specifically, concerns several recombinant Filovirus structural proteins produced from eukaryotic cells. In the invention, proteins are post- translationally modified and are purified so that native structure is retained, thereby providing material for use as immunogens to elicit protection against virally induced disease. [004] "Subunit protein" is defined here as any protein derived or expressed independently from the complete organism that it is derived from. Furthermore, a subunit protein may represent a full length native protein sequence or any fraction of the full length native protein sequence. Additionally, a subunit protein may contain in addition to the full length or partial protein sequence, one or more sequences, which may contain sequences that are homologous or heterologous to the organism from which the primary sequence was derived. This definition is significantly broader than the concept of a subunit protein as a single protein molecule that co- assembles with other protein molecules to form a multimeric or oligomeric protein. The Description Page 1 of 50 Recombinant Proteins from Filoviruses A.

The nucleoprotein (NP) of Marburg virus self-assembles tubule-like structures when recombinantly expressed in mammalian cells. These aggregates resemble structures observed in sections of viral inclusions of Marburg virus infected cells (Kolesnikova et al. 2000). In association with cellular RNA (when expressed in Sfll cells), Marburg virus NP forms regularly structured loose coils (Mavrakis et al. 2002). High salt treatment tightens the coiling. Ebola virus NP expressed recombinantly in mammalian cells forms intact nucleocapsids only if expressed in combination with Ebola VP35 and VP24 (Huang et al. 2002). Other virus proteins are not necessary for nucleocapsid formation, but post-translational O-glycosylation of NP is required to facilitate VP35-binding. NP increases the release of Ebola virus-like particles if co- expressed with VP40 in mammalian cells. Further addition of Ebola GP potentiates this effect.

The structure and function of the minor matrix protein VP24 are not fully understood. While co-expression of VP24 and VP40 in mammalian cells does not increase VLP release, co- expression of VP40, NP and VP24 in mammalian cells yields a higher amount of Ebola VLPs than co-expression of VP40 and NP alone, suggesting that VP24 may interact with NP (Licata et al. 2004). In cells infected with Ebola virus, as well as upon recombinant expression, VP24 appears to be concentrated in the perinuclear region (Han et al. 2003). VP24 from virus infected mammalian cells is not glycosylated via N-linked sugar residues and appears to tetramerize. Studies have further shown that VP24 strongly interacts with lipid bilayers and is potentially located within lipid rafts. Recent studies have shown that VP24 is essential for the formation of a functional ribonucleoprotein complex (Hoenen et al. 2006). In addition, it has been shown that VP24 seems to block proper IFN signaling giving another potential explanation of how Filoviruses evade the host immune responses (Reid et al. 2006).

The two small proteins contained in the nucleocapsid complex have different functions. VP35 is an essential part of the Filovirus nucleocapsid (Huang et al. 2002, Mϋhlberger et al. 1998) but the exact role in capsid assembly has not been shown yet. VP35 is a type I IFN antagonist and therefore a major contributor to Filovirus pathogenicity (Basler et al. 2003, Hartman et al. 2004). The role of the phosphoprotein VP30 is linked to transcription activation of the nucleocapsid complex. It was shown that phosphorylation on two serine residues (40 and 42) is essential for binding to NP and therefore inclusion into the nuclear core complex (Modrof et al. 2001). Phosphorylation negatively regulates transcription activation mediated by VP30.