BACKGROUND

The use of virus-like particles (“VLPs”) made from virus capsid-derived proteins and their use to deliver or expose antigens has been disclosed in the art.

For instance, VLPs have been made from animal viruses such as retroviruses or AAVs. Such VLPs, however, require complex structures and are not always convenient to produce.

VLPs derived from plant viruses have also been described. For instance, Sainsbury et al. (Annual Review of Phytopathology 2010 48:437-455) reports the production of VLPs from Cowpea Mosaic virus. Such constructs, however, require co-expression and assembly of two distinct subunits (L and S) or expression of a precursor polypeptide and a protease, rendering resulting VLPs difficult to correctly fold and produce.

Denis et al. (Virology 363 (2007) 59-68) have used the capsid protein ofPapayamosaic virus to expose short HCV viral epitopes. Similarly, Natilla and Hammond (Journal of Virological Methods 178 (2011) 209-215) have prepared VLPs from a capsid protein of a Maize Rayado Fino virus conjugated to a short (8 amino acids) peptide. Other examples were reported with Tomato Bushy stunt virus (Kumar et al. 2009, Virology 388, 185-190), Potato virus Y (Kalnciema et al 2012 Molecular Biotechnology 52 2,129) or Artichoke mottled crinckle virus (Arcangeli et al Journal of Biomolecular Structure and Dynamics Volume 32, Issue 4, 2014).

In each of these constructs, however, the resulting VLPs allowed coupling of generally only small molecules, and/or required assembly of distinct sub-units, and/or allowed only exposure of a peptide to the exterior of the VLP and/or generated large filamentous rather than icosahedral VLP structures comprising or not nucleic acids.

The present invention provides novel fusion molecules and VLPs derived from nepoviruses, having improved and unexpected properties.

SUMMARY OF THE INVENTION

The present invention relates to fusion molecules and VLPs derived from nepoviruses coat proteins and the uses thereof. The present invention shows that the coat protein of nepoviruses may be used to produce stable VLPs. The invention further shows that nepovirus coat protein is a very versatile protein which allows the fusion of large foreign compounds to the N- and/or C-terminal thereof without losing its ability to form VLPs. In addition, the present invention shows that the fusion of a compound at the C-terminus of the coat protein results in the exposure of the compound on the surface of VLPs, whereas a compound fused to the N-terminal leads to its internalization into the VLPs, making the compound inaccessible to antibodies (“caging”). It is therefore possible to produce VLPs having two distinct properties: the surface exposure and/or the protection by internalization (caging) of compounds of interest. Moreover, the VLPs are produced with a single type of coat protein, are quite small in size and simple in structure and nucleic acid-free. Also and advantageously, the coat proteins of nepoviruses may be fused to very large proteins (above 200 amino acids) without losing their ability to assemble into VLPs. To our knowledge, no viral protein has been described in the art having all of these properties simultaneously.

An object of the invention thus resides in virus-like particles comprising or consisting of nepovirus coat proteins.

A further object of the invention is a virus-like particle comprising or consisting of nepovirus coat proteins conjugated to a compound. Conjugation may be covalent (e.g., through genetic fusion or chemical coupling) and/or non-covalent (e.g., through ligand mediated binding).

Another object of the invention resides in a molecule comprising or consisting of a nepovirus coat protein conjugated to a compound.

Another object of the invention resides in a pharmaceutical composition comprising one or more conjugated nepovirus coat proteins or one or more virus-like particles as defined above.

The invention also relates to the use of nepovirus coat proteins to make virus-like particles.

The present invention also provides a method of producing a molecule as defined above, comprising providing a nepovirus coat protein and conjugating said protein to a compound. In a particular embodiment, the method comprises providing a nucleic acid construct encoding said conjugated molecule and expressing said nucleic acid in a host cell or an in vitro expression system.

The invention also provides a method of producing virus-like particles comprising providing a nepovirus coat protein and allowing said protein to assemble into virus-like particles. In a particular embodiment, the method comprises providing a nepovirus coat protein conjugated to a compound of interest and using such conjugated protein (possibly in mixture with other coat proteins) to make the virus-like particle. Alternatively, or in addition, the method further comprises a step of adding a reactive or active group to the virus-like particle or of incorporating a reactive or active group in the virus-like particle during production or assembly.

The invention also concerns the use of a nepovirus coat protein to deliver a compound to a subject.

Further objects of the invention include a nucleic acid molecule encoding a nepovirus coat protein conjugated to a compound of interest, a vector comprising such a nucleic acid, as well as a host cell containing the same.

The invention has wide utility in the pharmaceutical industry, to produce e.g., vaccines, adjuvants, drugs or imaging agents, for instance, for human or veterinary applications, as well as for research uses.

