Synthetic chimeric poxviruses
OwnerDavid Evans, Ryan Noyce +1inventors
InventorsDavid Evans, Ryan Noyce, Seth Lederman
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Abstract
The invention relates, in general, to synthetic chimeric poxviruses, compositions comprising such viruses, and the development and use of systems and methods for producing such synthetic chimeric poxviruses. The synthetic chimeric poxviruses are well suited for live virus vaccines and pharmaceutical formulations.
Claims20
A synthetic chimeric orthopoxvirus (scOPV), wherein the scOPV is replicated and reactivated from DNA derived from chemically synthesized DNA, wherein the wild type genome corresponding to said scOPV is selected from the group consisting of a horsepox virus, a vaccinia virus, a camelpox virus, a cowpox virus, an ectromelia virus, a monkeypox virus, a variola virus, a rabbitpox virus, a raccoonpox virus, a skunkpox virus, a taterapox virus, and a volepox virus, wherein the scOPV comprises a right terminal hairpin loop and a left terminal hairpin loop, one or both of the loops being heterologous to the corresponding wild type genome, and wherein the scOPV comprises one or more modifications to the corresponding wild type genome, the one or more modification being selected from the group consisting of modifications that eliminate one or more restriction sites and modifications that introduce one or more unique restriction sites.
A synthetic chimeric horsepox virus (scHPXV), wherein the scHPXV is replicated and reactivated from DNA derived from chemically synthesized DNA, wherein the genome of said scHPXV corresponds to a wild type genome of HPXV, wherein the scHPXV comprises a right terminal hairpin loop and a left terminal hairpin loop, one or both of the loops being heterologous to the corresponding wild type genome, wherein the scHPXV comprises one or more modifications to the corresponding wild type genome, the one or more modifications being selected from the group consisting of modifications that eliminate one or more restriction sites and modifications that introduce one or more unique restriction sites.
A synthetic chimeric orthopox virus (scOPV) generated by a method comprising the steps of:
- (i) chemically synthesizing overlapping DNA fragments that correspond to substantially all of a wild type genome is selected from the group consisting of a horsepox virus, a vaccinia virus, a camelpox virus, a cowpox virus, an ectromelia virus, a monkeypox virus, a variola virus, a rabbitpox virus, a raccoonpox virus, a skunkpox virus, a taterapox virus, and a volepox virus, wherein one or more of the overlapping DNA fragments are characterized by one or more modifications selected from the group consisting of modifications that eliminate one or more restriction sites and modifications that introduce one or more unique restriction sites,
- (ii) chemically synthesizing terminal hairpin loops of a poxvirus and ligating the terminal hairpin loops to the overlapping DNA fragments derived from the chemically synthesized DNA that comprise the left and right termini of the viral genome,
- wherein one or both the terminal hairpin loops are derived from one or more of a vaccinia virus, a camelpox virus, a cowpox virus, an ectromelia virus, a monkeypox virus, a variola virus, a rabbitpox virus, a raccoon poxvirus, a skunkpox virus, a Taterapox virus, or a volepox virus;
- (iii) transfecting the overlapping DNA fragments derived from the overlapping chemically synthesized DNA fragments of steps (i) and (ii) into helper virus-infected cells;
- (iv) culturing said cells to produce a mixture of helper virus and scOPV particles in said cells;
- and
- (v) plating the mixture on OPV-specific host cells to recover the scOPV.
A synthetic chimeric horsepox virus (scHPXV) generated by a method comprising the steps of:
- (i) chemically synthesizing overlapping DNA fragments that correspond to substantially all of a wild type HPXV genome, wherein one of more of the overlapping DNA fragments are characterized by one or more modifications selected from the group consisting of modifications that eliminate one or more restriction sites and modifications that introduce one or more unique restriction sites;
- (ii) chemically synthesizing terminal hairpin loops of a poxvirus and ligating the terminal hairpin loops to the overlapping DNA fragments derived from the chemically synthesized DNA that comprise the left and right termini of the viral genome, wherein one or both of the terminal hairpin loops are derived from one or more of a vaccinia virus, a camelpox virus, a cowpox virus, an ectromelia virus, a monkeypox virus, a variola virus, a rabbitpox virus, a raccoon poxvirus, a skunkpox virus, a Taterapox virus, or a volepox virus;
- (iii) transfecting the overlapping DNA fragments derived from the overlapping chemically synthesized DNA fragments of steps (i) and (ii) into helper virus-infected cells;
- (iv) culturing said cells to produce a mixture of helper virus and scHPXV particles in said cells;
- and
- (v) plating the mixture on HPXV-specific host cells to recover the scHPXV.
Dependencies are inferred from the claim text.