Flexible vaccine assembly and vaccine delivery platform

A method for making a virus-like particle (VLP) containing multiple, different in composition, peptides or proteins, said method comprising the steps of:

• a) disassembling separate virus or VLP populations, each displaying a distinct peptide or protein via genetic fusion;
• b) forming encapsidation intermediate populations each displaying a distinct peptide or protein;
• c) mixing encapsidation intermediates from different populations;
• d) forming intact VLP surrounding a nucleic acid core that is composed of different encapsidation intermediates such that the VLP displays one or more peptides or proteins.

A method for making a virus-like particle (VLP) containing multiple, different composition peptides, proteins or nucleic acid moieties, said method comprising the steps of:

• a) disassembling a virus or VLP population that has a surface residue for chemical conjugation, provided by genetic fusion;
• b) forming encapsidation intermediate populations;
• c) effecting chemical conjugation of unique peptide, protein or nucleic acid moieties to each of several separate encapsidation intermediate populations;
• d) mixing encapsidation intermediates from different populations in the presence of a nucleic acid scaffold;
• e) forming intact VLP surrounding a nucleic acid core that is composed of different encapsidation intermediates such that the VLP displays more than one moiety, such as a peptide, protein or nucleic acid, or combination thereof.

A method for making a TMV virus containing one or more, different composition peptides or proteins displayed, said method including the steps of:

• a) constructing a TMV expression vector with
• i) a gene for expression in mammalian cells placed downstream, 3′, of a internal ribosome initiation sequence (IRES) that lies either within the TMV 30 kDa movement protein or separately placed downstream, 3′, of the 30 kDa movement protein gene;
• ii) a coat protein expressed from a non-native subgenomic promoter downstream of the gene for expression in mammalian cells;
• iii) the coat protein having either a genetic fusion for the expression of a peptide sequence, protein and/or a surface residue for chemical conjugation, provided by genetic fusion;
• b) purifying the TMV viruses expressing peptide, protein and/or surface residue;
• c) effecting chemical conjugation of unique peptide, protein or nucleic acid moieties to purified TMV virus;
• d) using TMV virus with genetic and/or chemical fusion peptides for the stabilization and delivery of the RNA expression construct into mammalian cells or tissues.

A method for making a TMV virus-like particle (VLP) containing multiple, different composition peptides, proteins or nucleic acids, the method comprising the steps of:

• a) disassembling separate TMV populations, each having a surface residue for chemical conjugation provided by genetic fusion;
• b) forming encapsidation intermediate, 20S disk, populations from the separate TMV populations;
• c) effecting chemical conjugation of unique peptide, proteins or nucleic acid moieties to separate populations of the encapsidation intermediate 20S disks;
• d) mixing at least two different encapsidation intermediates, 20S disks, from different populations in the presence of a nucleic acid scaffold;
• e) forming intact TMV VLP surrounding a nucleic acid core that is composed of the at least two different encapsidation intermediates, 20S disks, such that the VLP displays more than one moiety, be it peptide, protein or nucleic acid, or some combination thereof.

A method for making a virus-like particle (VLP) containing multiple, different composition peptides or proteins displayed by a process comprising the steps of:

• a) disassembling separate VLP populations, each displaying a distinct peptide or protein via genetic fusion;
• b) disassembling a separate VLP population that has a surface residue for chemical conjugation, provided by genetic fusion;
• c) forming encapsidation intermediate populations such that:
• i) each displays a distinct peptide or protein and
• ii) each displays a surface residue for chemical conjugation;
• d) effecting chemical conjugation of unique peptide, protein or nucleic acid moieties to separate populations of the encapsidation intermediate displaying surface residue for chemical conjugation;
• e) mixing encapsidation intermediates from different populations displaying peptides or proteins by genetic fusion or displaying peptides, proteins or nucleic acids by chemical conjugation;
• f) forming intact VLP surrounding a nucleic acid core that is composed of different encapsidation intermediates such that the VLP displays more than one moiety, be it peptide, protein or nucleic acid, or some combination of these moieties.

A method for purification of TMV or virus like particles (VLP) displaying a peptide fusion or whole protein, comprising the steps of:

• a) adding PEG to a virus containing S1 or S2 supernatant, at an empirically determined concentration of 1% to 10%, depending on the epitope displayed;
• b) adding salt to 4% w/v if not already present in the S1 or S2 supernatant;
• c) chilling the supernatant to 4° C., for 30 minutes to 1 hour;
• d) recovering the precipitated virus by centrifugation;
• and
• c) re-suspending the virus and repeating the process to increase purity.