WO2017165317A2 - Thérapies, vaccins et procédés prédictifs pour flavivirus - Google Patents

Thérapies, vaccins et procédés prédictifs pour flavivirus Download PDF

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Publication number
WO2017165317A2
WO2017165317A2 PCT/US2017/023260 US2017023260W WO2017165317A2 WO 2017165317 A2 WO2017165317 A2 WO 2017165317A2 US 2017023260 W US2017023260 W US 2017023260W WO 2017165317 A2 WO2017165317 A2 WO 2017165317A2
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WIPO (PCT)
Prior art keywords
replikin
zika virus
zikv
sequence
peptide
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PCT/US2017/023260
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English (en)
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WO2017165317A3 (fr
Inventor
Samuel Bogoch
Elenore S. Bogoch
Anne-Elenor BOGOCH BORSANYI
Samuel Winston Bogoch
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Samuel Bogoch
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Publication of WO2017165317A2 publication Critical patent/WO2017165317A2/fr
Publication of WO2017165317A3 publication Critical patent/WO2017165317A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to therapies for preventing and treating Flaviviruses, including Zika virus, Dengue virus, and Japanese encephalitis virus, methods of differentiating virulence of strains of Flaviviruses and of predicting outbreaks of Flaviviruses, and compounds for diagnostic, therapeutic, and/or preventive purposes in Flaviviruses.
  • Flavivirus is a genus of viruses that includes the West Nile virus, dengue virus, tick- borne encephalitis virus, yellow fever virus, Zika virus (ZIKV), Japanese encephalitis virus (JEV), and a handful of other viruses understood to cause encephalitis.
  • Human diseases caused by Flaviviruses include Japanese B encephalitis, Kyasanur Forest disease, St. Louis encephalitis, tick-borne encephalitis, and West Nile encephalitis.
  • Mosquitoes are the most common vector for human disease from Flaviviruses. Some species of virus are tick-borne and some have no known vector.
  • Flaviviruses are understood to have positive-sense, single stranded RNA genomes of around 10,000 - 11,000 bases, enveloped, icosahedral nucleocapsid, and range from about 40 to about 65 nm.
  • Flaviviruses are transmitted by the bite from an infected mosquito or tick and are classified as arboviruses. Human infections with many Flaviviruses do not produce sufficient titers to be passed back to the vector arthropod. However, at least yellow fever, dengue, and zika viruses (each vectored by a mosquito to humans) apparently do not necessarily depend upon non-human animal hosts.
  • Zika virus is a mosquito-born pathogen in humans and others animals.
  • An epidemic of human infection in Brazil in 2015 and 2016 has now been linked to a fetal deformation known as microcephaly in thousands of babies born to mothers infected in Brazil. Infected mothers have born infants with smaller-than-expected brains resulting in brain damage.
  • Zika virus Human infection with Zika virus is generally understood to cause a mild illness in the infected patient.
  • the illness is known as Zika fever or disease and has been compared to mild forms of dengue fever. Current recommended treatment is rest. Until now, it was understood that Zika could not be prevented by drugs or vaccines.
  • the virus was first identified in a rhesus macaque in Kenya in 1947. It was first isolated from a human in Nigeria in 1968. The virus has been understood to historically be present in Southeast Asia, the Pacific Islands, and parts of Africa.
  • ZIKV is an icosahedral, enveloped virus with a single-stranded, non-segmented, positive-sense RNA genome. It has been classified in the Flaviviridae virus family and the Flavivirus genus. The virus is understood to be related to the Spondweni virus and has been designated as one of the two viruses in the Spondweni virus clade.
  • the positive-sense RNA genome of ZIKV has been identified as having about 10794 bases, including two non-coding flanking regions known as the 5' NCR and the 3' NCR.
  • ZIKV open reading frame has been characterized as follows: 5'-C-prM-E-NSl-NS2A-NS2B-NS3- NS4A-NS4B-NS5-3'.
  • the polyprotein coded in the open reading from is understood to be subsequently cleaved into a capsid (C) protein, a precursor membrane (prM) protein, an envelope (E) protein, and non-structural (NS) proteins.
  • the envelope protein comprises a majority of the surface of the virion and is understood to be involved host cell binding and membrane fusion in replication.
  • Non- structural proteins NS1, NS3, and NS5 have been identified as highly conserved and large.
  • the non-structural proteins NS2A, NS2B, NS4A, and NS4B are understood to be hydrophobic proteins of smaller size.
  • Within in the 3' NCR 428 nucleotides have been identified. These have been presented as playing a part in translation, RNA packaging, cyclization, genome stabilization, and recognition.
  • Zika virus vector is the female Ae aegypti but also has been isolated from many arboreal mosquito species in the Aedes genus. These include, A. africanus, A. apicoargenteus, A. furcifer, A. hensilli, A. luteocephalus and A. vittatus. Zika virus has also been isolated from Anopheles coustani, Mansonia uniformis, and Culex perfuscus.
  • the dengue virus (DEN) is the cause of dengue fever and is vectored to humans through mosquitos. At least five serotypes of the virus have been identified as disease-causing in humans and these five serotypes have additionally been considered to exist on a continuum.
  • the virus genome is around 11000 bases coding for capsid protein C, membrane protein M, envelope protein E (three structural proteins) and NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5 (seven non- structural proteins) with short non-coding regions on both the 5' and 3' ends.
  • Mosquito vectors of DEN include Ae. aegypti and Ae. albopictus,
  • JEV Japanese encephalitis virus
  • serocomplex which is understood to include 9 genetically and antigenically related viruses. Members of this complex are particularly severe in horses, and four are known to infect humans, including West Nile virus.
  • Wild birds including herons and pigs are understood to be reservoirs of the virus. Infection in humans may be severe.
  • Vectors of JEV include the mosquitoes Culex
  • Replikin peptides are a family of small peptides that have been correlated with the phenomenon of rapid replication in SARS, influenza, malaria, West Nile virus, foot and mouth disease, and many other pathogens. See, e.g., WO 2008/143717. Replikin peptides have likewise been generally correlated with the phenomenon of rapid replication in viruses, organisms, and malignancies.
  • the present invention provides compounds for diagnostic, therapeutic, and/or preventive purposes against Flaviviruses and methods of predicting outbreaks of Flaviviruses.
  • the present invention likewise provides compounds for diagnostic, therapeutic, and/or preventive purposes against Zika virus (ZIKV), Dengue virus (DEN), and Japanese Encephalitis virus (JEV) and methods of predicting outbreaks of ZIKV, DEN, and JEV (among other Flaviviruses).
  • ZIKV Zika virus
  • DEN Dengue virus
  • JEV Japanese Encephalitis virus
  • a first non-limiting aspect of the present invention provides an isolated or synthesized peptide consisting of no more than 50 amino acid residues comprising a Replikin sequence from an isolate of ZIKV or a homologue sharing at least 50%, 60%>, 70%, 80%>, 90%, or 95%o or more homology with a Replikin sequence from an isolate of ZIKV, an isolate of DEN or a homologue sharing at least 50%, 60%>, 70%, 80%, 90%, or 95% or more homology with a Replikin sequence from an isolate of DEN, or an isolate of JEV or a homologue sharing at least 50%), 60%), 70%), 80%), 90%), or 95% or more homology with a Replikin sequence from an isolate of ZIKV.
  • the Replikin sequence is identified as shared among isolates of at least two of ZIKV, DEN, and JEV or homologues of the Replikin sequence are identified as shared among isolates of at least two of ZIKV, DEN, and JEV, wherein said homologues share at least 50%, 60%, 70%, 80%, 90%, or 95% or more homology.
  • the Replikin sequence is identified as shared among isolates of all three of ZIKV, DEN, and JEV or homologues of the Replikin sequence are shared among all three of ZIKV, DEN, and JEV, wherein said homologues share at least 50%, 60%, 70%, 80%, 90%, or 95% or more homology.
  • a non-limiting embodiment of the first aspect of the invention provides a
  • composition comprising at least one peptide consisting of no more than 50 amino acid residues comprising a Replikin sequence from an isolate of ZIKV, DEN, or JEV or a homologue sharing at least 50%, 60%, 70%, 80%, 90%, or 95% or more homology with a Replikin sequence from an isolate of ZIKV, DEN, or JEV.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition is a blocking composition.
  • the pharmaceutical composition is an immunogenic composition.
  • the pharmaceutical composition further comprises a pharmaceutically-acceptable carrier, excipient, and/or adjuvant.
  • the Replikin sequence is identified as shared among isolates of at least two of ZIKV, DEN, and JEV or homologues of the Replikin sequence are identified as shared among isolates of at least two of ZIKV, DEN, and JEV.
  • the Replikin sequence is identified as shared among isolates of all three of ZIKV, DEN, and JEV or homologues of the Replikin sequence are shared among all three of ZIKV, DEN, and JEV.
  • the Replikin sequence is SEQ ID NO: 5.
  • a composition may comprise at least two peptides, each consisting of no more than 50 amino acid residues comprising a Replikin sequence from an isolate of ZIKV, DEN, or JEV or a homologue sharing at least 50%, 60%>, 70%, 80%>, 90%, or 95% or more homology with a Replikin sequence from an isolate of ZIKV, DEN, or JEV, wherein that at least two peptides are covalently linked.
  • the composition may comprise at least three or more peptides that are covalently linked.
  • covalent linkage may be through a peptide bond.
  • the linkage may be through PEGylation.
  • linkage may be through an Ahx spacer. Linkage may be done by any method known to one of skill in the art now or hereafter.
  • a non-limiting embodiment of the first aspect of the invention provides at least one peptide consisting of no more than 50 amino acid residues comprising at least one sequence of any one of SEQ ID NO(s): 1-7 or at least one homologue sharing at least 50%, 60%, 70%, 80%, 90%, or 95% or more homology with any one of SEQ ID NO(s): 1-7.
  • the at least one peptide consists essentially of any one of SEQ ID NO(s): 1-7.
  • the at least one peptide consists of any one of SEQ ID NO(s): 1-7.
  • the at least one peptide consists of a homologue of any one of SEQ ID NO(s): 1-7, wherein said homologue shares at least 50%, 60%, 70%, 80%, 90%, or 95% or more homology with any one of SEQ ID NO(s): 1-7.
  • a composition may comprise at least one peptide consisting of no more than 50 amino acid residues comprising at least one sequence of any one of SEQ ID NO(s): 1-7 or at least one homologue sharing at least 50%, 60%>, 70%, 80%>, 90%, or 95% or more homology with any one of SEQ ID NO(s): 1-7.
  • a non-limiting embodiment of the first aspect of the invention provides at least one described peptide that is lyophilized.
  • a non-limiting embodiment of the first aspect of the invention provides at least one described peptide that is freeze-dried.
  • a non-limiting embodiment of the first aspect of the invention provides at least one described peptide that is freeze-dried.
  • inventions provides at least one described peptide that is dissolved in an aqueous solution.
  • the aqueous solution may be pure deionized water or may be sterilized water.
  • a non-limiting embodiment of the first aspect of the invention provides a
  • composition administrable to a subject susceptible to or suffering from a ZIKV infection, said composition comprising at least one peptide consisting of no more than 50 amino acid residues comprising a sequence of any one of SEQ ID NO(s): 1-7 or homologue thereof and a pharmaceutically-acceptable carrier or adjuvant.
  • a non-limiting embodiment of the first aspect of the invention provides a
  • a pharmaceutical composition comprising a mixture of at least two peptides each consisting of no more than 50 amino acid residues, wherein each of said two peptide comprises at least one amino acid sequence of SEQ ID NO(s): 1-7 that is different from the other of said two peptides.
  • a pharmaceutical composition comprises a mixture of at least three, four, five, six, or seven or more different peptides of SEQ ID NO(s): 1-7.
  • administering stimulates an immune response against any one of the amino acid sequences of SEQ ID NO(s): 1-41.
  • a non-limiting second aspect of the present invention provides a vaccine against a Flavivims.
  • the vaccine is against at least ZIKV, DEN, or JEV.
  • the vaccine is against at least two of ZIKV, DEN, or JEV.
  • the vaccine is against all three of ZIKV, DEN, or JEV.
  • the vaccine comprises a pharmaceutical composition of the first aspect of the invention.
  • a non-limiting third aspect of the present invention provides a method of preventing or treating a ZIKV, DEN, or JEV infection comprising administering a pharmaceutical composition of the first non-limiting aspect of the invention or a vaccine of the second non- limiting aspect of the invention to a subject susceptible to or suffering from a ZIKV, DEN, or JEV infection.
  • administration of the pharmaceutical composition or vaccine stimulates an immune response in the subject against a ZIKV, DEN, or JEV.
  • administration of the pharmaceutical composition or vaccine provides a blocking effect against a ZIKV, DEN, or JEV.
  • a non-limiting fourth aspect of the present invention provides a method of stimulating the immune system of a subject against ZIKV, DEN, or JEV comprising
  • the subject is suitable for providing antibodies against a ZIKV, DEN, or JEV.
  • the subject is suitable for production of polyclonal antibodies.
  • the subject is suitable for production of monoclonal antibodies.
  • the polyclonal antibodies or monoclonal antibodies are useful for providing passive immunity in a different subject.
  • the polyclonal antibodies or monoclonal antibodies are useful for identifying ZIKV, DEN, or JEV and/or are useful for diagnosing ZIKV, DEN or JEV.
  • a non-limiting fifth aspect of the present invention provides an isolated, chemically- synthesized, or recombinantly-generated binding molecule that specifically binds a Replikin sequence of a ZIKV, DEN, or JEV and/or that specifically binds a homologue of a Replikin sequence of a ZIKV, DEN, or JEV, wherein the homologue is at least 50%, 605, 70%, 80%, 90%), 95%), or more homologous with a Replikin sequence of a ZIKV, DEN, or JEV.
  • the binding molecule binds at least one sequence of SEQ ID NO(s): 1-7 or a homologue of at least one sequence of SEQ ID NO(s): 1-7, wherein said homologue is at least 50%, 60%, 70%, 80%, 90%, 95%), or more homologous.
  • the binding molecule is an antibody or antibody fragment.
  • the antibody is a monoclonal antibody.
  • the antibody is a humanized antibody.
  • the antibody is an optimized antibody.
  • the antibody is an fc- optimized antibody.
  • the antibody is selected using phage display.
  • the antibody is produced from a microorganism such as E. coli.
  • a non-limiting sixth aspect of the present invention provides methods of delivering passive immunity to a patient suffering from a ZIKV, DEN, or JEV infection comprising administering to the patient at least one isolated, chemically-synthesized, or recombinantly- generated binding molecule of the fifth aspect of the invention.
  • a non-limiting seventh aspect of the present invention provides a method of making a pharmaceutical composition comprising: selecting at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide comprising at least one peptide sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or 100% homologous with at least one Replikin peptide sequence identified in a ZIKV, DEN, or JEV as a component of a pharmaceutical composition; and making said pharmaceutical composition.
  • the at least one Replikin peptide sequence is identified as shared among at least two of ZIKV, DEN, and JEV.
  • the at least one Replikin peptide sequence is identified as shared among all three of ZIKV, DEN, and JEV.
  • a Replikin sequence shared among ZIKV, DEN, and/or JEV is a homologue that is at least 50%, 60%, 70%, 80%, 90%, or 95% homologous.
  • the method of making a pharmaceutical composition comprises selecting at least one isolated or synthesized peptide of SEQ ID NO(s): 1-7, as at least one component, and making said vaccine with the at least one component.
  • the isolated or synthesized peptide comprises any one of SEQ ID NO(s): 1-7 and is up to 50, 60, 70, 80, 90, 100, 150, 200, or 250 residues in length.
  • the pharmaceutical composition is comprised in a vaccine.
  • a method of making a pharmaceutical composition comprises selecting at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or twenty or more isolated or synthesized Replikin peptide sequences identified in ZIKV, DEN, or JEV and/or isolated or synthesized functional fragments of Replikin peptide sequences identified in ZIKV, DEN, or JEV and/or isolated or synthesized homologues of Replikin peptide sequences identified in ZIKV, DEN, or JEV and making a pharmaceutical composition comprising said selected peptide sequences or functional fragments thereof.
  • the isolated or synthesized Replikin peptide sequences, homologs, or functional fragments thereof comprise at least one peptide sequence of SEQ ID NO(s): 1-7, at least one functional fragment of at least one peptide sequence of SEQ ID NO(s): 1-7, or at least one functional fragment of at least one Replikin peptide sequence identified in a ZIKV, DEN, or JEV and may consist of said sequences and may comprise said sequences and be up to 50, 60, 70, 80, 90, 100, 150, 200, 250 or more residues in length.
  • the at least one isolated or synthesized protein, protein fragment, polypeptide, or peptide has the same amino acid sequence as at least one protein, protein fragment, polypeptide or peptide identified in a relatively virulent strain of ZIKV up to seven days, one month, six months, one year, two years, or three years prior to making said vaccine.
  • An eighth non-limiting aspect of the present invention provides a method of determining an increased probability of an outbreak of ZIKV, DEN, or JEV within about six months to about one year following an increase in Replikin concentration in an isolate of ZIKV, DEN, or JEV comprising identifying an increase in the concentration of Replikin sequences in at least one first isolate of ZIKV, DEN, or JEV as compared to at least one other isolate of ZIKV, DEN, or JEV wherein said at least one first isolate is isolated at a later time period than said one other isolate and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of ZIKV, DEN, or JEV within about six months to about one year following the increase in the concentration of Replikin sequences.
  • a method of prediction comprises: (1) obtaining a plurality of isolates of a ZIKV, DEN, or JEV wherein at least one of said isolates is isolated about six months to about 3 years later than at least one other of said isolates; (2) analyzing the amino acid sequence of at least one protein or protein fragment in each isolate of the plurality of isolates for the presence and concentration of Replikin sequences; (3) comparing the
  • the ZIKV, DEN, or JEV outbreak is predicted within about six months.
  • the ZIKV, DEN, or JEV outbreak is predicted within about one year to about three years.
  • the method of prediction further comprises processing at least one step of the method on a computer.
  • An ninth non-limiting aspect of the present invention provides a nucleic acid sequence that is antisense to a nucleic acid that encodes for any Replikin peptide present in or identified in an isolate ZIKV, DEN, or JEV.
  • This may include one of SEQ ID NO(s): 1-7 or a small interfering nucleic acid sequence that interferes with a nucleic acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes any Replikin peptide of a ZIKV, DEN, or JEV including, for example, any one of SEQ ID NO(s): 1-7 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any one of SEQ ID NO(s): 1-7.
  • the nucleic acid sequence is 21 to 150 nucleotides in length. In a non-limiting embodiment, the nucleic acid sequence is up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. In a non-limiting embodiment, the nucleic acid encodes for or is antisense to a nucleic acid that encodes for a homologue of a Replikin peptide of ZIKV, DEN, or JEV.
  • Figure 1 illustrates the annual mean Replikin concentration of all Zika virus sequences from animal and human subjects available to applicants in a given year when queried at the NCBI PubMed database website for isolates from 1947 through 2015 and number of microcephaly cases reported from 2010 through January 2016.
  • mean Replikin concentration 179 sequences were available in the NCBI database when queried.
  • Dark blue lines reflect mean Replikin concentration.
  • Light blue lines reflect standard deviation from the annual mean. The two dark blue lines with light blue standard deviation illustrated in 2015 are from queries through May 2015 and through October 2015. Dark red lines from 2010 through 2016 illustrate cases of reported microcephaly times 200.
  • FIG. 2 illustrates the number of appearance over time of SEQ ID NO: 5, a particular, single, 100% identical, Replikin sequence encoded in the genes of three related Flaviviruses, namely, Zika virus (ZIKV), Dengue virus (DEN), and Japanese Encephalitis virus (JEV) as reported in the NCBI PubMed database in the given year.
  • ZIKV Zika virus
  • DEV Dengue virus
  • JEV Japanese Encephalitis virus
  • the blue lines represent the number of isolates of JEV identified as having the SEQ ID NO: 5 Replikin sequence encoded in the genome
  • the green lines represent the number of isolates of DEN identified as having the SEQ ID NO: 5 Replikin sequence encoded in the genome
  • the black lines represent the number of isolates (times 10) of ZIKV identified as having the SEQ ID NO: 5 Replikin sequence encoded in the genome.
  • a "protein fragment” as used in this specification is any fragment of an expressed whole protein, which is any portion of an expressed whole protein where a "portion" of a protein is less than an expressed whole protein.
  • a protein fragment is not naturally produced as part of the biological function of the virus.
  • a protein fragment reflects an expressed whole protein with one or more amino acids removed from the amino acid sequence of the expressed whole protein.
  • a protein fragment may also reflect an amino acid sequence that is at least 30%, 40%, 50%, 60%), 70%), 80%), 90%), 95%o, or more homologous with any portion of an expressed whole protein so long as the protein fragment does not reflect the entire amino acid sequence of the expressed whole protein or the functional protein following biological processing.
  • polypeptide is any portion of a protein fragment and is less than an expressed whole protein.
  • a protein fragment may reflect 50, 100, 150, 200, or more amino acid residues (or any size in between) so long as the protein fragment reflects less than the total length of the expressed whole protein.
  • a "functional fragment" of a Replikin sequence as described herein is a fragment, variant, analog, or chemical derivative of a Replikin sequence that retains at least a portion of the immunological cross reactivity with an antibody specific for the Replikin sequence.
  • a fragment of the Replikin sequence refers to any subset of the molecule.
  • Variant peptides of the sequence may be made by direct chemical synthesis, for example, using methods well known in the art.
  • An analog of a Replikin sequence to a non-natural protein or polypeptide is substantially similar to either the Replikin sequence of the protein or a fragment thereof.
  • Chemical derivatives of a Replikin sequence contain additional chemical moieties.
  • Amino acid analogues may include D- amino acid residues, or polypeptides with modified structural backbones, or polypeptides with non-natural chemical moieties added.
  • the term "preferentially binds" or “specifically binds” and related terms referencing the interaction of a binding molecule such as, for example, an antibody, and the structure to which it binds (antigen) means that the binding molecule preferentially recognizes the structure to which it binds even when present among other molecules (such as in a mixture of molecules).
  • Specific or preferential binding of a binding molecule to a binding structure or an immunogenic portion of a binding structure is specific and preferential when the binding molecule binds to the structure or portion thereof and does not bind with the same level of affinity to other structures.
  • Binding affinity may be determined by one of ordinary skill in the art using, for example, BIACORE, enzyme-linked immunosorbent assays, or radioimmuno assays.
  • a binding molecule may cross-react with related antigens and preferably does not cross- react with affinity to unrelated antigens. Binding between a binding molecule and the structure to which it binds may be mediated by covalent or non-covalent attachment, or both.
  • vaccine is any substance, compound, composition, mixture, or other therapeutic substance that, when administered to a human or animal via any method of administration known to the skilled artisan now or hereafter, produces an immune response, an antibody response, or a protective effect in the human or animal.
  • a "Replikin sequence” is an amino acid sequence of 7 to 50 amino acid residues comprising (1) a first lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues, where the sequence is the shortest sequence comprising the first and second lysine residues of element (1) and the at least one histidine of element (2).
  • a Replikin sequence may comprise more than two lysine residues and more than one histidine residue so long as at least two of the lysine residues and at least one histidine residue reflect the requirements of the definition of a Replikin sequence.
  • a Replikin sequence may or may not be the shortest sequence comprising the first and second lysine residues of element (1) and the at least one histidine residue of element (2).
  • a Replikin sequence may comprise a terminal lysine residue and a terminal lysine or histidine residue where the sequence is 7 to 50 amino acid residues in length and comprises (1) at least one lysine residue located six to ten residues from at least one other lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
  • Replikin sequence can also refer to a nucleic acid sequence encoding an amino acid sequence having 7 to about 50 amino acids comprising:
  • an "isolated" peptide may be synthesized by organic chemical methods.
  • An isolated peptide may also be synthesized by biosynthetic methods.
  • An isolated peptide may also refer to a peptide that is, after purification, substantially free of cellular material or other contaminating proteins or peptides from the cell or tissue source from which the peptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized by any method, or substantially free from contaminating peptides when synthesized by recombinant gene techniques or a protein or peptide that has been isolated in silico from nucleic acid or amino acid sequences that are available through public or private databases or sequence collections.
  • An isolated peptide may be synthesized by biosynthetic or organic chemical methods.
  • Proteins, protein fragments, polypeptides, or peptides in this specification may be chemically synthesized by any method known to one of skill in the art now and hereafter.
  • isolated proteins, protein fragments, polypeptides, or peptides may be synthesized by solid phase synthesis.
  • the production of these materials by chemical synthesis avoids the inclusion of (or the need to remove by purification) materials that are byproducts of other production methods such as recombinant expression or isolation from biological material.
  • Such byproducts may include, for example, avian proteins associated with vaccines produced using birds' eggs, bacterial proteins associated with recombinant production in bacteria, or proteins or contaminants associated with any recombinant activity such as with productions of proteins or other sequences in insect cells.
  • An "encoded” or “expressed” protein, protein sequence, protein fragment sequence, or peptide sequence is a sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence with any codon known to one of ordinary skill in the art now or hereafter. It should be noted that it is well known in the art that, due to redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon and still result in an identical amino acid sequence.
  • a method of identifying a Replikin amino acid sequence also encompasses a method of identifying a nucleic acid sequence that encodes a Replikin amino acid sequence wherein the Replikin amino acid sequence is encoded by the identified nucleic acid sequence.
  • “Homologous” or “homology” or “sequence identity” as used in this specification indicate that an amino acid sequence or nucleic acid sequence exhibits substantial structural equivalence with another sequence, namely any Replikin peptide sequence (including SEQ ID NO(s): 1-7) identified in an isolate of ZIKV or any nucleotide sequence encoding a Replikin peptide sequence in an isolate of ZIKV (a redundancy in a coding sequence may be considered identical to a sequence encoding the same amino acid).
  • a sequence is aligned for optimal comparison purposes with any one of possible basis sequences.
  • a basis sequence is a Replikin sequence identified in an isolate of ZIKV. Where gaps are necessary to provide optimal alignment, gaps may be introduced in the identified sequence or in the basis sequence. When a position in the identified sequence is occupied by the same amino acid residue or same nucleotide as the corresponding position in the basis sequence, the molecules are considered identical at that position (as used herein amino acid or nucleic acid "identity” is equivalent to amino acid or nucleic acid "homology”). To determine percent homology, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared between the identified sequence and the basis sequence.
  • the total number of amino acid residues or nucleotides in the identified sequence that are identical with amino acid residues or nucleotides in the basis sequence is divided by the total number of residues or nucleotides in the basis sequence (if the number of residues or nucleotides in the basis sequence is greater than the total number of residues or nucleotides in the identified sequence) or by the total number of amino acid residues or nucleotides in the identified sequence (if the number of residues or nucleotides in the identified sequence is greater than the total number of residues or nucleotides in the basis sequence).
  • the final number is determined as a percentage.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps (where a gap must be introduced for optimal alignment of the two sequences) and the length of each gap. Any structural or functional differences between sequences having sequence identity or homology will not affect the ability of the sequence to function as indicated in the desired application.
  • SEQ ID NO: 8 (KHATVLK) is considered 70% homologous with SEQ ID NO: 9 (KVKKHATVLK).
  • the 70% homology between SEQ ID NO: 8 and SEQ ID NO: 9 is determined as follows: SEQ ID NO: 8 is the identified sequence. SEQ ID NO: 9 is the basis sequence. Upon alignment, SEQ ID NO: 8 is identical to SEQ ID NO: 9 in seven of the ten sequences of SEQ ID NO: 9. To determine percent homology, then, the 7 aligned identical residues are divided by the total number of residues in SEQ ID NO: 9, namely 10 residues, giving 0.70 or more than 70% homology.
  • the polypeptide, protein fragment, or protein must first be optimally aligned with the basis sequence. Upon alignment of the sequences, the residue in the identified sequence that is furthest to the amino-terminus of the polypeptide, protein fragment, or protein and identical to a residue in the basis sequence that is furthest to the amino-terminus of the basis sequence is considered the amino-terminal residue of the identified sequence.
  • the residue in the identified sequence that is furthest to the carboxy -terminus of the polypeptide, protein fragment, or protein and identical to a residue in the basis sequence that is furthest to the carboxy-terminus of the basis sequence is considered the carboxy -terminal residue of the identified sequence.
  • the number of gaps in either the basis sequence or the identified sequence should be limited to the number of gaps allowable without significantly compromising the function of the identified sequence as compared to the basis sequence.
  • many gaps in the sequence of the basis peptide or in the sequence of the identified peptide are allowed based on homology as defined herein. Relatively more gaps are allowed if the lysines and histidines that create the definition of the Replikin peptide are identically shared between the basis peptide and the identified peptide.
  • lysines and histidines that create the definition of the Replikin peptide are shared at least in close position (for example within ten, nine, eight, seven, six, five, four, three, two, or one amino acid residue). If some of the lysine residues and histidine residues that create the definition of the Replikin peptide are not present in the identified peptide, fewer gaps may be allowed. Nevertheless, if the identified peptide functions similarly to the basis peptide, any number of gaps is allowed.
  • three or more gaps are allowed in the sequence of the basis peptide or in the sequence of the identified peptide within ten amino acid residues of the basis peptide if no lysines or histidines are present in the identified peptide. Two or more gaps or one or more gaps are also allowed. Nevertheless, if the identified sequence provides the same or a similar function to the basis sequence, more gaps are allowed up to the number of gaps that will provide a homology of 30%, 40%, 50%, 60%), 70%), 80%), 90%), 95%), or more homology. Additionally, where the lysines and histidines of the Replikin definition are present in both the identified peptide and the basis peptide, there should be no limit on how many gaps are allowed.
  • Replikin Count or “Replikin concentration” refers to the number of Replikin sequences per 100 amino acids in a protein, protein fragment, virus, or organism. A higher Replikin concentration in a first strain of a virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier-arising or later-arising strain of the virus or organism having a lower Replikin concentration.
  • Replikin concentration is determined by counting the number of Replikin sequences in a given sequence, wherein a Replikin sequence is a peptide of 7 to 50 amino acid residues comprising (1) a first lysine residue six to ten residues from a second lysine residue, (2) at least one histidine residue, (3) and 6% or more lysine residues where the Replikin sequence is the shortest sequence comprising the first and second lysine residues of element (1) and the at least one histidine residue of element (2).
  • a Replikin sequence may comprise more than two lysine residues and more than one histidine residue so long as there is at least one lysine residue six to ten residues from a second lysine residue and at least one histidine residue.
  • a Replikin sequence for the purpose of determining Replikin concentration as described in this paragraph may also be a nucleic acid that encodes a Replikin peptide sequence defined according to this paragraph.
  • Zika viruses are structurally and functionally related to rapid replication, virulence, and viral outbreaks.
  • Examples 2 through 8 demonstrate that Replikin sequences are highly conserved across time and regions. Identification of these structures, therefore, allows for targeting of the function of rapid replication (related to virulence) in ZIKV by stimulating an immune response against these functional targets and by administering blocking compounds (including Replikin sequences, binding molecules against Replikin sequences, and antisense nucleic acid sequences against Replikin sequences) that interfere with these functional targets.
  • blocking compounds including Replikin sequences, binding molecules against Replikin sequences, and antisense nucleic acid sequences against Replikin sequences
  • One non-limiting aspect of the invention therefore, provides compounds and compositions that stimulate an immune response against or otherwise mechanically block Replikin sequences resulting in inhibition of replication in the virus and diminishment of virulence.
  • Pharmaceutical compositions that target highly-conserved sequences and/or sequences present in a current outbreak are useful for treating and preventing ZIKV.
  • compositions may be produced, for example, by solid-phase synthesis of peptide sequences.
  • These peptide sequences may be freeze dried, or lyophilized, providing for long term stability of the peptides without refrigeration or other stabilizers or preservatives. Because the peptides are short, they are soluble in water and may be dissolved in water for administration as a vaccine or blocking agent without additional solubilizing compounds.
  • These compositions need not contain biologies and need not be refrigerated. Production may be ramped up easily with economies of scale to meet large populations (even the global population). Compositions may be manufactured in as little as seven days. Studies in influenza and taura syndrome virus have demonstrated both an immune response and blocking responses. See, e.g., US 2009/0041795 and US 2010/0215675 (each incorporated herein by reference).
  • Administration of a pharmaceutical composition to a subject having an immune system is understood to result in an immune response and/or blocking response against Replikin sequences in ZIKV including the amino acid sequences of SEQ ID NO(s): 1-7 or homologues thereof.
  • the pharmaceutical composition is useful for targeting replication and virulence in ZIKV.
  • a non-limiting immunogenic and/or blocking compound comprising at least one protein, protein fragment, polypeptide, or peptide of any one of the proteins, protein fragments, polypeptides, or peptides described herein including and not limited to comprising at least one Replikin peptide sequence identified in a ZIKV or at least one homologue of said at least one Replikin peptide sequence, or at least one functional fragment of at least one Replikin peptide sequence.
  • a Replikin sequence may be shared among different isolates of ZIKV.
  • the sequence may be shared among any two or more of isolates of ZIKV.
  • the Replikin sequences may be shared as identical or as close homologues.
  • the Replikin sequences may differ by a single amino acid residue.
  • the Replikin sequences may be conserved in ZIKV over one, two, three, or more years.
  • Replikin sequences identified in ZIKV may be altered by a single amino acid sequence to create a sequence not present in ZIKV in nature.
  • Replikin peptide sequences may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 1-7.
  • Peptidomimetic versions of Replikin peptide sequences or homologues thereof may likewise be used for immunogenic or blocking compositions.
  • Poly-N-substituted glycines, D-peptides, or beta-peptides may be constructed to function as any identified Replikin peptide sequence.
  • Linkage may be by any method known to art now or hereafter including but not limited to PEGylation, peptoid bonds, Ahx spacers, etc.
  • Replikin sequences may be targeted to inhibit or control replication and virulence in ZIKV.
  • a method of preventing or treating ZIKV infection comprising administering a pharmaceutical composition or a vaccine comprising a immunological and/or blocking composition to a subject susceptible to or suffering from a ZIKV infection.
  • Administration of a pharmaceutical composition or vaccine stimulates an immune response in the subject against ZIKV.
  • Administration of a pharmaceutical composition or vaccine provides a blocking response against ZIKV.
  • compositions of the invention alone or in various combinations
  • peptides in a pharmaceutical composition may be administered to a subject by any manner known to one of ordinary skill in the art including by intravenous or intramuscular injection, ocular swab or spray, nasal spray and/or inhalation spray, or any other method of administration in order to stimulate the immune system of the subject to produce an immune response or in order to provide a direct or otherwise indirect blocking effect.
  • the dosage of peptides in a pharmaceutical composition is in the range of from about 0.1 ⁇ g to about 10 mg, about 10 ⁇ g to about 1 mg, and about 50 ⁇ g to about 500 ⁇ g.
  • a non-limiting dosage may be 0.01 to 5 mg of peptide per gram of body mass of a subject.
  • a non-limiting dosage may be 0.05 to 1 mg of peptide per gram of body mass of a subject.
  • a non-limiting dosage may be 0.1 to 0.5 mg of peptide per gram of body mass of a subject. The skilled practitioner can readily determine the dosage and number of doses needed to produce an effective immune response or an effective blocking effect, or both.
  • the identification herein of function Replikin sequences conserved in ZIKV provides a method of stimulating the immune system of a subject against ZIKV comprising administering a pharmaceutical composition or a vaccine to a subject.
  • the subject may be a suitable subject for providing antibodies against ZIKV.
  • the subject may be suitable for stimulation and production of polyclonal antibodies or for stimulation and production of monoclonal antibodies.
  • the polyclonal antibodies and monoclonal antibodies are useful for providing passive immunity in a patient.
  • the polyclonal antibodies and monoclonal antibodies are likewise useful for identifying ZIKV and/or are useful for diagnosing ZIKV.
  • a vaccine is provided against ZIKV.
  • the vaccine targets Replikin structures in ZIKV thereby limiting replication and virulence.
  • a vaccine may comprise a Replikin peptide or homologue or function fragment of a Replikin peptide identified in a ZIKV.
  • a vaccine may comprise a pharmaceutical composition described herein.
  • a vaccine may comprise a mixture of a plurality of peptide sequences or homologues of any of SEQ ID NO(s): 1-7, or a mixture of a plurality of peptide sequences and/or homologues of any of SEQ ID NO(s): 1-7.
  • a vaccine may comprise an approximately equal molar mixture of isolated or synthesized peptides of any two or more of SEQ ID NO(s): 1-7, an approximately equal molar mixture of isolated or synthesized peptides of SEQ ID NO(s): 1-7, or an
  • a pharmaceutical composition or vaccine may comprise a pharmaceutically- acceptable carrier and/or adjuvant and/or excipient.
  • An adjuvant may be a UTOPE.
  • a TUOPE adjuvant may be covalently attached to an isolated or synthesized peptide at the C-terminus, the N-terminus, or both termini.
  • a UTOPE is a peptide sequence of 6 to 10 residues comprising one histidine residue with all other residues being lysine residues.
  • a non-limiting acceptable carrier, adjuvant, or excipient may include sterile water, oil and water emulsion, keyhole limpet hemocyanin.
  • a non-limiting carrier, excipient, or adjuvant may include a sterile diluent such as water (for dermal, nasal, or ocular application, spraying, or injection), saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for use typically include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • a relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • Binding molecules are provided as an aspect of the invention to target Replikin structures in ZIKV.
  • Replikin peptides may be used to generate antibodies, antibody fragments, or to generate or identify other binding agents, which may be used, for example for diagnostic purposes or to provide passive immunity in an individual. See, e.g., US 2007/0026009 and US 2009/0017052 (each incorporated herein by reference in their entirety).
  • Various procedures known in the art may be used for the production of antibodies to Replikin sequences or to proteins, protein fragments, polypeptides, or peptides comprising Replikin sequences.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Antibodies that are linked to a cytotoxic agent may also be generated.
  • Antibodies may also be administered in combination with an antiviral agent.
  • combinations of antibodies to different Replikins may be administered as an antibody cocktail.
  • Monoclonal antibodies to Replikins may be prepared using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, ⁇ Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • such fragments include but are not limited to F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be generated (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • a binding molecule may be any molecule that preferentially binds a Replikin sequence or homologue thereof.
  • a binding molecule may be ligand.
  • a binding molecule may be an antibody or antibody fragment.
  • An antibody fragment may be an F(ab')2 fragment or Fab fragment or any fragment of an antibody capable of specifically binding a Replikin sequence or homologue thereof.
  • An antibody may be a monoclonal antibody.
  • An antibody may be a humanized antibody.
  • An antibody may be an optimized antibody. In a non-limiting
  • the antibody may be an fc-optimized antibody.
  • the antibody is selected using phage display.
  • the antibody is produced from a microorganism such as E. coli.
  • an antibody, antibody fragment, or binding molecule binds to at least a portion of an amino acid sequence of at least one protein, protein fragment, polypeptide, or peptide comprising a peptide sequence that is 30%, 40%>, 50%>, 60%>, 70%>, 80%>, 90%), or 95%o or more homologous with at least one Replikin peptide sequence identified in ZIKV.
  • An antibody, antibody fragment, or binding agent may be isolated or synthesized.
  • An antibody or antibody fragment may be a monoclonal antibody or monoclonal antibody processed to create an antibody fragment.
  • An antibody or antibody fragment may be made from any biological or chemical method. Methods of predicting outbreaks of ZIKV
  • a non-limiting embodiment provides a method of differentiating between relatively more virulent and relatively less virulent forms of ZIKV.
  • a first non-limiting embodiment provides a method of identifying and/or diagnosing a relatively more virulent form of ZIKV comprising determining the Replikin concentration of at least one portion of at least one protein of at least one isolate of ZIKV or at least one portion of at least one gene that expresses at least one protein of the at least one isolate of ZIKV and comparing the Replikin concentration of the at least one isolate of ZIKV to a comparable Replikin concentration in at least one other isolate of ZIKV .
  • the at least one portion of at least one protein comprises the entirety of at least one protein expressed in a ZIKV and the comparable Replikin concentration is the Replikin concentration of the entirety of the same protein expressed in a ZIKV from the at least one other isolate of the same ZIKV.
  • the Replikin concentration of the at least one isolate of ZIKV is a mean of Replikin concentrations determined in a plurality of isolates of the same ZIKV.
  • the Replikin concentration of the at least one other isolate of ZIKV is a mean of Replikin concentration determined in a plurality of other isolates of the same ZIKV.
  • the plurality of isolates of ZIKV is a collection of isolates isolated in a given year and the plurality of other isolates of ZIKV is a collection of isolates of the same ZIKV isolated in a different year.
  • the Replikin concentration of the more virulent isolate of ZIKV is 3.0 or greater, 4.0 or greater, 5.0 or greater, 6.0 or greater, 8.0 or greater, 10.0 or greater, or 12.0 or greater per 100 amino acid residues.
  • the Replikin concentration of the more virulent isolate of ZIKV is 8.0 or greater per 100 amino acid residues.
  • a vaccine is manufactured following the differentiation between relatively more virulent and relatively less virulent forms of ZIKV.
  • a vaccine is manufactured following prediction of an outbreak of ZIKV following identification of a more virulent form of a ZIKV.
  • the vaccine comprises at least one structure of the isolate of ZIKB differentiated as relatively more virulent.
  • the vaccine comprises at least one Replikin peptide sequence identified in the isolate of ZIKB differentiated as relatively virulent.
  • the Replikin concentration of the at least one isolate of ZIKV is greater than the Replikin concentration of the at least one other isolate of ZIKV.
  • the Replikin concentration is a mean Replikin concentration of a plurality of isolates with standard deviation from the mean and the standard deviation from the mean is greater than the standard deviation from the mean Replikin concentration of a plurality of other isolates.
  • Another non-limiting embodiment provides a method of determining an increased probability of an outbreak of ZIKV within about one year following an increase in Replikin concentration in an isolate of ZIKV comprising identifying an increase in the concentration of Replikin sequences in at least one first isolate of a ZIKV as compared to at least one other isolate of the same kind of ZIKV wherein said at least one first isolate is isolated at a later time period than said one other isolate and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of the ZIKV within about one year following the increase in the concentration of Replikin sequences.
  • a method of prediction comprises: (1) obtaining a plurality of isolates of a ZIKV wherein at least one of said isolates is isolated about six months to about 3 years later than at least one other of said isolates; (2) analyzing the amino acid sequence of at least one protein or protein fragment in each isolate of the plurality of isolates for the presence and concentration of Replikin sequences; (3) comparing the concentrations of Replikin sequences in the at least one protein or protein fragment in each isolate of the plurality of isolates one to another; (4) identifying an increase in the concentration of Replikin sequences in said plurality of isolates over at least one time period of about six months or greater; and (5) predicting an outbreak of the ZIKV within about one month to about three years following said identified increase in the concentration of Replikin sequences.
  • the outbreak of ZIKV is predicted within about six months. In a further embodiment of the invention, the outbreak of ZIKV virus is predicted within about one year to about three years. In a further non-limiting embodiment, the method of prediction further comprises processing at least one step of the method on a computer.
  • the method of prediction further comprises comparison of the standard deviation from the mean of Replikin concentrations of isolates of ZIKV from a given time period, such as a given month, a given year, or any other given time period.
  • the Replikin concentration is a mean Replikin concentration of a plurality of isolates with standard deviation from the mean and the standard deviation from the mean is greater than the standard deviation from the mean Replikin concentration of a plurality of other isolates.
  • a further non-limiting embodiment provides a computer readable medium having stored thereon or signal containing instructions which, when executed, cause a processor to perform a method of predicting an expansion of a strain of ZIKV or an increase in virulence or morbidity of ZIKV.
  • the processor reports a prediction to a display, user, researcher, or other machine or person.
  • the processor identifies to a display, user, researcher, or other machine or person, a portion of a pathogen predicted to be an expanding ZIKV or predicted to increase in virulence or morbidity wherein said portion may be employed as a therapeutic or diagnostic compound.
  • Said portion may be a Replikin peptide or plurality of Replikin peptides or any other structure or portion of said genome of said pathogen including a Replikin Peak Gene.
  • said portion is synthesized and prepared in a pharmaceutical composition.
  • Another non-limiting embodiment provides a computer system, including a processor coupled to a network and a memory coupled to the processor, the memory containing a plurality of instructions to perform a method of predicting the relative virulence of at least one first group of ZIKV as compared to at least one second group of ZIKV.
  • Another non-limiting embodiment provides a machine-readable storage medium having stored thereon executable instructions that, when executed by a processor, cause the processor to provide sufficient data to a user, a display, or a printout such that said user or a user of said display or said printout may predict the virulence of a ZIKV based on regression analysis.
  • Another non-limiting embodiment provides a computer system, comprising: a processor coupled to a network; a memory coupled to the processor, the memory containing a plurality of instructions to perform a method of predicting the virulence of an ZIKV based on regression analysis.
  • a non-limiting aspect of the present invention provides a nucleic acid sequence that is antisense to a nucleic acid that encodes for any Replikin peptide present in or identified in a ZIKV isolate.
  • This may include one of SEQ ID NO(s): 1-7 or a small interfering nucleic acid sequence that interferes with a nucleic acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%), or 95% or more homologous with a nucleic acid that encodes any Replikin peptide of a ZIKV including, for example, any one of SEQ ID NO(s): 1-7 or is 30%, 40%, 50%, 60%, 70%, 80%), 90%), 95%) or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any one of SEQ ID NO(s): 1-7.
  • a nucleic acid sequence may be 21 to 150 nucleotides in length.
  • a nucleic acid sequence may be up to
  • the Replikins Global Surveillance SystemTM found a spike increase in ZIKV gene Replikin concentration in May and October of 2015, which correlates with and predicts the outbreak of ZIKV infection and microcephaly cases in Brazil in 2015 and early 2016.
  • Figure 1 and related analyses were generated from data accessed at the PubMed NCBI database website for Zika virus isolates from 1947 through 2015. The information for all accessed individual isolates is provided in Table 1 below including calculated Replikin concentration and year reported for each sequence.
  • zika virus strains geographic expansion of the asian lineage
  • the ankyrin repeat-containing adaptor protein Tvl-1 is a novel substrate and regulator of Raf-
  • RFXANK gene in North African major histocompatibility complex class II-deficient patients belonging to complementation group B Major histocompatibility complex class II expression deficiency caused by a RFXANK founder mutation a survey of 35 patients Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B - deficient cells
  • New functions of the major histocompatibility complex class Il-specific transcription factor RFXANK revealed by a high-resolution mutagenesis study RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome; an MHC class II immunodeficiency
  • a gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients
  • Zika viruses Biological transmission of arboviruses: reexamination of and new insights into components; mechanisms; and unique
  • Figure 1 similarly illustrates that a rise in Replikin concentration occurs before the ZIKV outbreak and increase in virulence and morbidity (as demonstrated by a spike in cases of microcephaly) in the virus population.
  • SEQ ID NO: 1 is a Replikin peptide sequence identified as conserved in ZIKV across many years. Replikin peptide sequences are associated with rapid replication and virulence in ZIKV (see Figure 1) and are understood to be immunogenic because of the positioning of the lysine residues in the sequence. The histidine in the Replikin peptide sequence likewise is associated with rapid replication and virulence. As a result, SEQ ID NO: 1 and homologues of SEQ ID NO: 1 are useful in a vaccine or in blocking composition for preventing and/or treating ZIKV infection. SEQ ID NO: 1 may be used alone or in combination with other Replikin sequences or homologues of Replikin sequences. SEQ ID NO: 1 may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 2-7.
  • SEQ ID NO: 1 Peptidomimetic versions of SEQ ID NO: 1 or homologues thereof may likewise be used for immunogenic or blocking compositions.
  • Poly-N-substituted glycines, D-peptides, or beta- peptides may be constructed to function as SEQ ID NO: 1.
  • the peptide KHWLVHK (SEQ ID NO: 2) was identified as conserved in reported isolates of ZIKV from the following years in the listed accession numbers. All occurrences identified by applicants in a given year at NCBI PubMed database website are listed.
  • AHL43467 position 79 AHL43466 position 79 , AHL43465 position 79 , AHL43463 position 79
  • AHL43446 position 79 AHL43445 position 79 , AHL43444 position 79
  • SEQ ID NO: 2 is a Replikin peptide sequence identified as conserved in ZIKV across many years. Replikin peptide sequences are associated with rapid replication and virulence in ZIKV (see Figure 1) and are understood to be immunogenic because of the positioning of the lysine residues in the sequence. The histidine in the Replikin peptide sequence likewise is associated with rapid replication and virulence.
  • SEQ ID NO: 2 and homologues of SEQ ID NO: 2 are useful in a vaccine or in blocking composition for preventing and/or treating ZIKV infection.
  • SEQ ID NO: 2 may be used alone or in combination with other Replikin sequences or homologues of Replikin sequences.
  • SEQ ID NO: 2 may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 1 and 3-7.
  • SEQ ID NO: 2 Peptidomimetic versions of SEQ ID NO: 2 or homologues thereof may likewise be used for immunogenic or blocking compositions.
  • Poly-N-substituted glycines, D-peptides, or beta- peptides may be constructed to function as SEQ ID NO: 2.
  • SEQ ID NO: 3 is a Replikin peptide sequence identified as conserved in ZIKV across many years. Replikin peptide sequences are associated with rapid replication and virulence in ZIKV (see Figure 1) and are understood to be immunogenic because of the positioning of the lysine residues in the sequence. The histidine in the Replikin peptide sequence likewise is associated with rapid replication and virulence. As a result, SEQ ID NO: 3 and homologues of SEQ ID NO: 3 are useful in a vaccine or in blocking composition for preventing and/or treating ZIKV infection. SEQ ID NO: 3 may be used alone or in combination with other Replikin sequences or homologues of Replikin sequences.
  • SEQ ID NO: 3 may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 1, 2, and 4-7.
  • Peptidomimetic versions of SEQ ID NO: 3 or homologues thereof may likewise be used for immunogenic or blocking compositions.
  • Poly-N-substituted glycines, D-peptides, or beta- peptides may be constructed to function as SEQ ID NO: 3.
  • the peptide KGRLSSGHLK (SEQ ID NO: 4) was identified as conserved in reported isolates of ZIKV from the following years in the listed accession numbers. All occurrences identified by applicants in a given year at NCBI PubMed database website are listed.
  • SEQ ID NO: 4 is a Replikin peptide sequence identified as conserved in ZIKV across many years. Replikin peptide sequences are associated with rapid replication and virulence in ZIKV (see Figure 1) and are understood to be immunogenic because of the positioning of the lysine residues in the sequence. The histidine in the Replikin peptide sequence likewise is associated with rapid replication and virulence. As a result, SEQ ID NO: 4 and homologues of SEQ ID NO: 4 are useful in a vaccine or in blocking composition for preventing and/or treating ZIKV infection. SEQ ID NO: 4 may be used alone or in combination with other Replikin sequences or homologues of Replikin sequences.
  • SEQ ID NO: 4 may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 1, 2, 3, and 5-7. Peptidomimetic versions of SEQ ID NO: 4 or homologues thereof may likewise be used for immunogenic or blocking compositions. Poly-N-substituted glycines, D-peptides, or beta- peptides may be constructed to function as SEQ ID NO: 4.
  • HLKCRLKMDK SEQ ID NO: 5
  • All occurrences identified by applicants in a given year at NCBI PubMed database website are listed.
  • SEQ ID NO: 5 is a Replikin peptide sequence identified as conserved in ZIKV across many years. Replikin peptide sequences are associated with rapid replication and virulence in ZIKV (see Figure 1) and are understood to be immunogenic because of the positioning of the lysine residues in the sequence. The histidine in the Replikin peptide sequence likewise is associated with rapid replication and virulence. As a result, SEQ ID NO: 5 and homologues of SEQ ID NO: 5 are useful in a vaccine or in blocking composition for preventing and/or treating ZIKV infection. SEQ ID NO: 5 may be used alone or in combination with other Replikin sequences or homologues of Replikin sequences.
  • SEQ ID NO: 5 may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 1-4, 6, and 7.
  • Peptidomimetic versions of SEQ ID NO: 5 or homologues thereof may likewise be used for immunogenic or blocking compositions.
  • Poly-N-substituted glycines, D-peptides, or beta- peptides may be constructed to function as SEQ ID NO: 5.
  • HWNNKEAL VEFK (SEQ ID NO: 6) was identified as conserved in reported isolates of ZIKV from the following years in the listed accession numbers. All occurrences identified by applicants in a given year at NCBI PubMed database website are listed.
  • SEQ ID NO: 6 is a Replikin peptide sequence identified as conserved in ZIKV across many years. Replikin peptide sequences are associated with rapid replication and virulence in ZIKV (see Figure 1) and are understood to be immunogenic because of the positioning of the lysine residues in the sequence. The histidine in the Replikin peptide sequence likewise is associated with rapid replication and virulence. As a result, SEQ ID NO: 6 and homologues of SEQ ID NO: 6 are useful in a vaccine or in blocking composition for preventing and/or treating ZIKV infection. SEQ ID NO: 6 may be used alone or in combination with other Replikin sequences or homologues of Replikin sequences. SEQ ID NO: 6 may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 1-5 and 7.
  • SEQ ID NO: 6 Peptidomimetic versions of SEQ ID NO: 6 or homologues thereof may likewise be used for immunogenic or blocking compositions.
  • Poly-N-substituted glycines, D-peptides, or beta- peptides may be constructed to function as SEQ ID NO: 6.
  • the peptide HRTLALAVIKYTYQNK (SEQ ID NO: 7) was identified as conserved in reported isolates of ZIKV from the following years in the listed accession numbers. All occurrences identified by applicants in a given year at NCBI PubMed database website are listed.
  • SEQ ID NO: 7 is a Replikin peptide sequence identified as conserved in ZIKV across many years. Replikin peptide sequences are associated with rapid replication and virulence in ZIKV (see Figure 1) and are understood to be immunogenic because of the positioning of the lysine residues in the sequence. The histidine in the Replikin peptide sequence likewise is associated with rapid replication and virulence.
  • SEQ ID NO: 6 and homologues of SEQ ID NO: 7 are useful in a vaccine or in blocking composition for preventing and/or treating ZIKV infection.
  • SEQ ID NO: 7 may be used alone or in combination with other Replikin sequences or homologues of Replikin sequences.
  • SEQ ID NO: 7 may be covalently linked to other Replikin sequences, such as any one or more of SEQ ID NO(s): 1-6.
  • SEQ ID NO: 7 Peptidomimetic versions of SEQ ID NO: 7 or homologues thereof may likewise be used for immunogenic or blocking compositions.
  • Poly-N-substituted glycines, D-peptides, or beta- peptides may be constructed to function as SEQ ID NO: 7.
  • HLKCRLKMDK (SEQ ID NO: 5)
  • HWNNKEALVEFK (SEQ ID NO: 6)
  • the peptides are synthesized by solid-state synthesis and lyophilized.
  • the lyophilized peptides are mixed together. A sufficient amount of mixture is dissolved in deionized water to prepare a solution having a concentration of 4 g/L.
  • a single rabbit is prepared for testing of the vaccine.
  • the rabbit is bled and tested for antibodies to the mixture of peptides at various stages before, during, and after administration of the vaccine.
  • Antibody response is determined via ELISA.
  • the ELISA plate is prepared by adherence of the mixture of the peptides to the surface of the wells of the plate. Concentration of binding antibodies on the plate is determined via optical density.
  • a 25 ⁇ aliquot of 4 g/L aqueous peptide mixture is administered to the rabbit (a 100 ⁇ g dose) intranasally on day 1.
  • a 25 ⁇ aliquot of 4 g/L aqueous peptide mixture is administered to the rabbit (a 100 ⁇ g dose) intranasally on day 15.
  • a 25 ⁇ aliquot of 4 g/L aqueous peptide mixture is administered to the rabbit (a 100 ⁇ g dose) intranasally on day 22 along with a 50 ⁇ aliquot of 4 g/L aqueous peptide mixture (a 200 ⁇ g dose) given via intramuscular administration. Blood is drawn from the rabbit on day 22 and ELISA tested for antibody response against the mixture of peptides (1 st Bleed).
  • a 25 ⁇ ⁇ aliquot of 4 g/L aqueous peptide mixture is administered to the rabbit (a 100 ⁇ g dose) intranasally on day 80 along with a 50 ⁇ ⁇ aliquot of 4 g/L aqueous peptide mixture (a 200 ⁇ g dose) intramuscular administration.
  • the peptides of the vaccine are conserved Replikin peptides of ZIKV.
  • Figure 1 demonstrates that Replikin sequences of ZIKV are structurally and functionally related to rapid replication and virulence in ZIKV and are conserved across strains of ZIKV, including most virulent strains. Because Replikin sequences are structurally and functionally related to rapid replication and virulence, targeting Replikin structures provides a mechanism for reducing rapid replication and virulence. The vaccine is therefore useful for preventing and treating ZIKV infection by targeting these replication mechanisms.
  • the vaccine of the example (as well as individual peptides of the vaccine or various mixtures of the individual peptides of the vaccine) is likewise useful as an immunogenic composition for stimulating the immune system of a subject against ZIKV, against an infection of an ZIKV, or to prevent a ZIKV infection.
  • Antibodies produced from the immune response are likewise useful for diagnosing ZIKV contamination and infection.
  • the vaccine of the example (as well as individual peptides of the vaccine or various mixtures of the individual peptides of the vaccine) is useful as a blocking composition to block replication of virus within infected cells.
  • a prediction of expansion or retraction of virulence of ZIKV population may be performed by a processor.
  • a prediction may be output to a user or display.
  • a particular Replikin peptide or Replikin Peak Gene within an isolate or population of isolates of KIKV may be predicted to be expanding or retracting in replication or virulence and this prediction may be output to a user or display.
  • a machine-readable storage medium may contain executable instructions that, when executed by a processor, cause the processor to provide sufficient data to a user, a printout, or a display such that the user or a user of the printout or display may predict expansion or retraction of population of ZIKV.
  • a process for prediction may comprise: comparing a Replikin concentration of at least one first isolate of ZIKV with a Replikin concentration of at least one second isolate of ZIKV; and predicting the population of the first isolate to be expanding if the Replikin concentration of the first isolate is greater than the Replikin concentration of the second isolate. In another embodiment, predicting the population of the first isolate to be expanding if the Replikin concentration of the first isolate is greater than eight or greater than ten Replikin sequences per 100 amino acid residues.
  • a computer system may include a processor coupled to a network, and a memory coupled to a processor, wherein the memory contains a plurality of instruction to perform the methods of prediction discussed herein.
  • a user of outputted data from a processor, storage medium, machine-readable medium, or computer system may include any person or any machine that records or analyzes the outputted data.
  • a display or printout may include any mechanism by which data is outputted so that any person or any machine may record or analyze the outputted data, including a printed document, a visual impulse, an aural impulse, or any other perceivable impulse, a computer monitor, a set of numbers, or any other display or printout of data including a digital recording medium.
  • a computer program product may have computer program logic arranged to put into effect a method of predicting an outbreak of ZIKV. The program may be contained in a signal or non-transitory signal.
  • Trivalent vaccine based on 100% identical Replikin sequence identified in ZIKV, DEN, and JEV
  • the new trivalent vaccine and blocker was designed based on the finding of the inventors following examination of all sequences on PubMed as of February 2016, that: 1) identical and homologous gene Replikins sequences occur in, that is, are shared by, different Flaviviruses, 2) that some of these gene Replikins are conserved back to 1947, and 3) evidence has been found of sharing, for example, due to independent appearance or possible sequential transfer between 2010 and 2014 of an identical Replikin structure between the Japanese
  • Encephalitis virus gene and the Dengue virus gene then first to appear in the Zika gene in 2013, and to peak in the Zika gene in 2014, giving one year early warning before the outbreak in 2015 .
  • Encephalitis virus and Dengue virus is of interest with regard to the increase 20-fold in fetal microcephaly, increase in Guillain Barre Syndrome, and other nervous system disorders reported to be associated with Zika virus infection.
  • Table 2 below provides the number of isolates in which SEQ ID NO: 5 appeared in a given year in three Flavivirus genes (JEV, DEN, and ZIKV) in specimens reported at the NCBI PubMed database. The data are illustrated in Figure 2. The number of isolates of ZIKV identified with SEQ ID NO: 5 is multiplied by 10 in Figure 2 so that the ZIKV data appears in the same visual range as the JEV and DEN data. As is understood by the artisan, the number of ZIKV isolates reported to the NCBI PubMed database has been relatively small compared to the number of JEV and DEN isolates.

Abstract

La présente invention concerne des thérapies, des vaccins et des procédés prédictifs pour des flavivirus, comprenant le virus Zika, le virus de la dengue et le virus de l'encéphalite japonaise, et permet d'obtenir des composés pour le diagnostic, la prévention et le traitement d'épidémies du virus Zika, du virus de la dengue et du virus de l'encéphalite japonaise.
PCT/US2017/023260 2016-03-20 2017-03-20 Thérapies, vaccins et procédés prédictifs pour flavivirus WO2017165317A2 (fr)

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US10273269B2 (en) 2017-02-16 2019-04-30 Modernatx, Inc. High potency immunogenic zika virus compositions
US10449244B2 (en) 2015-07-21 2019-10-22 Modernatx, Inc. Zika RNA vaccines
US10653767B2 (en) 2017-09-14 2020-05-19 Modernatx, Inc. Zika virus MRNA vaccines
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US20050002968A1 (en) * 2002-01-15 2005-01-06 Monath Thomas P. Flavivirus vaccines
CN103088038B (zh) * 2004-07-12 2015-06-24 美国天甲生物医药有限公司 黄病毒疫苗
US20090269367A1 (en) * 2008-04-23 2009-10-29 Samuel Bogoch Methods and compounds for mitigating pathogenic outbreaks using replikin count cycles
US20100144589A1 (en) * 2008-08-08 2010-06-10 Samuel Bogoch Methods of predicting cancer lethality using replikin counts
WO2015077522A1 (fr) * 2013-11-22 2015-05-28 Samuel Bogoch Thérapies, vaccins et procédés de prévision pour le syndrome d'effondrement des essaims d'abeilles

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US11278611B2 (en) 2015-10-22 2022-03-22 Modernatx, Inc. Zika virus RNA vaccines
US10273269B2 (en) 2017-02-16 2019-04-30 Modernatx, Inc. High potency immunogenic zika virus compositions
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US11207398B2 (en) 2017-09-14 2021-12-28 Modernatx, Inc. Zika virus mRNA vaccines

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