Virus-like particles and process for preparing same
AssigneeFolia Biotech Inc.
InventorsDenis Leclerc, Pierre Savard
Life of this patent
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Abstract
An in vitro process of preparing virus-like particles (VLPs) from recombinant papaya mosaic virus coat protein and ssRNA, which allows for large scale production of VLPs in high yields, is provided. Also provided are VLPs comprising ssRNA prepared by the in vitro process. The VLPs can be used as adjuvants and when fused to an antigen, as vaccines. The use of the VLPs for stimulation of the innate immune response is also provided.
Claims32
An in vitro process for preparing papaya mosaic vims (PapMV) virus-like particles (VLPs), comprising the steps of:
- a) combining recombinant PapMV coat protein and ssRNA at a protein:RNA ratio of between about 1:1 and 50:1 by weight, at a pH between about 6.0 and about 9.0, and a temperature between about 2° C. and about 37° C., for a time sufficient to allow assembly of VLPs, wherein the recombinant PapMV coat protein is predominantly in the form of low molecular weight species of less than 20-mers, and wherein the ssRNA is between about 250 nucleotides and about 5000 nucleotides in length and comprises a sequence identical to a reference sequence of about 250 nucleotides of the PapMV 5′-end genome, except wherein said sequence is polymutated in that all AUG codons originally present in the reference sequence have been mutated to UAA stop codons;
- b) treating the VLPs with nuclease to remove any RNA protruding from the particles, and
- c) separating the VLPs from other process components.
A papaya mosaic virus (PapMV) virus-like particle (VLP) prepared by an in vitro process comprising the steps of:
- a) combining recombinant PapMV coat protein and ssRNA at a protein:RNA ratio of between about 1:1 and 50:1 by weight, at a pH between about 6.0 and about 9.0, and a temperature between about 2° C. and about 37° C., for a time sufficient to allow assembly of VLPs, wherein the recombinant PapMV coat protein is predominantly in the form of low molecular weight species of less than 20-mers, and wherein the ssRNA is between about 250 nucleotides and about 5000 nucleotides in length and comprises a sequence identical to a reference sequence of about 250 nucleotides of the PapMV 5′-end genome, except wherein said sequence is polymutated in that all AUG codons originally present in the reference sequence have been mutated to UAA stop codons;
- b) treating the VLPs with nuclease to remove any RNA protruding from the particles, and
- c) separating the VLPs from other process components,
- wherein said PapMV VLP delivers said ssRNA to endosomally-located TLR7 to stimulate the innate immune response in a subject.
A papaya mosaic virus (PapMV) virus-like particle (VLP) comprising recombinant PapMV coat protein and ssRNA, wherein the ssRNA is between about 250 nucleotides and about 5000 nucleotides in length and comprises:
- a. a sequence identical to the nucleic acid sequence as set forth in SEQ ID NO:5, or
- b. a sequence identical to the nucleic acid as set forth in SEQ ID NO:
- 6, or
- c. a sequence identical to a fragment of at least about 250 nucleotides from the 5′ end of SEQ ID NO:
- 5 or 6,
- wherein every thymine (T) residue in said sequence is replaced by a uracil (U) residue, and wherein said PapMV VLP delivers said ssRNA to endosomally-located TLR7 to stimulate the innate immune response in a subject.
Dependencies are inferred from the claim text.