# US 11,345,896 B2 · Synthetic chimeric poxviruses > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,345,896 B2 - Document ID: US11345896B2 - Status: Granted - Kind: B2 - Application number: 15/802,189 - Filed: 2017-11-02 - Published: 2018-09-06 - Granted: 2022-05-31 - Earliest claimed: 2016-11-02 - Applicants: David Evans; Ryan Noyce; Seth Lederman - Inventors: David Evans; Ryan Noyce; Seth Lederman - Web page: https://patentgrep.com/patent/US11345896B2 ## CPC - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - A61K 39/12: Viral antigens - A61K 39/275: Poxviridae, e.g. avipoxvirus - A61P 31/20: for DNA viruses - A61K 2039/5254: {avirulent or attenuated} - C12N 2710/24021: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2710/24034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - C12N 2710/24121: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2710/24134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The scOPV of claim 1, wherein the one or both of the terminal hairpin loops are derived from a vaccinia virus. ### Claim 3 (depends on 2) The scOPV of claim 2, wherein the terminal hairpin loops are independently selected from the group consisting of a slow form and a fast form of the vaccinia virus terminal hairpin loop. ### Claim 4 (depends on 1) The scOPV of claim 1, wherein the chemically synthesized DNA comprises overlapping chemically synthesized DNA fragments corresponding to substantially all of the corresponding wild type genome and wherein one or both of the terminal hairpin loops are derived from 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. ### Claim 5 (depends on 4) The scOPV of claim 4, wherein the scOPV is replicated and reactivated from DNA derived from 1-14 overlapping chemically synthesized DNA fragments. ### Claim 6 (depends on 1, 2, 3, 4) The scOPV of any one of claims 1, 2, 3, 4, or 5, wherein the scOPV is reactivated using leporipox virus-catalyzed recombination and reactivation. ### Claim 7 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. ### Claim 8 (depends on 7) The scHIPXV of claim 7, wherein the one or more modifications eliminate one or more of the AarI or BsaI restriction sites. ### Claim 9 (depends on 7) The scHIPXV of claim 7, wherein one or both of the terminal hairpin loops are derived from 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. ### Claim 10 (depends on 9) The scHPXV of claim 9, wherein the one or both terminal hairpin loops comprise left and right terminal hairpin loops derived from a vaccinia virus. ### Claim 11 (depends on 10) The scHPXV of claim 10, wherein the terminal hairpin loops are independently selected from the group consisting of a slow form and a fast form of the vaccinia virus terminal hairpin loop. ### Claim 12 (depends on 11) The scHPXV of claim 11, wherein the slow form comprises a nucleotide sequence that is at least 90% identical to the sequence of SEQ ID NO: 11 and the fast form comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 12. ### Claim 13 (depends on 11) The scHPXV of claim 11, wherein the slow form consists of the nucleotide sequence of SEQ ID NO: 11 and the fast form consists of the nucleotide sequence of SEQ ID NO: 12. ### Claim 14 (depends on 7) The scHPXV of claim 7, wherein the chemically synthesized DNA comprises overlapping chemically synthesized DNA fragments that correspond to substantially all of the corresponding wild type HPXV genome and wherein the one or both 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. ### Claim 15 (depends on 14) The scHPXV of claim 14, wherein the scHPXV is replicated and reactivated from DNA derived from 1-14 overlapping chemically synthesized DNA fragments. ### Claim 16 (depends on 7) The scHPXV of claim 7 wherein the scHPXV is reactivated using leporipox virus-catalyzed recombination and reactivation. ### Claim 17 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. ### Claim 18 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. ### Claim 19 (depends on 1, 17) A composition comprising the scOPV of claim 1 or 17 and a pharmaceutically acceptable carrier. ### Claim 20 (depends on 7, 18) A composition comprising the scHPXV of claim 7 or 18 and a pharmaceutically acceptable carrier.