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BR-122026003071-A2 - Synthetic Chimeric Poxviruses, Their Production Method, Use, and Composition

BR122026003071A2BR 122026003071 A2BR122026003071 A2BR 122026003071A2BR-122026003071-A2

Abstract

The present invention relates generally to synthetic chimeric poxviruses, compositions comprising such viruses, and to the development and use of systems and methods for producing such synthetic chimeric poxviruses. Synthetic chimeric poxviruses are particularly suitable for live virus vaccines and pharmaceutical formulations.

Inventors

  • David Evans
  • Ryan Noyce
  • Seth Lederman

Assignees

  • DAVID EVANS
  • RYAN NOYCE
  • SETH LEDERMAN

Dates

Publication Date
20260317
Application Date
20171102
Priority Date
20161102

Claims (15)

  1. 1. Synthetic chimeric poxvirus (scPV), characterized in that it is replicated and reactivated from DNA derived from synthetic DNA, wherein the viral genome of said virus differs from a wild-type genome of said virus by having one or more modifications, the modifications being derived from a group comprising chemically synthesized DNA, cDNA, or genomic DNA.
  2. 2. Synthetic chimeric poxvirus (scPV), according to claim 1, characterized in that the viral genome of the scPV is based on an orthopoxvirus.
  3. 3. Synthetic chimeric poxvirus (scPV), according to claim 1 or 2, characterized in that one or more modifications comprise one or more modifications to introduce or eliminate one or more unique restriction sites.
  4. 4. Synthetic chimeric poxvirus (scPV), according to any one of claims 1 to 3, characterized in that the viral genome comprises heterologous terminal hairpin loops.
  5. 5. Synthetic chimeric poxvirus (scPV), according to any one of claims 1 to 3, characterized in that the viral genome comprises hairpin loops derived from vaccinia viruses.
  6. 6. Synthetic chimeric poxvirus (scPV), according to any one of claims 1 to 5, characterized in that the virus is replicated and reactivated from chemically synthesized overlapping DNA fragments that correspond to substantially the entire viral genome of the scPV.
  7. 7. Synthetic chimeric poxvirus (scPV), according to any one of claims 1 to 6, characterized in that the virus is reactivated using recombination and reactivation catalyzed by leporipox virus.
  8. 8. A method for producing a synthetic chimeric poxvirus (scPV), as defined in any one of claims 1 to 7, characterized in that it comprises the steps of: (i) chemically synthesizing overlapping DNA fragments that correspond substantially to the entire viral genome of the poxvirus; (ii) transfecting the overlapping DNA fragments into helper virus-infected cells; (iii) culturing said cells to produce a mixture of helper virus particles and synthetic chimeric poxviruses in said cells; and (iv) plating the mixture onto scPV-specific host cells to recover the scPV.
  9. 9. Method according to claim 8, characterized in that the auxiliary virus is a leporipox virus.
  10. 10. Method according to claim 8 or 9, characterized in that step (i) further comprises chemically synthesizing terminal hairpin loops from a poxvirus and linking them to fragments comprising the left and right ends of the viral genome.
  11. 11. Method, according to any one of claims 8 to 10, characterized in that the overlapping DNA fragments comprise: i) nucleotide sequences that are at least 85% identical to the sequences of SEQ ID NOs: 1-10; ii) nucleotide sequences that are at least 90% identical to the sequences of SEQ ID NOs: 1-10; iii) nucleotide sequences that are at least 95% identical to the sequences of SEQ ID NOs: 1-10; or iv) nucleotide sequences that consist of the sequences of SEQ ID NOs: 1-10.
  12. 12. Synthetic chimeric poxvirus (scPV), characterized in that it is generated by the method as defined in any one of claims 8 to 11.
  13. 13. Composition, characterized in that it comprises a pharmaceutically acceptable vehicle and the scPV as defined in any one of claims 1 to 7 and 12.
  14. 14. Use of scPV, as defined in any of claims 1 to 7 and 12, characterized in that it is for the preparation of a composition to activate or enhance the immune response against smallpox, vaccinia virus or monkeypox virus.
  15. 15. Use of scPV, as defined in any of claims 1 to 7 and 12, characterized in that it is for the preparation of a composition to induce an oncolytic response in an individual.

Description

BACKGROUND OF THE INVENTION [001] The present invention relates to Poxviruses (members of the family Poxviridae) which are double-stranded DNA viruses that can infect both humans and animals. Poxviruses are divided into two subfamilies based on host range. The subfamily Chordopoxviridae, which infects vertebrate hosts, consists of eight genera, of which four genera (Orthopoxvirus, Parapoxvirus, Molluscipoxvirus and Yatapoxvirus) are known to infect humans. Smallpox is caused by infection with smallpox virus (VARV), a member of the genus Orthopoxvirus (OPV). The OPV genus comprises several genetically related and morphologically identical viruses, including camelpox virus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, "mousepox agent"), horsepox virus (HPXV), monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoon poxvirus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus, vaccinia virus (VACV), smallpox virus (VARV), and volepox virus (VPV). With the exception of VARV, at least three other OPVs, including VACV, MPXV, and CPXV, are known to infect humans. To date, vaccination with "live" VACV is the only proven protection against smallpox. An aggressive vaccination program led to the eradication of smallpox in 1980, and routine smallpox vaccination of the public was discontinued. However, the need remains to find safe and effective means of vaccinating individuals against VARV and other OPVs. [002] A variety of VACV preparations have been used as smallpox vaccines. Most of these comprised several related viruses (e.g., Dryvax), and one comprises a unique molecular clone, ACAM2000. However, like Dryvax and other VACV vaccines, even ACAM2000 is associated with serious side effects including cardiomyopathy and pericarditis. To reduce risks, the ACAM2000 vaccine, like other vaccines, has several contraindications that preclude individuals with cancer, immunodeficiencies, organ transplant recipients, patients with atopic dermatitis, eczema, psoriasis, cardiac conditions, and patients using immunosuppressants. It is estimated that 15-50% of the United States population falls into one of these categories, thus confirming the need for the development of a safer vaccine or vaccination protocol (Kennedy et al., 2007; Kennedy, R., Poland, G.A. 2007. T-Cell epitope discovery for variola and vaccinia viruses. Rev. Med. Viroll.7: 93-113). Therefore, there is a need for the development of a vaccine that is equivalent in efficacy to Dryvax or ACAM2000™, but safer. [003] The present invention provides chimeric poxviruses assembled and replicated from chemically synthesized DNA. Because chemical genome synthesis is not dependent on a natural template, a wealth of structural and functional modifications of the viral genome is possible. Chemical genome synthesis is particularly useful when a natural template is not available for replication or genetic modification through conventional molecular biology methods. SUMMARY OF THE INVENTION [004] The present invention provides synthetic chimeric poxviruses (e.g., synthetic chimeric OPV or scOPV), methods for producing such viruses and the use of such viruses, for example, as immunogens, in immunogenic formulations, in in vitro assays, as vehicles for heterologous gene expression, or as oncolytic agents. The synthetic chimeric poxviruses of the invention are characterized by one or more modifications relative to a wild-type poxvirus. [005] In part, the present invention relates to the finding that a synthetic chimeric poxvirus (e.g., scOPV) can be produced from chemically synthesized overlapping fragments of the poxviral genome. In this way, the present invention provides, in part, synthetic chimeric poxviruses (e.g., scOPV) replicated and assembled from chemically synthesized nucleic acids. The description also provides compositions comprising such viruses. The description further provides methods for using the poxviruses produced according to the methods of the description. [006] In another aspect, the invention provides a method for protecting individual humans and human populations against the consequences of infection with smallpox, smallpox virus pseudotypes, and other OPVs using the synthetic chimeric poxviruses of the invention. In another aspect, the invention is a method for protecting individual humans and human populations against the consequences of infection with smallpox (VARV) and smallpox virus pseudotypes using the synthetic chimeric poxviruses of the invention, with less toxicity, morbidity, and mortality than available VACV-based vaccines. In certain aspects, the invention provides a synthetic chimeric poxvirus (scPV) that is replicated and reactivated from derived DNA and synthetic DNA, the viral genome of said virus differing from a wild-type genome of said virus in that it is characterized by one or more modifications, the modifications being derived from a group comprising chemically synthesized DNA, cDNA, or genomic DNA. [007] In some embodime