Multi-antigenic alphavirus replicon particles and methods
AssigneeAlphaVax, Inc.
InventorsJonathan F. Smith, Kurt Kamrud, Sergey Dryga, Ian Caley
Life of this patent
Estimating the term…
Abstract
Viral replicon selected nucleic acid expression libraries are useful for analyzing multiple antigens associated with a parasite, pathogen or neoplasia or for preparing immunogenic compositions for generating immune responses specific for the parasite, pathogen or neoplasia. Alphavirus replicon particles representative of the nucleic acid expression library are preferred. The nucleic acid library can be a random library, or it can be prepared after a selection step, for example, by differential hybridization prior to cloning into the replicon vector.
Claims19
A method for preparing heparin-binding Venezuelan equine encephalitis virus (VEE) replicon particles (VRPs) encoding and expressing a plurality of antigens of a neoplastic cell, pathogen or parasite, said method comprising the steps of:
- a) introducing a plurality of modified Venezuelan equine encephalitis virus (VEE) replicon nucleic acids into a plurality of cells, wherein said cells are permissive for VEE replication and packaging, wherein said modified VEE replicon nucleic acids comprise at least a virus packaging signal and at least one antigen coding sequence of a neoplastic cell, pathogen or parasite expressible in said modified VEE replicon nucleic acids and wherein said modified VEE replicon nucleic acids lack a coding sequence for at least one VEE structural protein, wherein said cells comprise at least one helper nucleic acid encoding the at least one VEE structural protein lacking in the modified VEE replicon nucleic acids, and wherein the plurality of modified VEE replicon nucleic acids encode a plurality of antigens of a neoplastic cell, pathogen or parasite, to produce a plurality of modified cells, wherein the step of introducing the nucleic acids is by electroporation of said cells to produce a plurality of modified cells;
- b) culturing said plurality of modified cells of step (a) under conditions allowing (i) expression of the at least one helper nucleic acid encoding the structural protein lacked by the modified VEE replicon nucleic acids and (ii) replication of said modified VEE replicon nucleic acids and packaging of said VEE replicon nucleic acids to form heparin-binding VRPs;
- c) contacting the modified cells after step (b) with an aqueous solution having a salt concentration from 0.2M to 5M to release the heparin-binding VRPs into the aqueous solution to produce a VRP-containing solution;
- and
- d) collecting VRPs from the VRP-containing solution of step (c).
A method for preparing Venezuelan equine encephalitis virus (VEE) replicon particles (VRPs) encoding and expressing a plurality of antigens of a neoplastic cell, pathogen or parasite, said method comprising the steps of:
- a) introducing a plurality of modified Venezuelan equine encephalitis virus (VEE) replicon nucleic acids into a plurality of cells, wherein said cells are permissive for VEE replication and packaging, wherein said modified VEE replicon nucleic acids comprise at least a virus packaging signal and at least one antigen coding sequence of a neoplastic cell, pathogen or parasite expressible in said modified VEE replicon nucleic acids and wherein said modified VEE replicon nucleic acids lack a coding sequence for at least one VEE structural protein, wherein said cells comprise at least one helper nucleic acid encoding the at least one VEE structural protein lacking in the modified VEE replicon nucleic acids, and wherein the plurality of modified VEE replicon nucleic acids encode a plurality of antigens of a neoplastic cell, pathogen or parasite, to produce a plurality of modified cells, wherein the step of introducing the nucleic acids is by electroporation of said cells, wherein the cells are electroporated at a density from 5×107 to 5×108 cells per mL, to produce a plurality of modified cells;
- b) culturing said plurality of modified cells of step (a) under conditions allowing expression of the at least one helper nucleic acid encoding the structural protein lacked by the modified VEE replicon nucleic acids and replication of said modified VEE replicon nucleic acids and packaging of said VEE replicon nucleic acids to form VRPs;
- c) contacting the modified cells after step (b) with an aqueous solution having a salt concentration from 0.2M to 5M to release the VRPs into the aqueous solution to produce a VRP-containing solution;
- and
- d) collecting VRPs from the VRP-containing solution of step (c).
Dependencies are inferred from the claim text.