EBOLA is USA PATENT

Well, Ebola was patented in USA, in 2010 – see below the patent application. It is the property of the USA government. They had the idea and own all the rights on Ebola. Nobody can manufacture it, or whatever, without paying for the patent rights. Why did they created it in the first place?

Ah, I forgot, maybe lots of money for who manufactures the “antidote”, sadistic extermination of “some stupid, unnecessary, common slaves”,… , to make big profits, and have some petrodollars to share/pay the “elected”…, more “castles”, more islands to buy, maybe a few more mistresses, …. you got it.  

This is so sick. But why do I wonder, it’s just another crime in the big chain of crimes.

Well, just one message to all of them: Your time is up ! :-)

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“What the global “health agencies” and the UN are doing has nothing to do with health. They are manufacturing disease and attempting to make them as virulent and deadly as possible.

“Author of Emerging Viruses: AIDS And Ebola: Nature, Accident or Intentional?, Horowitz says the swine-bird-human flu strain, reported to be found first in Mexico in late-March 2009, could have only come from Dr James S. Robertson and his colleagues in association with the US Centre for Disease Control and vaccine manufacturer Novavax, Inc, which was ready to profit from the release he says. Nobody else takes H5N1 Asian-flu infected chickens, takes them to Europe, extracts their DNA, combines their proteins with H1N1 viruses from the 1918 Spanish flu isolate, additionally mixes in some swine-flu genes from pigs, then reverse engineers them to infect humans, he said.”

Source: Patents (look for yourself on Google  http://www.google.com/patents/CA2741523A1)

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Publication numberCA2741523 A1
Publication typeApplication
Application numberCA 2741523
PCT numberPCT/US2009/062079
Publication dateApr 29, 2010
Filing dateOct 26, 2009
Priority dateOct 24, 2008
Also published asEP2350270A2, 4 More »
InventorsJonathan S. Towner, 4 More »
ApplicantJonathan S. Towner, 5 More »
Export CitationBiBTeX, EndNote, RefMan
External Links: CIPO, Espacenet
Human ebola virus species and compositions and methods thereof
CA 2741523 A1
ABSTRACT
Compositions and methods including and related to the Ebola Bundibugyo virus (EboBun) are provided.
Compositions are provided that are operable as immunogens to elicit and immune response or protection from EboBun challenge in a subject such as a primate. Inventive methods are directed to detection and treatment of EboBun infection.
CLAIMS(30)
1. An isolated hEbola virus comprising a nucleic acid molecule comprising a nucleotide sequence of:
a) a nucleotide sequence set forth in SEQ ID NOS: 1 or 10;
b) a nucleotide sequence hybridizing under stringent conditions to SEQ ID NOS:
1 or 10; or c) a nucleotide sequence of at least 70%-99% identity to the SEQ ID NOS: 1 or 10.
2. An isolated hEbola virus having Centers for Disease Control Deposit Accession No.
200706291.
3. The hEbola virus of any one of claims 1 or 2 which is killed.
4. The hEbola virus of claim 1 which is an attenuated hEbola virus.
5. The virus of claim 4 wherein at least one property of the attenuated hEbola virus is reduced from among infectivity, replication ability, protein synthesis ability, assembling ability or cytopathic effect.
6. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ
ID
NOS: 1 or 10 or a complement thereof.
7. An isolated nucleic acid molecule comprising a nucleotide sequence of between 4 and 4900 contiguous nucleotides of the nucleotide sequence of SEQ ID NOS: 1 or 10, or a complement thereof; with the proviso that said nucleotide sequence is not comprised by the nucleotide sequence set forth in SEQ ID NO: 20; or between 5500 and 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NOS: 1 or 10, or a complement thereof.
8. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2-9, 59, or SEQ ID NO: 11-19 or a complement of said nucleotide sequence.
9. An isolated RNA or DNA nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID
NOS: 1 or 10 or a complement thereof.
10. An isolated polypeptide encoded by the nucleic acid molecule of any one of claims 7-9.
11. An isolated polypeptide comprising the amino acid of:
a) an amino acid sequence set forth in any of SEQ ID NOS: 2-19, or 59; or b) an amino acid sequence that has 70% – 99% homology to the amino acid sequence of (a).
12. An isolated polypeptide comprising the amino acid sequence having to 250 contiguous amino acid residues of the amino acid sequence of SEQ ID
NOS: 5 or 18 (VP24);
5 to 280 contiguous residues of the amino acid sequence of SEQ ID NOS: 6 or 17 (VP30);
5 to 320 contiguous residues of the amino acid sequence of SEQ ID NOS: 8 or 13 (VP40);
5 to 340 contiguous residues of the amino acid sequence of SEQ ID NOS: 7 or 12 (VP35);
5 to 370 contiguous residues of the amino acid sequence of SEQ ID NOS: 4 or 15 (SGP);
5 to 370 contiguous residues of the amino acid sequence of SEQ ID NOS: 59 or 16 (SSGP);
5 to 670 contiguous residues of the amino acid sequence of SEQ ID NOS: 9 or 14 (GP);
5 to 730 contiguous residues of the amino acid sequence of SEQ ID NOS: 3 or 11 (NP); or 5 to 2200 contiguous residues of the amino acid sequence of SEQ ID NOS: 2 or 19 (L).
13. An isolated antibody or an antigen-binding fragment thereof which immunospecifically binds to the hEbola virus of any one of claims 1or 2 or neutralizes the virus.
14. An isolated antibody or an antigen-binding fragment thereof which immunospecifically binds to the polypeptide of any one of claims 11 or 12 or neutralizes an hEbola virus.
15. A method for detecting the presence of a the hEbola virus or a nucleic acid molecule derived therefrom of claim 1 in a biological sample, said method comprising:(a) contacting the sample with an agent that selectively binds to the virus or the nucleic acid molecule derived therefrom; and (b) detecting whether the compound binds to the virus or the nucleic acid molecule derived therefrom in the sample.
16. The method of claim 15, wherein the agent is an antibody.
17. The method of claim 15, wherein the agent is a nucleic acid molecule comprising a nucleotide sequence having between 4 and 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NOS: 1 or 10, or a complement thereof.
18. A method for detecting the presence of the polypeptide of claim 11 in a biological sample, said method comprising:
(a) contacting the biological sample with an agent that selectively binds to said polypeptide; and (b) detecting whether the compound binds to said polypeptide in the sample.
19. The method of claim 18, wherein the agent is an antibody or an antigen-binding fragment thereof.
20. A formulation comprising the hEbola virus of any one of claims 3 or 4, and a pharmaceutically acceptable carrier.
21. A formulation comprising an amount of a protein extract of the hEbola virus of claim 3 or 4 or a subunit thereof, and a pharmaceutically acceptable carrier.
22. A formulation comprising an amount of a nucleic acid molecule of the nucleotide sequence of SEQ ID NOS: 1 or 10 or a complement thereof, and a pharmaceutically acceptable carrier.
23. A formulation comprising an immunogenically effective amount of the nucleic acid molecule of claim 9 or a complement thereof, and a pharmaceutically acceptable carrier.
24. A vaccine formulation comprising a therapeutically or prophylactically effective amount of the hEbola virus of claim 3 or 4 or a protein extract therefrom, and a pharmaceutically acceptable carrier.
25. A vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule SEQ ID NOS: 1 or 10 or a complement thereof, and a pharmaceutically acceptable carrier.
26. A vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule of claim 9 or a complement thereof, and a pharmaceutically acceptable carrier.
27. A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of an anti-hEbola agent of an antibody or an antigen-binding fragment thereof which immunospecifically binds to the hEbola virus of Deposit Accession No.
200706291, or polypeptides or protein derived therefrom and optionally has the nucleotide sequence of SEQ ID
NOS: 1 or 10, or a fragment thereof.
28. A kit comprising a container containing the formulation of any one of claims 24-26.
29. A method for identifying a subject infected with the virus of claim 1 or 2, comprising:
(a) obtaining total RNA from a biological sample obtained from the subject;
(b) reverse transcribing the total RNA to obtain cDNA; and (c) amplifying the cDNA using a set of primers derived from a nucleotide sequence of the virus of claim 1 or 2.
30. A primer that has the nucleotide sequence of one of SEQ ID NOS: 24-57.
DESCRIPTION  (OCR text may contain errors)

HUMAN EBOLA VIRUS SPECIES AND COMPOSITIONS AND METHODS THEREOF

DEPOSIT STATEMENT
[0001] The invention provides the isolated human Ebola (hEbola) viruses denoted as Bundibugyo (EboBun) deposited with the Centers for Disease Control and Prevention (“CDC”;
Atlanta, Georgia, United States of America) on November 26, 2007 and accorded an accession number 200706291. This deposit was not made to an International Depository Authority (IDA) as established under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and is a non-Budapest treaty deposit. The deposited organism is not acceptable by American Type Culture Collection (ATCC), Manassas, Virginia, an International Depository Authority (IDA) as established under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Samples of the stated Deposit Accession No. 200706291 will be made available to approved facilities for thirty years from the date of deposit, and for the lifetime of the patent issuing from, or claiming priority to this application.

RELATED APPLICATIONS

[0002] This application claims priority benefit of U.S. Provisional Application 61/108,175 filed 24 October 2008; the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0003] The invention is related to compositions and methods directed to a novel species of human Ebola (hEbola) virus.

BACKGROUND OF THE INVENTION

[0004] The family Filoviridae consists of two genera, Marburgvirus and Ebolavirus, which have likely evolved from a common ancestor’. The genus Ebolavirus includes four species: Zaire, Sudan, Reston and Cote d’Ivoire (Ivory Coast) ebolaviruses, which have, with the exception of Reston and Cote d’Ivoire ebolaviruses, been associated with large hemorrhagic fever (HF) outbreaks in Africa with high case fatality (53-90%)2.

[0005] Viruses of each species have genomes that are at least 30-40% divergent from one another, a level of diversity that presumably reflects differences in the ecologic niche they occupy and in their evolutionary history. Identification of the natural reservoir of ebolaviruses remains somewhat elusive, although recent PCR and antibody data suggest that three species of arboreal fruit bats may be carriers of Zaire ebolavirus3. No data has yet been published to suggest reservoirs for the Sudan, Reston and Cote d’Ivoire ebolavirus species. However, a cave-dwelling fruit bat has been recently implicated as a natural host for marburgvirus4′ s, supporting the hypothesis that different bat species may be the reservoir hosts for the various filoviruses.

[0006] Filovirus outbreaks are sporadic, sometimes interspersed by years or even decades of no apparent disease activity. The last new species of ebolavirus was discovered 14 years ago (1994), in Cote d’Ivoire (Ivory Coast), and involved a single non-fatal case, a veterinarian who performed an autopsy on an infected chimpanzee found in the Tai Forest6. No further disease reports have been associated with Cote d’Ivoire ebolavirus, in contrast to Zaire and Sudan ebolaviruses which have each caused multiple large outbreaks over the same time period.

[0007] In late November 2007, HF cases were reported in the townships of Bundibugyo and Kikyo in Bundibugyo District, Western Uganda. The outbreak continued through January 2008, and resulted in approximately 149 cases and 37 deaths. Laboratory investigation of the initial 29 suspect-case blood specimens by classic methods (antigen capture, IgM and IgG
ELISA) and a recently developed random-primed pyrosequencing approach identified this to be an Ebola HF
outbreak associated with a new discovered ebolavirus species. These specimens were negative when initially tested with highly sensitive real-time RT-PCR assays specific for all known Zaire and Sudan ebolaviruses and Marburg viruses. This new species is referred to herein as “the Bundibugyo species”, abbreviated “EboBun”.

[0008] Accordingly, compositions and methods directed to the new Ebola virus species are described herein and the most closely related Ebola Ivory Coast species, which compositions and methods are useful for diagnosis and prevention of human Ebola virus infection; including related vaccine development, and prevention of hemorrhagic fever in a human population.

SUMMARY OF THE INVENTION

[0009] The present invention is based upon the isolation and identification of a new human Ebola virus species, EboBun. EboBun was isolated from the patients suffering from hemorrhagic fever in a recent outbreak in Uganda. The isolated virus is a member of the Filoviridae family, a family of negative sense RNA viruses. Accordingly, the invention relates to the isolated EboBun virus that morphologically and phylogenetically relates to known members filoviridae.

[0010] In one aspect, the invention provides the isolated EboBun virus deposited with the Centers for Disease Control and Prevention (“CDC”; Atlanta, Georgia, United States of America) on November 26, 2007 and accorded an accession number 200706291, as stated in the paragraph entitled “DEPOSIT STATEMENT” supra.

[0011] In another aspect, the invention provides an isolated hEbola EboBun virus comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence set forth in SEQ ID NO: 1; b) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 1 under stringent conditions; and c) a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the SEQ ID NO:
1. In another aspect, the invention provides the complete genomic sequence of the hEbola virus EboBun.

[0012] In a related aspect, the invention provides nucleic acid molecules isolated from EboBun, or fragments thereof.

[0013] In another aspect, the invention provides proteins or polypeptides that are isolated from the EboBun, including viral proteins isolated from cells infected with the virus but not present in comparable uninfected cells; or fragments thereof. In one embodiment of the present invention, the amino acid sequences of the proteins or polypeptides are set forth in SEQ ID
NOS: 2-9 and 59, or fragments thereof.

[0014] In a related aspect, the invention provides an isolated polypeptide encoded by the nucleic acid molecule of the inventive hEbola EboIC (Sequence ID No. 10) virus described above.

[0015] In another aspect, the invention provides an isolated hEbola EboIC
virus comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence set forth in SEQ ID NO: 10; b) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 10 under stringent conditions; and c) a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the SEQ ID NO:
10. In another aspect, the invention provides the complete genomic sequence of the hEbola virus EboIC.

[0016] In a related aspect, the invention provides nucleic acid molecules isolated from EboIC, or fragments thereof.

[0017] In another aspect, the invention provides proteins or polypeptides that are isolated from the EboIC, including viral proteins isolated from cells infected with the virus but not present in comparable uninfected cells; or fragments thereof. In one embodiment of the present invention, the amino acid sequences of the proteins or polypeptides are set forth in SEQ ID
NOs: 11-19, or fragments thereof.

[0018] In a related aspect, the invention provides an isolated polypeptide encoded by the nucleic acid molecule of the inventive hEbola EboIC virus described above.

[0019] In other aspects, the invention relates to the use of the isolated hEbola virus for diagnostic and therapeutic methods based on EbBun, EboIC, or a combination thereof. In one embodiment, the invention provides a method of detecting in a biological sample an antibody immunospecific for the genus of West Afrin Ebola Species constituting hEbola EbBun and EboIC
virus using at least one the inventive isolated hEbola virus described herein, or any of the inventive proteins or polypeptides as described herein. In another specific embodiment, the invention provides a method of screening for an antibody which immunospecifically binds and neutralizes hEbola EboBun. Such an antibody is useful for a passive immunization or immunotherapy of a subject infected with hEbola.

[0020] In another aspect, the invention provides an isolated antibody or an antigen-binding fragment thereof which immunospecifically binds to the hEbola virus of the invention described above.

[0021] In other aspects, the invention provides methods for detecting the presence, activity or expression of the Glade of Bundibungyo-Ivory Coast hEbola virus in a biological material, such as cells, blood, saliva, urine, feces and so forth; and specifically at least one of EbBun or EboIC.

[0022] In a related aspect, the invention provides a method for detecting the presence of the inventive hEbola virus described above in a biological sample, the method includes (a) contacting the sample with an agent that selectively binds to a West African hEbola virus; and (b) detecting whether the compound binds to the West African hEbola virus in the sample.

[0023] In another aspect, the invention provides a method for detecting the presence of the inventive polypeptide described above, in a biological sample, said method includes (a) contacting the biological sample with an agent that selectively binds to the polypeptide;
and (b) detecting whether the agent binds to the polypeptide in the sample. In another aspect, the invention provides a method for detecting the presence of a first nucleic acid molecule derived from the inventive hEbola virus described above in a biological sample, the method comprising: (a) contacting the biological sample with an agent that selectively binds to the polypeptide; and (b) detecting whether the agent binds to the polypeptide in the sample.

[0024] In another aspect, the invention provides a method for propagating the hEbola virus in host cells comprising infecting the host cells with the inventive isolated hEbola virus described above, culturing the host cells to allow the virus to multiply, and harvesting the resulting virions.
Also provided by the present invention are host cells infected with the inventive hEbola virus 5 described above.

[0025] In another aspect, the invention provides a method of detecting in a biological sample the presence of an antibody that immunospecifically binds hEbola virus, the method comprising: (a) contacting the biological sample with the inventive host cell host described above; and (b) detecting the antibody bound to the cell.

[0026] In another aspect, the invention provides vaccine preparations, comprising the inventive hEbola virus, including recombinant and chimeric forms of the virus, nucleic acid molecules comprised by the virus, or protein subunits of the virus. The invention also provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of the inventive hEbola virus described above, and a pharmaceutically acceptable carrier. In one embodiment, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a protein extract of the inventive hEbola virus described above, or a subunit thereof; and a pharmaceutically acceptable carrier. In another, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or a complement thereof, and a pharmaceutically acceptable carrier. In another, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising any of inventive the nucleotide sequences as described above, or a complement thereof, and a pharmaceutically acceptable carrier.

[0027] In a related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of the inventive hEbola virus described above, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a protein extract of the inventive hEbola virus described above or a subunit thereof, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or a complement thereof, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising the inventive nucleotide sequence as described above or a complement thereof, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of any of the inventive polypeptides described above.

[0028] In another aspect, the present invention provides pharmaceutical compositions comprising antiviral agents of the present invention and a pharmaceutically acceptable carrier. In a specific embodiment, the antiviral agent of the invention is an antibody that immunospecifically binds hEbola virus or any hEbola epitope. In another specific embodiment, the antiviral agent is a polypeptide or protein of the present invention or nucleic acid molecule of the invention.

[0029] In a related aspect, the invention provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of an anti-hEbola EboBun agent and a pharmaceutically acceptable carrier.

[0030] The invention also provides kits containing compositions and formulations of the present invention. Thus, in another aspect, the invention provides a kit comprising a container containing the inventive immunogenic formulation described above. In another aspect, the invention provides a kit comprising a container containing the inventive vaccine formulation described above. In another, the invention provides a kit comprising a container containing the inventive pharmaceutical composition described above. In another, the invention provides a kit comprising a container containing the inventive vaccine formulation described above. In another, the invention provides a method for identifying a subject infected with the inventive hEbola virus described above, comprising: (a) obtaining total RNA from a biological sample obtained from the subject; (b) reverse transcribing the total RNA to obtain cDNA; and (c) amplifying the cDNA using a set of primers derived from a nucleotide sequence of the inventive hEbola virus described above.

[0031] The invention further relates to the use of the sequence information of the isolated virus for diagnostic and therapeutic methods.

[0032] In another aspect, the present invention provides methods for screening antiviral agents that inhibit the infectivity or replication of hEbola virus or variants thereof.

[0033] The invention further provides methods of preparing recombinant or chimeric forms of hEbola.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 represents a Phylogenetic tree comparing full-length genomes of Ebolavirus and Marburg virus by Bayesian analysis;

[0035] FIG. 2 represents an alignment of genomes of novel hEbola EboBun (SEQ
ID NO: 1) referred to below as “Ebola Bundibugyo” or “EboBun”, and hEbola Zaire (SEQ ID
NO: 20);
referred to below as “Ebola Zaire `76″ or “EboZ” and hEbola Ivory Coast (SEQ
ID NO: 10) also referred to below as “EboIC”.

 

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