(19) & (11) EP 1 802 746 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 7/04 (2006.01) A61K 39/29 (2006.01) 04.05.2011 Bulletin 2011/18 A61K 9/127 (2006.01) A61K 39/145 (2006.01) (21) Application number: 05795050.3 (86) International application number: PCT/EP2005/011297 (22) Date of filing: 20.10.2005 (87) International publication number: WO 2006/045532 (04.05.2006 Gazette 2006/18) (54) VIROSOME PARTICLES COMPRISING ANTIGENS FROM INFLUENZA VIRUS AND HEPATITIS B VIRUS VIROSOMENPARTIKEL MIT ANTIGENEN AUS INFLUENZAVIRUS UND HEPATITIS-B-VIRUS PARTICULES DE TYPE VIROSOME COMPORTANT DES ANTIGÈNES DU VIRUS GRIPPAL ET DE VIRUS DE L’HÉPATITE B (84) Designated Contracting States: US-A- 5 879 685 US-A1- 2003 180 351 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • BRUSS V: "Envelopment of the hepatitis B virus SK TR nucleocapsid" VIRUS RESEARCH, AMSTERDAM, NL, [Online] vol. 106, no. 2, 29 (30) Priority: 27.10.2004 EP 04025573 September 2004 (2004-09-29), pages 199-209, XP004657025ISSN: 0168-1702 Retrieved from the (43) Date of publication of application: Internet: URL:http://www.sciencedirect.com/ 04.07.2007 Bulletin 2007/27 science?_ ob=MImg&_ imagekey=B6T32-4DF4D84-1-7&_cdi=4 934&_ (73) Proprietor: Crucell Switzerland AG user=987766&_orig=search&_coverDate=1 3018 Bern (CH) 2%2F01%2F2004&_qd=1&_ sk=998939997&view=c&w chp=dGLbVtz- (72) Inventors: zSkWb&md5=b3b8f71aafc7c6f3bc2f • MOSER, Christian f4df11af2dbe&ie=/sdarticle.pdf> 3122 Kehrsatz (CH) • HUCKRIEDE ANKE ET AL: "Influenza virosomes • ASSERO, Giovanna in vaccine development." METHODS IN 95125 Catania (IT) ENZYMOLOGY. 2003, vol. 373, 2003, pages 74-91, • FICHERA, Epifanio XP008042413 ISSN: 0076-6879 94100 Enna (IT) • GLÜCK REINHARD ET AL: "Influenza virosomes • VENTURA, Dario as an efficient system for adjuvanted vaccine 95030 Tremestieri Etneo CT (IT) delivery." EXPERT OPINION ON BIOLOGICAL • LEMPEREUR, Laurence THERAPY. JUL 2004, vol. 4, no. 7, July 2004 95129 Catania (IT) (2004-07), pages 1139-1145, XP008045248 ISSN: • FELNEROVA, Diana 1744-7682 3018 Bern (CH) • HAGLUND K ET AL: "Expression of Human Immunodeficiency Virus Type 1 Gag Protein (74) Representative: Hateboer, Guus et al Precursor and Envelope Proteins from a Crucell Holland B.V. Vesicular Stomatitis Virus Recombinant: High- P.O. Box 2048 Level Production of Virus-like Particles 2301 CA Leiden (NL) Containing HIV Envelope" VIROLOGY, ACADEMIC PRESS,ORLANDO, US, vol. 268, no. (56) References cited: 1, 1 March 2000 (2000-03-01), pages 112-121, EP-A- 1 346 727 WO-A-2004/045582 XP004436135 ISSN: 0042-6822 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 802 746 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 802 746 B1 • LATHAM T ET AL: "Formation of wild-type and • HILLEMAN M.R.: ’Critical overview and outlook: chimeric influenza virus-like particles following pathogenesis, prevention, and treatment of simultaneous expression of only four structural hepatitis and hepatocarcinoma caused by proteins." JOURNAL OF VIROLOGY. JUL 2001, hepatitis B virus’ VACCINE vol. 21, 2003, pages vol. 75, no. 13, July 2001 (2001-07), pages 4626 - 4649 6154-6165, XP002323947 ISSN: 0022-538X 2 EP 1 802 746 B1 Description [0001] The present invention relates to a virosome comprising a virosomal membrane comprising at least one lipid and envelope proteins of an enveloped virus and nucleocapsid particles of said enveloped virus located on the inside 5 and the outside of the virosome and attached to said envelope proteins. Furthermore, the invention relates to a vaccine comprising the virosome of the invention and a method for the production of a virosome of the invention. Moreover, the invention relates to a use of a virosome of the invention for the preparation of a vaccine, e.g. for the prevention or alleviation of a disease related to an HBV infection. [0002] The development of novel and increasingly safer vaccines frequently makes use of well- characterized antigens, 10 in particular highly purified recombinant proteins or synthetic peptides. In spite of some achievements, this approach is impeded by the fact that such antigens are often poor immunogens when administered alone. This fact has necessitated the development of suitable adjuvant and carrier systems that possess the ability to enhance the immunogenicity of a given antigen. One possible approach is the integration of antigens into a higher structure, e.g. a virus- like particle. The physical association of all vaccine components in a single particle ensures their simultaneous interaction with individual 15 immune cells, and thereby, maximal exploitation of synergistic potentials. This is of particular relevance if immuno- stimulatory or immuno-modulatory components (adjuvants) are included in the formulation. Furthermore, the particle structure itself can have immuno-stimulatory effects and increase both, the stability and the immunogenicity of the individual components. [0003] It is thus a problem to find a suitable approach of combining the relevant antigens in an industrial applicable 20 formulation, which leads to an efficient prophylactic and/or therapeutic application. [0004] During a virus replication in a host cell, copies of the viral genome are generated and viral proteins are expressed and processed before they assemble to mature virions while taking advantage of the cellular infrastructure. The common basis is that virus replication and assembly of progeny requires the environment of a living host cell and an ordered series of specific interactions between viral nucleic acids, viral and host cell proteins, and lipid membranes, that leads 25 to segregation and assembly of the macromolecular virion structure. The large number of different molecules required and in addition, the cellular structures involved illustrate the high complexity of any virion assembly. There are major differences between different virus classes, and in particular, between enveloped and non- enveloped viruses. Common to all enveloped viruses is the outer shell of the virus, composed of a lipid membrane with integrated viral proteins, and, as a consequence, the necessary interaction between membrane-associated and soluble viral proteins or protein- based 30 structures, e.g. the nucleocapsids in order to assemble a mature enveloped virus. Non-enveloped viruses lack a lipid- based membrane and assemble from protein and nucleic acid molecules only. [0005] Numerous approaches to reconstitute viral particles in vitro and in vivo have been described in the literature and can be divided into distinct categories: 35 (a) In vitro reconstitution of viral envelopes Virus-derived or recombinant envelope proteins can be purified and formulated with or without additional lipids into proteoliposomes. This pure in vitro approach achieves the generation of the outer shell of enveloped viruses, the envelope, but does not include the core of the virus, the nucleocapsid. There are examples of chimerical virosomal structures, which integrate envelope proteins from different viruses. Reconstituted viral envelopes have also been 40 used successfully for gene transfer (DNA or RNA) but these methods did not depend on packaging of a functional, protein-based nucleocapsid but rather an association of the nucleic acids directly with the reconstituted envelope. (b) Heterologous expression of one or more viral proteins Isolated recombinant viral proteins can self-assemble into virus-like structures (VLPs): HPV (yeast, baculo), HCV (baculo), HBs antigen (yeast, CHO), HBc (E. coli). Common to all these approaches is that the self- assembly takes 45 place in the heterologous cellular expression system and the virus-like particles are subsequently purified. Therefore, the assembly does not take place in vitro but relies on a cellular system. VLPs have been used as vaccines and as vaccine carriers. (Pumpens, P.; Grens, E. (2001) Intervirology 44 (2-3); 98-114; Noad R, Roy P. (2003) Trends Microbiol. 11(9): 438-44) (c) Reconstitution of non-enveloped (icosahedral) viruses or virus-like particles in vitro 50 This approach is based on separately purified components. Due to the absence of a lipid membrane- based envelope, non-enveloped viruses are simpler in their structure and can self-assemble under certain conditions in vitro if all necessary components are present in the correct stoechiometry. Similarly, the inner core of enveloped viruses, the lipid-free nucleocapsids, or subunits thereof have been reconstituted in vitro from purified recombinant components, e.g. of influenza virus (Martin-Benito J. et al. (2001) EMBO Rep. 2(4): 313-7). 55 (d) Purification of viral nucleocapsids Nucleocapsids have been extracted and purified from many different virus types in order to characterise their com- position. These preparations can also be used for transfection of susceptible cells aiming at virus rescue. Successful virus rescue implies that a functional nucleocapsid was isolated and delivered to the cytoplasm of a host cell. 3 EP 1 802 746 B1 However, this does not imply the successful reconstitution of a functional enveloped virus, because the natural way of infection, which depends on a functional envelope, is bypassed by the use of a transfectant, the latter mediating direct delivery of the nucleocapsid into the cytoplasm of a host cell. (e) Pseudotyping of enveloped viruses and viral vectors in a cell culture system. 5 This in vivo approach has been widely and successfully used for the production of chimerical viruses or vectors (e.g. Retroviruses, Lentiviruses, and AAV) at lab scale. The key element for the production of pseudotyped viruses is a helper cell that co-expresses all the proteins to be integrated into the virion and mediates assembly of the virions.