EP2300493A2 - Verfahren und verbindungen zur abschwächung von krankheitsausbrüchen durch replikin-zählzyklen - Google Patents

Verfahren und verbindungen zur abschwächung von krankheitsausbrüchen durch replikin-zählzyklen

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Publication number
EP2300493A2
EP2300493A2 EP20090734389 EP09734389A EP2300493A2 EP 2300493 A2 EP2300493 A2 EP 2300493A2 EP 20090734389 EP20090734389 EP 20090734389 EP 09734389 A EP09734389 A EP 09734389A EP 2300493 A2 EP2300493 A2 EP 2300493A2
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EP
European Patent Office
Prior art keywords
replikin
pathogen
cycle
virus
time period
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP20090734389
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English (en)
French (fr)
Inventor
Samuel Bogoch
Elenore S. Bogoch
Samuel Winston Bogoch
Anne Elenore Borsanyi
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Individual
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Individual
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Publication date
Priority claimed from US12/108,458 external-priority patent/US9408902B2/en
Application filed by Individual filed Critical Individual
Publication of EP2300493A2 publication Critical patent/EP2300493A2/de
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • infectious disease In surveys of global health, infectious disease often accounts for as many as five of the top ten causes of death in lower- and middle-income countries and respiratory infections are often assigned as the fourth leading cause of death in higher-income countries. Further, pathogenic outbreaks and pandemics continue to threaten human populations from previously unknown or otherwise mutated pathogenic diseases. Previously unknown or otherwise mutated pathogenic diseases often occur when a pathogen diverges from an established host, such as pigs or chickens, into a new host, such as humans. In view of this phenomenon, new strategies are continually needed for mitigating pathogenic outbreaks from previously-known or previously- unknown pathogens.
  • threatening pathogenic diseases include malaria, influenza, West Nile virus, foot-and-mouth disease, and other threats to global health in both humans and animals.
  • Therapies or methods of treatment that are useful across different pathogenic strains or even across pathogenic groups are especially helpful in improving the fight against mutable pathogenic disease and outbreaks of previously-unknown pathogens.
  • WNV West Nile virus
  • WNV West Nile virus
  • encephalitis causes encephalitis and other serious neuroinvasive diseases in a small percentage of human infections. In about four percent of reported cases, the resulting neuroinvasive disease results in death.
  • WNV is flaviviridae virus, first observed in North America in 1999 and now considered endemic in the United States. The virus is spread to humans through mosquito (and related insect) bites.
  • WNV is a single-stranded sense RNA virus and is a member of the Japanese encephalitis virus antigenic complex, which includes several medically important viruses associated with human encephalitis: Japanese encephalitis, St. Louis encephalitis, Murray Valley encephalitis, and Kunjin encephalitis, an Australian subtype of WNV.
  • pandemic influenza requires new and more effective methods of predicting and tracking lethal outbreaks of influenza.
  • Influenza vaccines remain the most effective defense against influenza virus, but because of the ability of the virus to mutate, and the availability of non-human host reservoirs, it is expected that influenza will remain an emergent or re-emergent infectious threat.
  • Global influenza surveillance indicates that influenza viruses may vary within a country and between countries and continents during an influenza season.
  • Viro logic surveillance is of importance in monitoring antigenic shift and drift.
  • Disease surveillance is also important in assessing the impact of epidemics. Both types of information have provided the basis of vaccine composition and use of antivirals.
  • the present invention provides a quantitative cyclic structure comprising Replikin peptide concentrations identified in a strain of microorganism through time, wherein said cyclic structure correlates in time with the expansion and/or contraction of a population of said strain of microorganism, the infectivity of said strain of microorganism, and/or the lethality of said strain of microorganism.
  • the present invention provides methods of preventing, mitigating, and treating outbreaks of a pathogen comprising predicting an expansion of a population of a strain of pathogen or an increase in the virulence, morbidity, and/or lethality of a strain of pathogen as compared to another strain of the same or a related pathogen and administering to an animal or patient a compound comprising an isolated or synthesized portion of the structure or genome of the pathogen to mitigate, prevent, or treat the predicted outbreak of the pathogen.
  • the present invention further provides methods of predicting an expansion of a strain of pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen comprising identifying a cycle in the Replikin Count in a protein fragment, protein, genome fragment, or genome of a pathogen and predicting an increase in the virulence, morbidity, and/or mortality of said pathogen within the identified cycle in Replikin Count.
  • the present invention further provides Replikin peptides identified within a pathogen predicted to be expanding or to have an increase in virulence, morbidity, and/or mortality as diagnostic, therapeutic, or preventive agents against an outbreak of the pathogen.
  • a first non-limiting aspect of the invention provides a method of preventing, mitigating, or treating an outbreak of a pathogen predicted to have an expansion of population comprising predicting an expansion of the population of a first pathogen comprising identifying at least one cycle of Replikin concentration in isolates of the pathogen and predicting that an expansion of the population of the first pathogen will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration, and administering to an animal or patient a compound comprising an isolated or synthesized portion of the structure or genome of the pathogen to mitigate, prevent, or treat the predicted outbreak of the pathogen.
  • a further embodiment of the first aspect of the invention provides a method of preventing, mitigating, or treating an outbreak of pathogen comprising predicting an expansion of the population or an increase in virulence, morbidity, and/or mortality of an isolate or plurality of isolates of a first strain of pathogen as compared to another isolate or plurality of isolates of the same or a related strain of pathogen comprising: (1) identifying a first cycle in the Replikin concentration of a plurality of isolates of said first strain of pathogen, (2) identifying a first peak in the Replikin concentration within the identified first cycle at a first time point or time period, and (3) predicting an increase in the virulence of an isolate of the same or related strain of pathogen isolated at a second time point or time period subsequent to the first time point or time period; and administering to an animal or a patient a compound comprising an isolated or synthesized portion of the structure or genome of the at least one isolate of the pathogen to prevent, mitigate, or treat the outbreak of the pathogen.
  • the pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, taura syndrome virus, white spot syndrome virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, Helicobacter pylori, Entamoeba invadens, L. legionella, S. aureus, maize streak virus, bovine herpes virus, feline immunodeficiency virus, human immunodeficiency virus, rous sarcoma virus, avian sarcoma virus, Sindbis virus, hepatitis virus, b. anthracis, or any other infectious agent.
  • the influenza virus is an HlNl, H2N2, H3N2, H5N1, H3N8, or H9N2 strain of influenza virus.
  • said expansion of a strain of pathogen or increase in virulence, morbidity, and/or mortality of an isolate or plurality of isolates of a strain of pathogen comprises identifying a second cycle in the Replikin concentration of a plurality of isolates of a second strain of pathogen that shares synchrony with said first cycle in the Replikin concentration of said plurality of isolates of said first strain of pathogen and identifying a first peak in the Replikin concentration within the identified first cycle at a first time point or time period and identifying a first peak in the Replikin concentration within the identified second cycle of said second strain of pathogen at a second time point or time period that is similar to said first time point or time period and predicting an increase in the virulence of said first strain of pathogen following the
  • said pathogen is an influenza virus.
  • said first strain of influenza is any strain different from said second strain of influenza.
  • said first strain of influenza is H5N1 and said second strain of influenza is H9N2, or vice versa.
  • said isolated or synthesized portion of the structure or genome of the at least one isolate of a pathogen is a protein or protein fragment comprising a Replikin peptide and/or a Replikin Peak Gene, a Replikin peptide identified within a Replikin Peak Gene, or any structure or portion of the structure of said pathogen.
  • said isolated or synthesized portion of the structure or genome is a nucleic acid encoding a Replikin Peak Gene, a Replikin peptide or a plurality of Replikin peptides within a Replikin Peak Gene, or a Replikin peptide or plurality of Replikin peptides.
  • the second time point or time period is up to three years after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about one year after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about six months after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of a pathogen following the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of influenza following the first time point or first time period.
  • the next influenza season is the next winter season in a geographic region following the first time point or time period.
  • the second time point or time period is the next season of malaria following the first time point or first time period.
  • the next season of malaria is the next rainy season.
  • the second time point or time period is the next season of West Nile virus.
  • the next season of West Nile virus is a summer season.
  • the identified peak in the cycle of Replikin concentration has a higher Replikin concentration than a chronologically earlier peak in the cycle of Replikin concentration.
  • the identified peak in the cycle of Replikin concentration is significantly higher than the earlier peak.
  • the identified peak is significantly higher than the earlier peak with a p value less than 0.01.
  • the identified peak is significantly higher than the earlier peak with a p value less than 0.001.
  • a second non- limiting aspect of the invention provides a method of predicting an expansion of the population of a first pathogen comprising identifying at least one cycle of Replikin concentration in isolates of the pathogen and predicting that an expansion of the population of the first pathogen will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration, wherein the at least one cycle is cycle A.
  • the rising portion comprises a peak wherein said expansion of the population of the first pathogen is predicted after the occurrence of the peak.
  • the cycle comprises at least a first rising portion and a second rising portion, wherein said first rising portion occurs prior in time to said second rising portion.
  • the cycle comprises at least three rising portions, wherein the at least three rising portions are at least rising portion A', rising portion B' and rising portion C.
  • the rising portion B' comprises a peak and the rising portion A' comprises a peak, and the peak of rising portion B' has a greater Replikin concentration than the peak of rising portion A'.
  • the method of prediction further comprises processing the method on a computer.
  • the cycle comprises more than one cycle including, for example, from peak to trough to peak to trough or from trough to peak to trough to peak.
  • the cycle comprises three peaks or three troughs or more.
  • the method of prediction comprises identifying at least one other cycle of Replikin concentration in isolates of at least one other strain of pathogen, wherein the at least one other cycle is cycle B, and wherein cycle B shares synchrony with cycle A; and predicting that an expansion of the population of the first pathogen will occur after the occurrence of a rising portion in cycle A that corresponds to a rising portion in cycle B.
  • the first pathogen is a first strain of influenza virus and the one other pathogen is a different strain of influenza virus.
  • the first pathogen is an H5N1 strain of influenza virus and the one other strain of pathogen is an H9N2 strain of influenza virus.
  • the expansion of the population of the first pathogen is predicted within three years after the peak. In a further embodiment, the expansion of the population of the first pathogen is predicted within one year after said peak. In a further embodiment, the expansion of the population of the first pathogen is predicted after the next virulence season of the pathogen.
  • a further embodiment of the second aspect of the invention provides a method of predicting an expansion of a population of a pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen relative to the population or the virulence, morbidity, and/or mortality of another pathogen of the same species or of another pathogen of a related species comprising: (1) identifying a cycle in the Replikin concentration of isolates of a plurality of the pathogen, (2) identifying a first peak in the Replikin concentration of isolates of a plurality of said pathogen within the identified cycle at a first time point or time period, and (3) predicting an expansion of the population of a pathogen of the same or a related species or an increase in the virulence, morbidity, and/or mortality of a pathogen of the same or a related species isolated at a second time point or time period subsequent to the first time point or time period.
  • the pathogen may be, but is not limited to, a malarial trypanosome, West Nile virus, influenza virus, equine influenza virus, coronavirus, foot and mouth disease virus, taura syndrome virus, white spot syndrome virus, or other pathogen or infectious agent.
  • the pathogen is a malarial trypanosome.
  • the trypanosome is P. falciparum, P. vivax, P. ovale, or P. malariae.
  • the trypanosome is P. falciparum.
  • the method predicts an increase in mortality from malarial infection.
  • the identified Replikin cycle represents Replikin concentrations identified in a histidine rich protein of P. falciparum. In another non-limiting embodiment of the present invention, the identified Replikin cycle represents Replikin concentrations identified in the histidine-rich protein of P. falciparum.
  • the pathogen is a West Nile virus. In a further embodiment, the identified Replikin cycle represents concentration identified in the envelope protein of West Nile virus. In another non- limiting embodiment, the pathogen is a foot and mouth disease virus. In a further embodiment, the identified Replikin cycle represents concentrations identified in the VPl protein of foot and mouth disease virus.
  • the pathogen is an influenza virus.
  • the identified Replikin cycle represents concentrations identified in the pBl gene area of influenza virus.
  • the influenza virus is an HlNl, H2N2, H3N2, H3N8, H5N1, or H9N2 strain of influenza virus.
  • the second time point or time period is up to three years after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about one year after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about six months after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of a pathogen following the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of influenza following the first time point or first time period.
  • the next influenza season is the next winter season in a geographic region following the first time point or time period.
  • the second time point or time period is following the next dry season after the first time point or time period.
  • the second time point or time period is the next season of malaria following the first time point or first time period.
  • the next season is the next rainy season.
  • the identified peak in the cycle of Replikin concentration has a higher Replikin concentration than a chronologically earlier peak in the cycle of Replikin concentration.
  • the identified peak in the cycle of Replikin concentration is significantly higher than the earlier peak.
  • the identified peak is significantly higher than the earlier peak with a p value less than 0.01.
  • the identified peak is significantly higher than the earlier peak with a p value less than 0.001.
  • predicting said expansion of population or said increase in virulence, morbidity, and/or mortality of an isolate of a pathogen comprises identifying a second cycle in the Replikin concentration of a plurality of isolates of a second strain or related strain of pathogen that shares synchrony with said first cycle in the Replikin concentration of said plurality of isolates of said first strain of pathogen and identifying a first peak in the Replikin concentration within the identified first cycle at a first time point or time period and identifying a first peak in the Replikin concentration within the identified second cycle of said second strain of pathogen or related strain of pathogen at a second time point or time period that is similar to said first time point or time period and predicting an expansion of the population or an increase in the virulence, morbidity, and/or mortality of said first strain of pathogen following the first time point or time period.
  • the pathogen is a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or any other infectious agent.
  • said pathogen is an influenza virus.
  • said first strain of influenza is any strain different from said second strain of influenza.
  • said first strain of influenza is H5N1 and said second strain of influenza is H9N2, or vice versa.
  • the strain is any influenza strain and the related strain is any other strain wherein a relationship with said first strain is determined by comparing the Replikin cycles of said strain and said related strain.
  • the strains are related because the Replikin cycles share synchrony.
  • a further non- limiting embodiment of the second aspect of the invention provides a method of predicting an expanding population of a pathogen or an increase in virulence, morbidity, and/or mortality in a pathogen comprising: (1) determining the mean Replikin Count in a plurality of isolates of at least two strains of pathogen at a plurality of successive time points; (2) comparing the mean Replikin Count at at least four successive time points for each strain and identifying at least one cycle of increasing mean Replikin Counts over the at least four time points for each of the at least two strains; (3) identifying at least partial synchrony between the at least one cycle of increasing mean Replikin Counts for each of the at least two strains; and (4) predicting an increase in virulence following in time the increase in mean Replikin Count in the at least one cycle in said at least two strains wherein said at least one cycle in said at least two strains occurs at a corresponding time period.
  • stepwise cycles are identified between successive time points.
  • specific conserved Replikin sequences are identified within the step-wise cycles.
  • Replikin sequences are identified at the peak of a stepwise cycle. The Replikin sequences identified at the peak of a stepwise cycle are useful for developing a vaccine or therapeutic composition of an isolated or synthesized Replikin peptide for use in preventing or treating outbreaks of malaria with relatively higher mortality.
  • the pathogen is influenza.
  • the at least two strains of influenza are H9N2 and H5Nl.
  • Another non-limiting embodiment of the second aspect of the invention provides a method of predicting a contraction or failure of a population of a strain of pathogen, wherein an isolate of said pathogen is isolated at a time point or time period subsequent to a decreasing portion of a Replikin cycle.
  • a further non- limiting embodiment of the second aspect of the invention provides a method for making a vaccine comprising predicting an expanding population of a pathogen or related strain of pathogen or an increase in virulence, morbidity, and/or mortality of a pathogen or a related strain of pathogen and identifying a portion of the structure or genome of said isolated influenza virus to be comprised in a vaccine.
  • a further non- limiting embodiment of the second aspect of the present invention provides an isolated or synthesized portion of the structure or genome of a pathogen wherein said pathogen is predicted to have an expansion of the population of the pathogen.
  • the isolated or synthesized portion is a protein, protein fragment, or peptide comprising a Replikin peptide or a Replikin Peak Gene.
  • the isolated or synthesized portion of the structure or genome of a pathogen consists of one or more Replikin peptides and/or one or more Replikin Peak Genes.
  • the one or more Replikin peptides are conserved during a cycle in Replikin concentration at at least two successive time points or time periods in the cycle.
  • Another non-limiting embodiment of the second aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified as conserved in an isolate of said pathogen from among a plurality of isolates of said pathogen, wherein said isolates are isolated during a cycle in Replikin concentration at at least two successive time points or time periods, and the cycle preferably includes at least two peaks or two troughs.
  • the pathogen is an influenza virus.
  • the Replikin peptide is at least one of HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1),
  • KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4), HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12).
  • the pathogen is a West Nile virus.
  • the Replikin peptide is at least one of KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK (SEQ ID NO: 15), or HNDKRADP AFVCK (SEQ ID NO: 16).
  • the pathogen is a foot and mouth disease virus.
  • the Replikin peptide is at least one of HKQKIIAP AK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18).
  • the pathogen is malaria.
  • the Replikin peptide is at least one of a Replikin peptide identified from at least one of the following accession numbers: ABU43157, CAD49281, CAD49281, or XP001349534.
  • Replikin peptides may be comprised in an immunogenic compound of the invention.
  • a further non- limiting embodiment of the second aspect of the invention provides a computer readable medium having stored thereon instructions which, when executed, cause a processor to perform a method of predicting an expansion of a strain of pathogen or an increase in virulence, morbidity, and/or mortality of a pathogen.
  • 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 pathogen or predicted to increase in virulence, morbidity, and/or mortality, 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.
  • a third non-limiting aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified in an isolate of a pathogen, wherein said isolate is isolated during a rising portion of a cycle in Replikin concentration from among a plurality of isolates of the pathogen, or is isolated at a peak in a cycle in Replikin concentration from among a plurality of isolates of a pathogen, or isolated subsequent to a peak in a cycle in Replikin concentration from among a plurality of isolates of a pathogen.
  • Another non- limiting embodiment of the third aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified as conserved in an isolate of a pathogen from among a plurality of isolates said pathogen, wherein said isolates are isolated during a cycle in Replikin concentration at at least two successive time points or time periods, and the cycle includes at least two peaks or two troughs.
  • the pathogen is a malarial trypanosome.
  • the identified cycle is in the histidine rich protein of P. falciparum.
  • the identified cycle is in the ATP-ase protein of P.
  • the identified cycle is in a Replikin Peak Gene of a trypanosome that causes malaria.
  • the pathogen is a West Nile virus.
  • the identified cycle is in the envelope protein of West Nile virus.
  • the pathogen is a foot and mouth disease virus.
  • the identified cycle is in a VPl protein of a foot and mouth disease virus.
  • the pathogen is an influenza virus.
  • influenza virus is an HlNl, H2N2, H3N2, H3N8, H5N1, or H9N2 influenza virus.
  • the identified cycle is in the neuraminidase or hemagglutinin protein of an influenza virus.
  • a fourth non-limiting aspect of the present invention provides an immunogenic composition comprising a Replikin peptide identified in an isolate of a pathogen, wherein said isolate is isolated during a rising portion of a cycle in Replikin concentration from among a plurality of isolates of said pathogen, or is isolated at a peak in the identified cycle in Replikin concentration from among a plurality of isolates of the pathogen, or is isolated subsequent to a peak in the identified cycle in Replikin concentration from among a plurality of isolates of the pathogen.
  • the immunogenic composition is a vaccine for prevention or treatment of an infection of a pathogen.
  • Another non-limiting embodiment of the present invention provides an antibody to a Replikin peptide identified in an isolate of the pathogen, wherein said isolate is identified during a rising portion of a cycle in Replikin concentration, or is identified at a peak in a cycle in Replikin concentration, or is identified subsequent to a peak in a cycle in Replikin concentration.
  • the pathogen is a West Nile virus.
  • the pathogen is a foot and mouth disease virus.
  • a fifth non- limiting aspect of the invention provides a method of preventing, mitigating, or treating an outbreak of a pathogen comprising predicting an expansion of a strain of pathogen comprising (1) determining a mean Replikin Count and a standard deviation of said mean Replikin Count for a plurality of isolates of a strain of pathogen for a first time period in a first geographic region, (2) determining a Replikin Count of at least one isolate of the same or a related strain of pathogen from a second time period and/or second geographic region wherein said second time period is different from said first time period and/or said second geographic region is different from said first geographic region, and (3) predicting an expansion of said strain of pathogen isolated in said second time period and/or second geographic region if the Replikin Count of said at least one isolate is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period and in said first geographic region; and administering to an animal or a patient a compound
  • said pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or any other kind of infectious agent.
  • said first time period is one year and said first geographic region is a country.
  • said second time period is one year.
  • said second geographic region is a country.
  • the pathogen is influenza virus
  • said first geographic region is China.
  • the pathogen is a malarial trypanosome
  • said first geographic region is India.
  • the pathogen is West Nile virus
  • said first geographic region is a state within the United States.
  • said plurality of isolates of a strain of pathogen for a first time period in a first geographic region is a plurality of isolates from all publicly available sequences in said first time period in said first geographic region.
  • said plurality of isolates is all isolates from a species of animal.
  • said plurality of isolates is all isolates from a particular species of bird such as swans, chickens, falcons, turkeys, ducks, or other domestic or wild birds.
  • said isolated or synthesized portion of the structure or genome of the at least one isolate of pathogen is a protein or protein fragment comprising a Replikin peptide.
  • said protein or protein fragment is a Replikin peptide.
  • said protein or protein fragment comprises a Replikin Peak Gene.
  • said protein or protein fragment is a Replikin Peak Gene.
  • said protein or protein fragment is a Replikin peptide identified within a Replikin Peak Gene.
  • said isolated or synthesized portion of the structure or genome is a nucleic acid encoding a Replikin Peak Gene, a nucleic acid encoding a Replikin peptide or plurality of Replikin peptides within a Replikin Peak Gene, or a nucleic acid encoding a Replikin peptide.
  • the at least one isolate of the same strain of pathogen from a second time period and/or second geographic region is a plurality of isolates from said second time period and/or second geographic region and the Replikin Count of each isolate of the plurality of isolates from said second time period and/or second geographic region is compared separately to said one standard deviation of said mean Replikin Count.
  • an expansion of said strain of pathogen isolated in said second time period and/or second geographic region is predicted if the number of Replikin Counts of said plurality of isolates from said second period and/or said second geographic region that is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period in said first geographic region, is greater than the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is less than said one standard deviation of the mean.
  • the Replikin Count is the concentration of Replikin peptides identified encoded in the genome of an isolate of the pathogen. In a further embodiment, the Replikin Count is the concentration of Replikin peptides identified in the expressed proteins of an isolate of the pathogen. In a further embodiment, the Replikin Count is the concentration of Replikin peptides identified in at least one protein or gene area of an isolate of the pathogen.
  • the gene area is the pBl gene area of the genome of influenza virus, the histidine-rich protein gene area of a malarial trypanosome, the VPl gene area of foot and mouth disease virus, or the envelope protein gene area of West Nile virus.
  • the Replikin Count is the concentration of Replikin peptides identified in at least one protein fragment of an isolate of the pathogen.
  • the Replikin Count is the concentration of Replikin peptides identified in a Replikin Peak Gene of an isolate of the pathogen.
  • the Replikin Peak Gene is identified in the polymerase area of an influenza virus genome.
  • the Replikin Peak Gene is identified in the pBl area of an influenza virus genome. In a further embodiment, the Replikin Peak Gene is identified in the histidine-rich protein area of a malarial trypanosome, the VPl area of a foot and mouth disease virus, or the envelope protein of a West Nile virus.
  • a sixth non-limiting aspect of the present invention provides a method of predicting an expansion of a strain of pathogen comprising
  • the method of predicting further comprises processing the method on a computer.
  • the pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or any other kind of infectious agent.
  • a further non- limiting embodiment of the sixth aspect of the invention provides a method for making a vaccine comprising predicting an expansion of said strain of pathogen isolated in said second time period and/or second geographic region and identifying a portion of the structure or genome of said isolated influenza virus to comprise a vaccine.
  • the at least one isolate of the same strain of pathogen from a second time period and/or second geographic region is a plurality of isolates from said second time period and/or second geographic region.
  • the Replikin Count of each isolate of the plurality of isolates from said second time period and/or second geographic region is compared separately to said one standard deviation of the mean.
  • an expansion of a strain of pathogen isolated in said second time period and/or said second geographic region is predicted if the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period in said first geographic region, is greater than the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is less than said one standard deviation of the mean.
  • an expansion of a strain of influenza virus isolated in said second time period and/or second geographic region is predicted if the ratio of the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is greater than said one standard deviation of the mean, divided by the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is less than said one standard deviation of the mean, is greater than one.
  • a further non- limiting embodiment of the sixth aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified in an isolate of a pathogen predicted to have an expanding population.
  • the Replikin peptides for diagnostic, therapeutic, and/or preventive purposes are conserved over time or across geographic regions.
  • Another non-limiting embodiment of the sixth aspect of the invention provides a method of predicting a contraction or failure of a population of a strain of pathogen, wherein a Replikin Count of at least one isolate of a strain of pathogen from a first time period and/or first geographic region is less than one standard deviation of the mean of the Replikin Count of a plurality of isolates of influenza from a second time period and second geographic region.
  • Another non-limiting embodiment provides a method of predicting a contraction or failure of a population of a strain of pathogen, wherein the number of Replikin Counts of a plurality of isolates from a first time period and/or a first geographic region greater than one standard deviation of the mean of the Replikin Count of a plurality of isolates from a second time period in a second geographic region, is less than the number of Replikin Counts of the plurality of isolates from the first time period and/or the first geographic region that is less than said one standard deviation of the mean.
  • said contraction or failure is predicted if the ratio of the number of Replikin Counts of said plurality of isolates from said first time period and/or said first geographic region that are greater than said standard deviation of the mean, divided by the number of Replikin Counts of said plurality of isolates from said first time period and/or said first geographic region that are less than said standard deviation of the mean, is less than one.
  • a further non- limiting embodiment of the sixth aspect of the invention provides a computer readable medium having stored thereon instructions which, when executed, cause a processor to perform a method of predicting an expansion of a strain of pathogen or the expansion of a virus or organism.
  • 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 pathogen, 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.
  • a seventh non- limiting aspect of the present invention provides an immunogenic composition comprising a portion of the structure or genome of an isolate of a pathogen, wherein said isolate of said pathogen is (1) an isolate having a Replikin Count greater than one standard deviation of a mean Replikin Count of a plurality of isolates of pathogen isolated in a different time period and/or in a different geographical region, (2) an isolate from a first time period and/or geographical region wherein the number of a plurality of isolates from the first time period and/or geographical region having a Replikin Count greater than said one standard deviation of the mean is greater than the number of isolates having a Replikin Count less than said one standard deviation of the mean, (3) isolated during a rising portion of a cycle or a set of two or more synchronous cycles in Replikin concentration from among a plurality of isolates of influenza, and/or (4) isolated at a peak in the identified cycle or set of synchronous cycles in Replikin concentration from among a plurality of isolates of influenza
  • the immunogenic composition is a vaccine for prevention or treatment of an infection of a pathogen.
  • Another non-limiting embodiment provides an antibody to a Replikin peptide identified in an isolate of pathogen predicted to have an increase in virulence, morbidity, and/or lethality or expansion of its population.
  • Figure 1 illustrates cycling between 1986 and 2007 of mean annual Replikin concentration in the histidine rich protein of Plasmodium falciparum for sequences available at www.pubmed.com for isolates from 1986 through 2007.
  • three rising portions of cycles of Replikin concentration and two decreasing portions of cycles of Replikin concentration are observable with peaks at 1987 and 1999.
  • a first rising portion and decreasing portion of a cycle is observed from 1986 to 1995.
  • a second rising portion and decreasing portion of a cycle is observed from 1996 to 2005.
  • a new cycle appears to have begun between 2005 and 2007.
  • the peak of the first rising portion was identified in 1987 with a mean annual Replikin Count of 38.2 and standard deviation of ⁇ 23.5.
  • Figure 2 illustrates that mortality rates per 1000 clinical cases of malaria in humans generally correlate with mean annual Replikin Count in sequences of the P. falciparum ATP-ase enzyme publicly available at www.pubmed.com. Mean annual Replikin Counts of P '. falciparum ATP-ase increased from 1997 to 1998 along with an increase in mortality per malaria case from 1997 and 1998 to 1999. The mean annual Replikin Count of P. falciparum ATP-ase decreased from 1998 to 2006 along with the mortality rates from 1999 to 2005 (consistent mortality data is considered presently available only through 2005). The data for Figure 2 may be seen in Table 6 below. Mortality rates in Figure 2 and Table 6 are recorded as declared by the World Health Organization. See www.who.int.
  • Figure 3 illustrates cycling of mean annual Replikin Count in West Nile virus in correlation with cycling of West Nile virus morbidity.
  • the mean annual Replikin Count of the Envelope Protein of WNV (black) and standard deviation (capped line) is compared to the annual number of human cases in the United States as reported by the Centers for Disease Control (CDC) (gray).
  • Mean annual Replikin Count was analyzed in envelope protein sequences from isolates isolated between 2000 and 2006 and publicly available at www.pubmed.com.
  • the standard deviation of the mean of the Replikin Count of the envelope protein is observed to increase markedly from 2000 to 2001 (p ⁇ 0.001).
  • Figure 3 illustrates two rising portions and one decreasing portion in a cycle of Replikin concentration and two rising portions and one decreasing portion in a cycle of WNV human morbidity, the first rising portion from 2000 to 2003 and the second rising portion from 2004 to 2006/2007.
  • conserveed viral Replikin structures within the envelope protein are observed throughout the illustrated cycles and the relationship between Replikin structure and rapid replication and virulence are observed through time.
  • FIG 4 illustrates cycling in Replikin concentration in the whole genome of foot and mouth disease virus (FMDV) type O isolated between 1999 and 2008 and reported at www.pubmed.com.
  • the data demonstrate that annual Replikin Counts (Mean and Standard Deviation (SD)) for isolates of FMDV type O occurred with two rising portions and one decreasing portion. A first rising portion and a first decreasing portion are observed between 1999 and 2005. A second rising portion is observed beginning in 2005 through 2008. The cycle is presently incomplete since a second trough is not yet observable.
  • SD Standard Deviation
  • Mean annual Replikin Count is further observed to provide advance warning signals (with p ⁇ 0.001) prior to severe FMDV outbreaks in the Middle East, Africa, India, and Asia (including China) in 2008-2009.
  • Replikin cycles are detectable because of repeating conserved virus structures and continuity of the Replikin phenomenon through time.
  • the data in Figure 4 demonstrate that the highest mean annual Replikin Counts over the ten year period reflected in Figure 4 were observed in 2007 and 2008.
  • Figure 5 illustrates cycles of mean annual Replikin Count in influenza sequences from the pBl gene area for isolates isolated between 1993 and 2008 and reported at www.pubmed.com.
  • the mean annual Replikin Count of the pBl gene area of isolates of H9N2 is shown in light gray columns with standard deviation shown above in dark gray columns.
  • the number of poultry flocks reported in Israel with H9N2 infection is provided in white columns.
  • the data illustrate an increase in mean annual Replikin Count that corresponds to an increase in influenza outbreaks in flocks of poultry in Israel between 2000 and 2004.
  • the standard deviation data further emphasize the extent of expanding Replikin Counts within the annual H9N2 influenza population.
  • Figure 6 illustrates synchronous cycles of mean annual Replikin Counts in the pBl gene area of H9N2 and H5N1 influenza isolates. The data represent analysis of sequences of isolates isolated between 1993 and 2008 and reported at www.pubmed.com.
  • annual mean Replikin Count for H9N2 is reported in light gray columns with standard deviation reported above in dark gray columns.
  • Annual mean Replikin Count for H5N1 is reported in black columns with standard deviation reported above in white columns.
  • Figure 6 visibly illustrates synchrony between the H9N2 and H5N1 Replikin Cycles.
  • Figure 7 illustrates cycling in mean annual Replikin Counts in the pB 1 gene area in the three influenza pandemics of the last century. Strain- specific high Replikin Counts accompany each of the three pandemics: 1918, 1957, and 1968. In each case, a first peak is followed by a decline (likely due to immunity in the hosts), then by a second recovery peak and a "rebound" epidemic. The probability is very low that these correlations are due to chance, since they are specific for each strain, specific for each of the three pandemic years out of the century, specific for each post-pandemic decline, and specific for each rebound epidemic.
  • the data supports a prediction of an increase in virulence and morbidity following a peak in a cycle of mean annual Replikin Count in influenza virus.
  • an increase in virulence and morbidity was accompanied by increased mortality in the pandemics of the 20 th Century.
  • Figure 8 illustrates the same data as Figure 7 but is expanded in size for better viewing of the data for individual years.
  • Figure 8(A-C) illustrates cycles in Replikin Count in strains of influenza related to outbreaks of influenza between 1917 and 2007.
  • the data illustrate an increase in Replikin Count before and accompanying each influenza A pandemic and outbreak since 1918 and low Replikin Counts during quiescent periods of influenza A infection and continually in non-lethal Influenza B.
  • the graph provides annual Replikin Counts from 1917-2007 for all Replikin Peak Genes isolated in silico in the pBl gene area of influenza strains having amino acid or nucleic acid sequences publicly available at PubMed.
  • Human Influenza A strains are (1) HlNl (thin solid line), (2) H2N2 (long-short- long dashed line), (3) H3N2 (medium dashed line), and (4) H5N1 (long dashed line). H5N1 strains isolated from chicken are illustrated by a short dashed line. The total number of sequences analyzed for the data (N) is 14,227.
  • pandemics, epidemics and outbreaks are the 1918 HlNl pandemic, the 1930's HlNl epidemic, the 1957 H2N2 pandemic, the 1968 H3N1 pandemic, the 1977-78 H3N2 outbreaks and the H5N1 outbreaks of 2001-2004 and 2007.
  • a 1997 outbreak of H5N1 is not shown in Figures 7 and 8.
  • pandemics, epidemics and outbreaks are associated with Replikin Counts of four or above in the RPG of influenza strains. Over the same period, constant low Replikin Counts of less than four may be observed during quiescent non-lethal periods of influenza A infections and low Replikin Counts of less than four may be observed in non-lethal Influenza B.
  • Figure 9 illustrates an immune response with protective effect following administration of a vaccine comprising a mixture of peptides of SEQ ID NO(s): 1-12 to chickens later challenged with Low-Path H5N1 virus.
  • Eighty chickens were divided into four groups of twenty chickens each on a first day after hatch.
  • Group 1 was a negative control subjected to neither vaccination nor infection with the Low-Path H5N1 virus.
  • Group 2 was a vaccine control subjected to vaccination intranasally on day 1 after hatch, intraocularly on day 7 after hatch, and via spray inhalation on day 14 after hatch.
  • Group 2 was not subjected to infection with the Low- Path H5N1 virus.
  • Group 3 was subjected to vaccination on the same schedule as Group 2 and Low-Path H5N1 was introduced to the cleft palate of the chickens on day 28.
  • Group 4 was a challenged control that was not vaccinated but was infected with H5N1 on day 28 via the cleft palate.
  • days 7, 14, and 21 between six and nine chickens from each group were tested for serum production of antibodies against H5N1 virus. The data from the serum antibody tests are contained in Table 15 and illustrated in Figure 9.
  • Figure 9 illustrates that only one of seven (14%) chickens tested in Group 3 (vaccinated and challenged with virus) was observed to produce antibody in serum seven days after challenge while four of seven chickens (57%) tested in Group 4 (not vaccinated but challenged) were observed to produce antibody in serum seven days after challenge.
  • Figure 9 further illustrates that only three of six chickens (50%) tested in Group 3 (vaccinated and challenged) were observed to produce antibody in serum fourteen days after challenge while seven of nine (78%) chickens tested in Group 4 (not vaccinated but challenged) were observed to produce antibody in serum fourteen days after challenge.
  • Figure 9 further illustrates that two of seven (29%) chickens tested in Group 3 were observed to produce antibody in serum twenty-one days after challenge while three of nine (33%) chickens tested in Group 4 were observed to produce antibody in serum twenty-one days after challenge.
  • the vaccine control Group 2 six of six (100%) chickens tested were observed to produce antibody in serum fourteen days after challenge while no chickens tested on days 7 or 21 were observed to produce antibody in serum.
  • the negative control Group 1
  • Example 10 In combination with data provided in Example 10 demonstrating that no H5N1 virus was observed by PCR detection excreted in feces or saliva from chickens in Groups 1, 2, and 3 (negative control, vaccine control, a vaccine/challenge groups, respectively) and that H5N1 virus was observed by PCR detection excreted in feces and saliva for all chickens in Group 4 (challenge control), one of ordinary skill in the art concludes that chickens in the vaccinated groups (Groups 2 and 3) produced an immune response to the vaccine and that chickens in the vaccinated and challenged group (Group 3) were provided a measure of protection from the Low-Path H5N1 challenge on day 28 following hatch.
  • a "Replikin cycle” or "a cycle of Replikin concentration” or “a cycle of Replikin Count” means Replikin concentrations of a plurality of isolates of a species of virus or organism wherein at least four of said plurality of isolates are isolated at successive time points or in successive time periods, wherein a Replikin concentration of a second individual isolate or second mean of a plurality of isolates at a second time point or time period is higher than a Replikin concentration of a first individual isolate or a first mean of a plurality of isolates at a first time point or time period, a Replikin concentration of a third individual isolate or a third mean of a plurality of isolates at a third time point or time period is lower than the Replikin concentration at a second time point or time period, and a Replikin concentration of a fourth individual isolate or fourth mean of a plurality of isolates at a fourth time point or time period is higher than the Replikin concentration at a third time point
  • any rising portion is predictive of an expansion in population or an increase in virulence, morbidity, and/or mortality of a pathogen in hosts and any decreasing portion is predictive of a contracting population or a decrease in virulence, morbidity, and/or mortality of pathogen in hosts.
  • a "step-wise" cycle is any set of cycles wherein a first Replikin cycle peak in time is lower than a second Replikin cycle peak in time or a first Replikin cycle peak in time is higher than a second Replikin cycle peak in time.
  • a step-wise cycle also occurs when successive peaks are observed to move lower.
  • a step-wise cycle may also be observed if successive troughs move higher or lower. Step-wise cycles provide additional predictive capacity for predictions of expansion or contraction of a population.
  • a Replikin cycle that is "synchronous,” shares “synchrony,” or any other related word, with another Replikin cycle means a cycle having a period or phase or any portion of the cycle that is similar to some period, phase, or portion of the cycle wherein said similarity may be determined visually, mathematically, statistically, or by any other method known or hereinafter known by one of skill in the art.
  • Synchronous cycles do not necessarily share portions that arise or occur at exactly the same time. Synchronous cycles in related pathogens will at times be shifted by some measure of time from one another and may shift in time from one another in any portion of either cycle.
  • a portion of a Replikin cycle "corresponds" in time with another Replikin cycle if there is a similarity between the portions of the cycle. Any correspondence need not be exact.
  • a "rising portion" of a Replikin cycle means the Replikin concentration of an isolate or the mean Replikin concentration of a plurality of isolates, wherein the isolate or isolates were isolated at a time point or time period of the Replikin cycle where the trend of Replikin concentration in the Replikin cycle is increasing from at least a first time point or time period to at least a second time point or time period. Additionally, the rising portion may include a peak.
  • a "decreasing portion" of a Replikin cycles means the opposite of a rising portion, wherein a decreasing portion may include a trough.
  • a "peak" in a Replikin cycle means a second time point or time period within a Replikin cycle, wherein the Replikin concentration at a first time point or time period sequentially preceding the second time point or time period is lower than the Replikin concentration at the second time point or time period, and the Replikin concentration at a third time point or time period sequentially following the second time point or time period is lower than the Replikin concentration at the second time point or time period.
  • a peak may include a general region of a cycle that is generally higher than a sequentially preceding region and generally higher than a sequentially following region rather than an exact time point or time period.
  • a "trough" in a Replikin cycle means the opposite of a peak in a
  • Replikin Count Expansion Index or “RCE Index” is the number of Replikin Counts of a plurality of isolates from a first time period and/or first geographic region that are greater than one standard deviation of the mean of the Replikin Count of a plurality of isolates isolated in a second time period and in a second geographic region, divided by the number of Replikin Counts of said plurality of isolates from said first time period and/or said first geographic region that are less than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said second time period in said second geographic region.
  • An RCE or RCVE Index predicts the expansion of a pathogen in a particular region and/or time period if the ratio of the RCE or RCVE Index is greater than one.
  • An RCE or RCVE Index predicts the contraction, retraction, reduction, or failure of a pathogen in a particular region and/or time period if the ratio of the RCE or RCVE Index is less than one.
  • An RCE or RCVE Index predicts equilibrium between expansion and contraction in the pathogen population if the ratio of the RCVE Index is equal to one.
  • a "related pathogen” means a first pathogen that is of the same species, genus, or family as a second pathogen for which a relationship is known now or hereafter by one of skill in the art.
  • a related pathogen may be a first pathogen that is of the same species but a different strain from a second pathogen.
  • a related pathogen may be a first pathogen that is the same or different species from a second pathogen and shares a host, reservoir, or vector with the second pathogen.
  • first pathogen Even if a first pathogen is not of the same species, genus, or family as a second pathogen, the first pathogen is related to the second pathogen if the first pathogen has a Replikin cycle that is synchronous with the Replikin cycle of the second pathogen.
  • a related pathogen may be within the same family as a first pathogen.
  • a related pathogen may be within the same genus as a first pathogen.
  • a related pathogen may be within the same species as a first pathogen.
  • a related pathogen may be within the same strain as a first pathogen.
  • time periods are any two time periods or time points that may be differentiated from each other.
  • an isolate of an organism or virus isolated during the year 2004 may be considered to be isolated in a different time period than an isolate of the same organism or virus isolated during the year 2005.
  • an isolate of an organism or virus isolated in May 2004 may be considered to be isolated in a different time period than an isolate of the same organism or virus isolated in June 2004.
  • Replikin concentrations of different isolates one may use comparable time periods.
  • an isolate from 2004 may be compared to at least one other isolate from some other year such as 2002 or 2005.
  • an isolate from May 2004 may be compared to at least one isolate from some other month of some year, for example, an isolate from December 2003 or from June 2004.
  • an "isolate” is any virus or organism isolated from a natural source wherein a natural source includes, but is not limited to, a reservoir of an organism or virus, a vector of an organism or virus, or a host of an organism or virus.
  • Obtaining,” “isolating,” or “identifying” an isolate is any action by which an amino acid or nucleic acid sequence within an isolate is obtained including, but not limited to, isolating an isolate and sequencing any portion of the genome or protein sequences of the isolate, obtaining any nucleic acid sequence or amino acid sequence of an isolate from any medium, including from a database such as PubMed, wherein the nucleic acid sequence or amino acid sequence may be analyzed for Replikin concentration, or any other means of obtaining the Replikin concentration of a virus isolated from a natural source at a time point or within a time period.
  • an earlier-arising virus or organism or a virus or organism isolated at “an earlier time point” or during “an earlier time period” is a specimen of a virus or organism collected from a natural source of the virus or organism on a date prior to the date on which another specimen of the virus or organism was collected from a natural source.
  • a “later-arising” virus or organism or a virus or organism isolated at a "later time point" or during a “later time period” is a specimen of a virus or organism collected from a natural source of the virus (including, but not limited to, a reservoir, a vector, or a host) or a natural source of the organism on a date subsequent to the date on which another specimen of the virus or organism was collected from a natural source.
  • the "next virulence season" of a pathogen is a time period in which an increase in morbidity of a pathogen is expected based on seasonal changes, such as a change from summer to winter or a change from a wet season to a dry season, wherein the pathogen was experiencing less morbidity in a previous sequential time period prior to the time period in which the increase in morbidity is expected to occur.
  • the term "dry season” or "winter season” with respect to malaria describes a season in any geographical region wherein mosquito activity (including feeding and reproduction) is significantly less than during other times of the year.
  • a peak in a Replikin cycle before a dry season or winter season predicts an increase in virulence, morbidity, and/or mortality in malaria in the following rainy season or summer season when mosquito activity is greatest.
  • trypanosome that causes malaria means any Plasmodium species or other species known now or hereafter to cause malaria.
  • Malarial trypanosomes include but are not limited to Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.
  • a "Replikin Peak Gene (RPG)" (or sometimes a Replikin Peak Gene Area-RPGA) means a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has a highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) when compared to other segments or named genes of the genome.
  • RPG Replikin Peak Gene
  • Replikin Peak Gene or sometimes a Replikin Peak Gene Area-RPGA
  • a whole protein or gene or gene segment that contains the amino acid portion having the highest concentration of continuous Replikin sequences is also referred to
  • An RPG may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine.
  • an RPG may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine or may likewise have neither a terminal lysine nor a terminal histidine so long as the terminal portion of the RPG contains a Replikin sequence or Replikin sequences defined by the definition of a Replikin sequence, namely, an amino acid sequence having about 7 to about 50 amino acids comprising:
  • an RPG may include the protein or protein fragment that contains an identified RPG.
  • a Replikin Count in the RPG may be used to track changes in virulence and lethality.
  • the RPG may be used as an immunogenic compound or as a vaccine.
  • Whole proteins or protein fragments containing RPGs are likewise useful for diagnostic, therapeutic and preventive purposes, such as, for example, to be included in immunogenic compounds, vaccines and for production of therapeutic or diagnostic antibodies.
  • a "Replikin sequence” is an amino acid sequence of 7 to about 50 amino acids comprising or consisting of a Replikin motif wherein the Replikin motif comprises: (1) at least one lysine residue located at a first terminus of said isolated peptide and at least one lysine residue or at least one histidine residue located at a second terminus of said isolated peptide;
  • Replikin sequence For the purpose of determining Replikin concentration, a Replikin sequence must have a lysine residue at one terminus and a lysine or a histidine residue at the other terminus. For diagnostic, therapeutic, and preventive purposes, a Replikin sequence may or may not have defined termini.
  • the term "Replikin sequence" can also refer to a nucleic acid sequence encoding an amino acid sequence having about 7 to about 50 amino acids comprising:
  • amino acid sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine.
  • peptide or “protein” refers to a compound of two or more amino acids in which the carboxyl group of one amino acid is attached to an amino group of another amino acid via a peptide bond.
  • isolated or “synthesized” peptide or biologically active portion thereof refers 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 “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.
  • outbreak is an increase in virulence, morbidity, and/or mortality in a pathogenic disease or an expansion in the population of pathogen as compared to a baseline of an earlier occurring epidemiological pattern of infection in the same disease.
  • One of ordinary skill in the art will know how to determine an epidemiological baseline.
  • morbidity is the number of cases of a disease caused by the virus, either in excess of zero cases in the past or in excess of a baseline of endemic cases in the past. Therefore the baseline of endemic cases, in epidemiological terms, may, for example, relate to whether none or some cases were present in a geographic region in the immediate past.
  • the past in epidemiological terms, may mean more than one year and can mean several years or more as understood by one of ordinary skill in the art. The past may also mean less than one year as determined by one of ordinary skill in the art.
  • the baseline often reflects an annual recurrence or expansion and contraction of these diseases.
  • "expanding" pathogen or population of pathogen means an increase in virulence, morbidity, and/or lethality of a pathogen (e.g., strain of P. falciparum, a strain of influenza virus, etc.) and/or an expansion of the population of a pathogen (e.g., strain of P. falciparum, a strain of influenza virus, etc.) wherein said expansion includes an increase in the occurrence of the pathogen in a given geographic region or in a given time period or both, or a spreading of the occurrence of the pathogen to another geographic region.
  • a pathogen e.g., strain of P. falciparum, a strain of influenza virus, etc.
  • an increase or decrease in "virulence” includes an increase or decrease in virulence, morbidity, lethality, host mortality, and/or expansion of a pathogen, such as an influenza virus.
  • geographic region or similar term is an area differentiated from another area by space. For example, China is a geographic region that may be differentiated from the geographic region of India. Likewise a geographic region may be a town, or city, or continent or any area differentiable from another area. A geographic region may encompass the entire earth if an isolate or plurality of isolates from a given time period is compared to isolates from another time period over the entire earth and no geographic differentiation is undertaken for the comparison.
  • 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 about 50 amino acid residues with a lysine residue on one end and a lysine residue or a histidine residue on the other end wherein the peptide comprises (1) a lysine residue six to ten residues from another lysine residue, (2) a histidine residue, (3) and 6% or more lysine residues, or wherein a Replikin sequence is a nucleic acid that encodes a Replikin peptide sequence.
  • continuous Replikin sequences means a series of two or more Replikin sequences that are overlapped and/or are directly covalently linked.
  • the present invention provides methods of preventing, mitigating, and treating outbreaks of a pathogen by predicting an expansion of a strain of pathogen or an increase in the virulence, morbidity, and/or lethality of a strain of pathogen as compared to another strain of the same pathogen and administering to an animal or patient a compound comprising an isolated or synthesized portion of the structure or genome of the pathogen to mitigate, prevent, or treat the predicted outbreak of the pathogen.
  • the present invention further provides methods of predicting an expanding population of a pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen comprising identifying a cycle in the Replikin Count in a protein fragment, protein, genome fragment, or genome of a pathogen and predicting an expansion of the population of the pathogen or an increase in the virulence, morbidity, and/or mortality of the pathogen within the identified cycle in Replikin Count.
  • An increase in the virulence, morbidity, or mortality of a pathogen relative to the virulence, morbidity, and/or mortality of another pathogen of the same species may be predicted by identifying a peak in a cycle or cycles in the concentration of Replikin sequences in the pathogen and predicting an expansion of the population of the pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen of the same or a related species isolated subsequent to the peak.
  • a Replikin cycle is a cycle in the concentration of Replikin sequences identified in at least four isolates of a species of virus or organism isolated at successive times where (1) the concentration in the first isolate-in-time is higher than the concentration in the second isolate-in-time, the concentration in the third isolate-in-time is higher than the concentration in the second isolate-in-time, and the concentration of the fourth isolate -in-time is lower than the concentration in the third isolate-in-time, or (2) the concentration in the first isolate -in-time is lower than the concentration in the second isolate-in-time, the concentration in the third isolate-in-time is lower than the concentration in the second isolate -in-time, and the concentration of the fourth isolate-in-time is higher than the concentration in the third isolate -in- time.
  • an increase in virulence, morbidity, and/or mortality of a pathogen may be predicted for a pathogen arising during a rising portion of the cycle or subsequent to the peak of a cycle.
  • An expanding population may represent an increase in population in a region or expansion from one region into another region.
  • Replikin Counts may represent individual isolates, or mean Replikin Counts of groups of isolates from a given region and/or time period.
  • step-wise cycles may be identified between successive time points.
  • specific conserved Replikin sequences are identified within the step-wise cycles.
  • An increase in virulence, morbidity, or mortality of a pathogen may be determined using the methods of the invention in any pathogen or infectious agent where a concentration of Replikins may be determined in the genome, a genome fragment, another nucleic acid sequence, a protein, a protein fragment, or other amino acid sequence from the pathogen.
  • a pathogen may be malaria, West Nile virus, foot and mouth disease virus, porcine circovirus, porcine respiratory and reproductive syndrome virus, taura syndrome virus, white spot syndrome virus, tomato leaf curl virus, bacillus anthracis, small pox virus, human immunodeficiency virus, Sindbis virus, hepatitis virus, staphylococcus, legionella, human papilloma virus, Helicobacter, Acetobacter, Aerobacter, Brivebacterium, Clostridium, Erinia, Esheria, Klebsiealla, Maemophilus, Mycoplasma, Psuedomonas, Salmonella, Candida, Entamoeba, or any other form of infectious agent including viruses, bacteria, protozoa, fungi, or other infectious agent.
  • Any Replikin sequence, Replikin Peak Gene, or protein fragment containing a Replikin sequence or Replikin Peak Gene identified in a strain of pathogen that is predicted to have an increase in virulence, morbidity, or mortality may be isolated and/or synthesized as a diagnostic, therapeutic, or prophylactic agent to mitigate the predicted outbreak of the pathogen.
  • a cycle of Replikin concentration or "Replikin cycle" of a trypanosome may be seen in Figure 1. Cycles of Replikin concentrations in West Nile virus, foot and mouth disease virus, and influenza virus may be seen in Figures 3-6, respectively.
  • a Replikin cycle is identified by initially isolating at least four isolates or groups of isolates from at least four time points or time periods, for example, an isolate or group of isolates may be obtained in 1999, 2001, 2002, and 2004, or may be obtained in January, May, September, and December of a given year. Isolates may be obtained from more than four time points or time periods and precision of a Replikin Cycle generally will improve with increases in the number of isolates per time point or time period and with increases in the number of time points or time periods. The Replikin Count of the genome or expressed proteins of each isolate is determined.
  • Replikin Count may be determined in a Replikin Peak Gene, in the entire genome, in a particular gene or gene segment, or in a particular protein or protein fragment of each of the isolates. Mean Replikin Count for a given time point or given time period is determined if a plurality of isolates has been obtained for the given time point or given time period. Replikin Count may then be analyzed per unit time.
  • a cycle in Replikin concentration is identified by four time points or time periods, where the Replikin Count at a second time point or time period is higher than at first time point or time period, the Replikin Count at a third time point or time period is lower than at second time point or time period, and Replikin Count at a fourth time point or time period is higher than at the third time point or time period; or where the Replikin Count at a second time point or time period is lower than at first time point or time period, the Replikin Count at a third time point or time period is higher than at second time point or time period, and Replikin Count at a fourth time point or time period is lower than at the third time point or time period.
  • a peak in a Replikin cycle is identified within the cycle at a second time point or time period within a Replikin cycle, wherein the Replikin concentration at a first time point or time period sequentially preceding the second time point or time period is lower than the Replikin concentration at the second time point or time period, and the Replikin concentration at a third time point or time period sequentially following the second time point or time period is lower than the Replikin concentration at the second time point or time period.
  • a peak may include a general region of a cycle that is generally higher than a sequentially preceding region and generally higher than a sequentially following region rather than an exact time point or time period.
  • a trough in a Replikin cycle is identified within the cycle is identified within the cycle at a second time point or time period within a Replikin cycle, wherein the Replikin concentration at a first time point or time period sequentially preceding the second time point or time period is higher than the Replikin concentration at the second time point or time period, and the Replikin concentration at a third time point or time period sequentially following the second time point or time period is higher than the Replikin concentration at the second time point or time period.
  • troughs may be identified as a general region of a cycle that is generally lower than a sequentially preceding region and generally lower than a sequentially following region rather than an exact time point or time period.
  • Replikin peptides of the invention identified at a peak of the Replikin cycle include Replikin peptides identified at or near the peak of the Replikin cycles including prior to and subsequent to the precise point of the peak.
  • a rising portion of a Replikin cycle is any point at which the trend of Replikin concentration in the Replikin cycle is increasing from at least a first time point or time period to at least a second time point or time period and can include a peak.
  • an increase in virulence, morbidity, or mortality may be predicted following a rising portion or peak in a Replikin cycle.
  • the invention now provides methods of tracking and predicting tracks of pathogens as they increase in virulence, expand in population within a region or into a region, or increase in morbidity or mortality by monitoring changes in Replikin concentration.
  • Replikins analysis provides the skilled artisan with information on population expansion, and increases in virulence, morbidity, and mortality months before or at the very beginning of an outbreak. This information is clearly important for the time needed to organize public health responses, including the testing and administration of specific vaccines.
  • the importance of prior information concerning pathogenic outbreaks may be analogized to the savings of life and property that have resulted from advance warning of hurricanes since information from weather satellites has become available.
  • the present invention also provides a method of predicting an expansion of a strain of pathogen by (1) determining a mean Replikin Count and a standard deviation of the mean Replikin Count for a plurality of isolates of a strain of pathogen for a first time period in a first geographic region; (2) determining a Replikin Count of at least one isolate of the same or a related strain of pathogen from a second time period and/or second geographic region wherein the second time period is different from the first time period and/or the second geographic region is different from the first geographic region; and (3) predicting an expansion of the strain of pathogen isolated in the second time period and/or second geographic region, if the Replikin Count of the at least one isolate is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in the first time period and in the first geographic region.
  • At least one isolate of the same or related strain of pathogen from a second time period and/or second geographic region may be a plurality of isolates from the second time period and/or second geographic region.
  • the Replikin Count of each isolate of the plurality of isolates from the second time period and/or second geographic region is compared separately to one standard deviation of the mean.
  • An expansion of pathogen isolated in the second time period and/or second geographic region may also be predicted if the number of Replikin Counts of a plurality of isolates from the second period and/or second geographic region that is greater than one standard deviation of the mean is greater than the number of Replikin Counts of said plurality of isolates from the second period and/or second geographic region that is less than one standard deviation of the mean.
  • the method may also employ a ratio of the number of Replikin Counts that are greater than one standard deviation of the mean divided by the number of Replikin Counts that are less than one standard deviation of the mean. The ratio is called a Replikin Count Expansion Index (RCE Index).
  • Another way to determine the RCE Index is to divide the percent of Replikin Counts in a plurality of isolates of influenza virus grouped by time and/or region that are higher than one standard deviation of the mean by the percent of Replikin Counts that are lower than one standard deviation of the mean.
  • An RCE Index may be used to quantify the future risk of an outbreak of pathogen by tracking Replikin Counts in strains of pathogen over time.
  • the mean Replikin Count of the plurality of isolates from the first time period and geographic region may be considered a control.
  • a control population preferably has a relatively large number of isolates with a relatively small variability in the Replikin Count of the isolates but any population may be deemed a control when a comparison between the control and a related isolate or plurality of isolates is desired.
  • a control may be related to the population that is being studied. For example, if an infection in a bird species, such as swans, is being studied, the control may be something closely related, such as chickens, wherein isolates from chickens may be relatively numerous (if available) and relatively stable (if possible) wherein stability in Replikin Count through the population demonstrates a level of equilibrium between the expansion and contraction of the strain or a related strain of influenza virus in chickens.
  • a control may reflect a highest number of isolates reported in a year or in several years in a geographic area.
  • An expansion of a strain of pathogen may be determined using the methods of the invention in any pathogen or infectious agent where a concentration of Replikins may be determined in the genome, a genome fragment, another nucleic acid sequence, a protein, a protein fragment, or other amino acid sequence from the pathogen.
  • a pathogen may be malaria, West Nile virus, foot and mouth disease virus, influenza virus, porcine circovirus, porcine respiratory and reproductive syndrome virus, taura syndrome virus, white spot syndrome virus, tomato leaf curl virus, bacillus anthracis, small pox virus, human immunodeficiency virus, Sindbis virus, hepatitis virus, staphylococcus, legionella, or any other form of infectious agent including viruses, bacteria, protozoa, fungi, or other infectious agent.
  • Any Replikin sequence, Replikin Peak Gene, or protein fragment containing a Replikin sequence or Replikin Peak Gene identified in a strain of pathogen that is predicted to have an increase in virulence, morbidity, or mortality may be isolated and/or synthesized as a diagnostic, therapeutic, or prophylactic agent to mitigate the predicted outbreak of the pathogen.
  • the present invention further provides the opportunity to identify Replikin sequences (including nucleic acid sequences and peptide sequences) for diagnostic, therapeutic, or preventive purposes (such as the construction of vaccines and other pharmaceuticals).
  • the present invention contemplates, for example, Replikin peptides identified within a pathogen where the pathogen is predicted to have an expanding population or a higher virulence, morbidity, and/or mortality than another pathogen of the same or a related species based on the predictive methods of the invention.
  • Replikin peptides identified in an isolate of a pathogen wherein said isolate is isolated during a rising portion of a cycle in Replikin concentration among a plurality of isolates of the pathogen or is isolated at a peak in a cycle in Replikin concentration among a plurality of isolates of the pathogen, are useful for diagnostic, therapeutic, and preventive purposes.
  • a Replikin peptide identified in the genome of an isolate identified in a rising portion of a cycle in Replikin concentration or identified at a peak in a cycle in Replikin concentration is useful as a peptide to stimulate the immune system of a human or animal to produce an immune response against infection by the pathogen or to produce antibodies against a pathogen predicted to have higher virulence, morbidity, and/or mortality.
  • antibodies against these pathogens are useful for diagnosing the more highly virulent or mortal disease in a subject or useful as therapies against the infection either as a prophylactic or after onset of the infection.
  • Replikin peptides identified during a rising portion in Replikin concentration in a Replikin cycle or identified at or near a peak in Replikin concentration in a Replikin cycle that are conserved during the rising portion of the Replikin cycle are useful as compounds for diagnostic, therapeutic, and preventive purposes.
  • Conservation of the Replikin peptides during a rise in virulence, morbidity, and/or mortality provides targets that are more constant and likely more involved in the mechanisms of rapid replication that provide the predicted increase in virulence, morbidity, and/or mortality.
  • these conserved Replikin peptides are of use as compounds or in compositions for stimulating the immune system of a subject to produce an immune response, an antibody response, and/or a protective effect in the subject.
  • Replikin peptides identified and isolated using the methods of the invention include influenza peptides such as HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1), KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4), HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 1),
  • the invention further contemplates use of the Replikin peptide as immunogenic compositions and contemplates the immunogenic compositions as vaccines, including vaccines that provide an immune response, vaccines that provide a humoral immune response, vaccines that provide an antigenic immune response, and vaccines that provide a protective effect.
  • the invention additionally contemplates an antibody to the Replikin peptides of the invention.
  • High Replikin Counts and RPGs have been shown to be related to rapid replication, viral outbreaks, epidemics, morbidity and host mortality, for example, in influenza virus strains, including H5N1, in SARS corona virus, in shrimp taura syndrome virus, and in foot and mouth disease virus.
  • Replikin sequences identified at or near the peak of the Replikin cycle or during a rising portion of the Replikin cycle in a pathogen are appropriate peptides for diagnostics, vaccines, and other treatments.
  • Replikin sequences are chemically defined, the sequences may be synthesized by organic chemistry rather than biological techniques, and thus are potentially more specific, more reproducible and more reliable.
  • the chemically defined Replikin sequences identified by Applicants are likewise potentially freer from adverse reactions that are characteristic of biologically derived vaccines and antibodies.
  • One aspect of the present invention provides methods of preventing, mitigating, or treating pathogenic outbreaks predicted through analysis of cycles of Replikin Counts or through analysis of controls using mean Replikin Counts and standard deviation (e.g., Replikin Count Expansion Index). For example, advance information concerning Replikin peptides and Replikin
  • Peak Genes in expanding strains of a pathogen allows for the rapid production of specific effective synthetic vaccines using one, or a combination, of Replikin peptides or using Replikin Peak Genes.
  • Such synthetic vaccines have been demonstrated in rabbits, chickens, and shrimp. See, e.g., Examples 6 and 7 of U.S. Appln. Ser. No. 11/355,120, filed February 16, 2006 and Example 2 of U.S. Appln. Ser. No. 12/108,458, filed April 23, 2008.
  • a mixture of Replikin peptides administered orally to shrimp provided up to a 91% protective effect for shrimp challenged with taura syndrome virus.
  • Taura syndrome virus is an often lethal rapidly- replicating pathogen that has a significant negative impact on the shrimp industry.
  • Example 10 For example, as may be seen in Example 10 below, three of six vaccinated chickens, when inoculated with H5N1 virus, produced no measurable amount of antibodies against H5N1 in their serum. Instead, the virus was apparently blocked by mucosal immunity from even entering the chickens' blood stream. For those three chickens in which a serum immune response was measured (that is, virus entered the host and was presented to antibody generating cells), the vaccine additionally provided a protective effect against replication of the virus in the chickens' system (no virus was excreted in the feces or saliva of the chickens). As such, mucosal immunity, in addition to other immunities, is an important aspect of the immunity imparted by Replikin-based vaccines.
  • Cyclic increases in Replikin concentration in the genome can be a mechanism of expansion of a pathogen into a territory.
  • the Replikin concentration in each Replikin Peak Gene of each Replikin cycle in an expanding population apparently may build on the previous one.
  • repeated analyses of cyclic changes in a virus' Replikin structure is useful to bring current the targets for the chemical synthesis of Replikin vaccines having a best fit for emerging pathogens having increased virulence, morbidity, and/or lethality.
  • These strain-specific vaccines may be manufactured in seven days as have been demonstrated with a 91% protection of shrimp against the lethal taura syndrome virus. See, e.g., U.S. Appln. Ser. No. 12/108,458, filed April 23, 2008 (incorporated herein in its entirety by reference).
  • the present invention provides methods of predicting an expansion of the population of a trypanosome that causes malaria or an increase in the virulence, morbidity, and/or mortality of a trypanosome that causes malaria as compared to another trypanosome of the same species or a related species.
  • An expanding population or increase in virulence, morbidity, and/or mortality of a trypanosome that causes malaria may be predicted by identifying a cycle of Replikin concentration among a plurality of isolates of the species of trypanosome and identifying a rising portion or peak in that cycle.
  • An increase in virulence, morbidity, and/or mortality is predicted following the time point or time period when the rising portion or peak is identified.
  • An expanding population may represent an increase in population in a region or expansion from one region into another region.
  • a further non-limiting embodiment of one aspect of the invention provides a method of predicting an increase in morbidity and mortality in malaria comprising: (1) determining the mean Replikin Count in a plurality of isolates of a malarial trypanosome at a plurality of successive time points; (2) comparing the mean Replikin Count at at least four successive time points and identifying at least one cycle of increasing mean Replikin Counts over the at least four time points; and (3) predicting an increase in morbidity and/or mortality following in time the increase in mean Replikin count in at least one of said cycles.
  • step-wise cycles are identified between successive time points.
  • FIG. 1 illustrates cycling between 1986 and 2007 of annual mean Replikin concentration in the histidine rich protein of Plasmodium falciparum. P. falciparum is a trypanosome that is most commonly associated with malaria. Cycles are observable with peaks in 1987 and 1999. A new cycle appears to have begun between 2005 and 2007.
  • changes in malaria virulence and mortality may be predicted by identifying a peak within an identified cycle in the Replikin concentration of isolates of a plurality of the trypanosome and predicting an increase in the virulence, morbidity, and/or mortality of a trypanosome of the same species isolated at a time point or time period subsequent to the time point or time period of the identified peak in the cycle of Replikin concentration.
  • morbidity data is not reflected in the analysis of malaria in Figure 1 and is also not contained in Figure 2, which compares Replikin Count in the ATP-ase protein of P.
  • Cyclic increases in Replikin concentration in the genome can be a mechanism of expansion of an infectious organism into a territory.
  • the Replikin concentration in each Replikin Peak Gene of each Replikin cycle apparently builds on the previous one.
  • this build-up probably occurs during winter seasons, dry seasons, or otherwise dormant periods.
  • repeated analyses of cyclic changes in the organism's Replikin structure is useful to bring current the targets for the chemical synthesis of Replikin vaccines having a best fit for emerging pathogens having increased virulence, morbidity, and/or mortality.
  • strain-specific vaccines may be manufactured in seven days as has been demonstrate with a 91% protection of shrimp against the lethal taura syndrome virus. See, e.g., U.S. Appln. Ser. No. 12/108,458, filed April 23, 2008 (incorporated herein in its entirety by reference).
  • the Replikin cycle may be identified in any trypanosome that causes malaria. For example, it may be identified in the genome of a trypanosome, including P. falciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.
  • the Replikin cycle may likewise be identified in the histidine rich protein or in the ATP-ase protein, including in these proteins in P. falciparum.
  • the Replikin cycle may likewise be identified in a Replikin Peak Gene of a trypanosome that causes malaria.
  • Replikin sequences were identified as conserved sequences in the histidine-rich protein of malaria in the rising portion and peak of the illustrated Replikin cycle. Such sequences are useful as diagnostic and therapeutic compounds for virulent malaria infections.
  • the sequences are useful in the production of immunogenic compounds including vaccines and may be comprised in immunogenic therapies including vaccines.
  • Replikin peptides identified in the ABU43157 isolate in 2007 are available as a diagnostic, therapeutic, or preventive compounds or compositions of the invention because they were identified in a rising portion of a Replikin cycle. See Figure 1 and Table 5.
  • Replikin peptides identified in the 1999 isolate at accession number CAD49281 are likewise Replikin peptides of the invention. The 1999 isolate is present at the peak of a Replikin cycle, as such, Replikin peptides identified in the isolate reported at CAD49281 may be used as immunogenic compounds.
  • the 1998 accession number XPOO 1349534 is identified as from an isolate from a rising portion in a Replikin cycle.
  • Replikin peptides identified in XPOO 1349534 are likewise useful as immunogenic compounds or vaccines or for diagnosis or treatment of malaria. See Figure 1 and Table 5 for all accession numbers discussed in this paragraph.
  • an expanding population of West Nile virus or an increase in virulence, morbidity, or morality of West Nile virus may be predicted by identifying a cycle of Replikin concentration in isolates of West Nile virus and predicting an expanding population of virus or an increase in virulence, morbidity, and/or mortality of West
  • Nile virus following a rising portion, or peak in the cycle of Replikin concentration An expanding population may represent an increase in population in a region or expansion from one region into another region.
  • a correlation between virus biochemical cycles and virus virulence, morbidity, and/or mortality cycles may be identified and used to predict expansions in a virus population or increases in virulence, morbidity, and/or mortality in a virus in a host population.
  • a non- limiting embodiment of the aspect of the invention provides a method of predicting an increase in morbidity in a viral disease such as West Nile virus comprising: (1) determining the mean Replikin Count in genomes of a plurality of isolates of a virus at a plurality of successive time points; (2) comparing the mean Replikin Count at at least four successive time points and identifying at least two peaks or two troughs in the trend of Replikin Counts over the at least four time points; and (3) predicting an increase in morbidity following in time the increase in mean Replikin count within said cycles.
  • step-wise cycles are identified between successive time points.
  • specific conserved Replikin sequences are identified within the step-wise cycles.
  • Table 2 below, provides data from analysis of envelope protein sequences in West Nile virus available at www.pubmed.com for isolates from 2000 through 2007.
  • the data which are illustrated in Figure 3, provide an example of cycling in mean annual Replikin Count in a virus wherein the cycle predicts morbidity.
  • the data additionally further support immunogenic compounds, diagnostic compounds, and, among other things, vaccines because they support the principles upon which such Replikin vaccines and other therapies are based including, in particular, the role Replikin sequences play in virulence and morbidity in pathogenic diseases, the correlation of Replikin Count with pathogenicity generally, and targeting of the Replikin structures for control of rapid replication and disease generally. See, e.g., U.S. Appln. Ser. No. 11/355,120, filed February 16, 2006 and U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008 (each incorporated herein by reference in their entirety).
  • Immunogenic compounds for therapeutic vaccines against West Nile virus include, for example, KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK (SEQ ID NO: 15), and HNDKRADPAFVCK (SEQ ID NO: 16).
  • These Replikin peptide sequences are conserved within the step-wise cycles of West Nile virus in Figure 3, which render them of particular use for therapies against expanding West Nile virus populations following the cyclic peaks identified in Figure 3.
  • the sequences may be administered to animals or humans as a vaccine.
  • a vaccine may comprise a pharmaceutically acceptable carrier and/or adjuvant.
  • a vaccine can be manufactured within seven days of the identification of sequences, such as these, that are conserved in step-wise cycles identified in the Replikin Count of a pathogen such as West Nile virus.
  • the sequences may likewise be used for diagnostic purposes to identify isolates of the expanding population of West Nile virus.
  • an expanding population of foot and mouth disease virus or an increase in virulence, morbidity, or mortality of West Nile virus may be predicted by identifying a cycle of Replikin concentration in isolates of foot and mouth disease virus and predicting an expanding population of virus or an increase in virulence, morbidity, and/or mortality of virus following a rising portion, or peak in the cycle of Replikin concentration.
  • An expanding population may represent an increase in population in a region or expansion from one region into another region.
  • a correlation between virus biochemical cycles and virus virulence, morbidity, and/or mortality cycles may be identified and used to predict expansions in a virus population or increases in virulence, morbidity, and/or mortality in a virus in a host population.
  • a non-limiting embodiment of the aspect of the invention provides a method of predicting an increase in morbidity in a viral disease such as foot and mouth disease virus comprising: (1) determining the mean Replikin Count in genomes of a plurality of isolates of a virus at a plurality of successive time points; (2) comparing the mean Replikin Count at at least four successive time points and identifying at least two peaks or two troughs in the trend of mean Replikin Counts over the at least four time points; and (4) predicting an increase in virulence and/or morbidity following in time an increase in mean Replikin count within a cycle.
  • step-wise cycles are identified between successive time points.
  • Increased Replikin Counts provide advance warnings of Foot and Mouth Disease outbreaks and the basis of a conserved synthetic FMDV Vaccine.
  • One aspect of the invention contemplates provision of advance warning of outbreaks of FMDV by identifying cycles in the Replikin Count of isolates of FMDV over time. As may be seen from the data in Figure 4, in 2000, an outbreak of Foot and Mouth Disease Virus (Type O) (FMDV) was predicted by a peak in annual mean Replikin Count. An outbreak in 2001-2002 was observed in the United Kingdom and in the Netherlands.
  • Replikin peptide structures found to be conserved over decades are now the basis of a synthetic Replikins vaccine for FMDV.
  • Replikin sequences identified as conserved within the Replikin cycles in Figure 4 include HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18). These sequences are also observed to be conserved over time in isolates of foot and mouth disease type A.
  • Figure 4 illustrates cycles of Replikin Count in Type O isolates of FMDV.
  • the data illustrated in Figure 4 are contained in Table 3.
  • Replikin peptides (1) were identified and counted automatically, with tests of statistical significance of changes, using a software program (ReplikinsForecastTM Replikins LLC, Boston, MA) designed to analyze the protein sequences of any organism, in this case FMDV published in PubMed.
  • Replikin structure in the virus was tracked for its occurrence in each virus specimen in each of the years for which virus sequence data was published, conservation of Replikin structures for decades was found. The structure of these conserved Replikins is the basis of synthetic Replikins vaccines for FMDV.
  • Replikin peptides conserved in FMDV over decades include HKQKIIAP AK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18).
  • Sequences identified within Replikin cycles and as conserved within Replikin cycles are particularly useful for diagnostic and therapeutic purposes.
  • the sequences identified as new and/or conserved in FMDV Replikin cycles are useful for (1) designing and chemically synthesizing vaccines that contain both older conserved Replikins as well as newer ones to provide the most accurate and maximal anti- organism immune stimulating properties, (2) designing and chemically synthesizing antibodies that contain reactive sites against both older conserved Replikins and newer ones to provide the most accurate and maximal anti-organism immune protective properties, and (3) designing and chemically synthesizing compounds that contain reactive sites against both older conserved Replikins and newer ones to provide the most accurate and maximal anti-organism protective properties.
  • One aspect of the present invention provides methods of predicting an outbreak of influenza by predicting an increase in the virulence, morbidity, and/or lethality of a strain of influenza virus or an expansion of the population of a strain of influenza virus using a Replikin Count Virus Expansion Index.
  • an increase in virulence, morbidity, and/or lethality or an expansion of a strain of influenza virus is predicted by (1) determining a mean Replikin Count and a standard deviation from the mean Replikin Count for a plurality of isolates of a strain of influenza virus for a first time period in a first geographic region, (2) determining a Replikin Count of at least one isolate of the same or a related strain of influenza virus from a second time period and/or a second geographic region different from the first time period and/or the second geographic region, and (3) predicting an increase in virulence, morbidity, and/or lethality or an expansion of the strain of influenza isolated in the second time period and/or second geographic region, if the Replikin Count of the at least one isolate from a second time period and/or a second geographic region is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in the first time period in the first geographic region.
  • At least one isolate of the same or related strain of influenza virus from a second time period and/or second geographic region may be a plurality of isolates from the second time period and/or second geographic region.
  • the Replikin Count of each isolate of the plurality of isolates from the second time period and/or second geographic region is compared separately to one standard deviation of the mean.
  • An expansion of influenza isolated in the second time period and/or second geographic region may also be predicted if the number of Replikin Counts of a plurality of isolates from the second period and/or second geographic region that is greater than one standard deviation of the mean is greater than the number of Replikin Counts of said plurality of isolates from the second period and/or second geographic region that is less than one standard deviation of the mean.
  • the method may also employ a ratio of the number of Replikin Counts that are greater than one standard deviation of the mean divided by the number of Replikin Counts that are less than one standard deviation of the mean.
  • the ratio is called a Replikin Count Virus Expansion Index (RCVE Index).
  • Another way to determine the RCVE Index is to divide the percent of Replikin Counts in a plurality of isolates of influenza virus grouped by time and/or region that are higher than one standard deviation of the mean by the percent of Replikin Counts that are lower than one standard deviation of the mean.
  • An RCVE Index may be used to quantify the future risk of an outbreak of influenza by tracking Replikin Counts in strains of influenza over time.
  • the mean Replikin Count of the plurality of isolates from the first time period and first geographic region is considered a control.
  • a control population preferably has a relatively large number of isolates with a relatively small variability in the Replikin Count of the isolates, but any population may be deemed a control when a comparison between the control and a related isolate or plurality of isolates is desired.
  • a control may be related to the population that is being studied.
  • influenza infection in a bird species such as swans
  • the control may be something closely related such as chickens, wherein isolates from chickens may be relatively numerous (if available) and relatively stable (if possible) and wherein stability in Replikin Count through the population demonstrates a level of equilibrium between the expansion and contraction of the strain or related strain of influenza virus in chickens.
  • a control may reflect a highest number of isolates reported in a year or in several years in a geographic area.
  • Influenza B may be a model control during the 20 th century for influenza strains because both Replikin Count and morbidity in all hosts are remarkably stable throughout some 40 years with a relatively small standard deviation and no lethal outbreaks recorded.
  • Replikin Count and replication rate appear to be just sufficient to balance losses for steady survival of the species. This is in contrast to H2N2, which disappeared at the end of the century after dropping Replikin Counts less than one standard deviation of the mean with no Replikin Counts greater than one standard deviation of the mean to balance the survival of the strain.
  • any measure of Replikin concentration may be used in influenza or in other pathogens.
  • Replikin Count may reflect the concentration of Replikin peptides identified encoded in the genome of an isolate.
  • Replikin Count may also reflect the concentration of Replikin peptides identified in the expressed proteins of an isolate or in at least one protein or protein fragment of an isolate.
  • Replikin Count may also reflect the concentration of Replikin peptides identified in a Replikin Peak Gene of an isolate.
  • the Replikin Peak Gene of an influenza virus may be any segment of the genome or of any expressed protein or protein fragment having the highest concentration of continuous and/or overlapping Replikin peptides identified.
  • the Replikin Peak Gene is identified in the polymerase area of the influenza virus genome. Within the polymerase area, the Replikin Peak Gene is often identified in the pBl gene area. Replikin Counts within the pBl gene may also be used.
  • Any Replikin peptide, Replikin Peak Gene, protein, protein fragment, or nucleic acid sequence encoding any Replikin peptide, Replikin Peak Gene, protein, or protein fragment in an isolate predicted by the methods of the invention to be expanding may be used for diagnostic, therapeutic, and/or preventive purposes.
  • a vaccine may be manufactured by identifying a portion of the structure or genome of an influenza isolate predicted to expand in population and using that portion in a vaccine composition.
  • Methods of the invention also provide methods of predicting a decrease in virulence, morbidity, and/or lethality of a strain of influenza and/or predicting a contraction or failure of a strain of influenza wherein a Replikin Count of at least one isolate of a strain of influenza from a second time period and/or second geographic region is less than one standard deviation of the mean of the Replikin Count of a plurality of isolates of influenza from a first time period and first geographic region.
  • a decrease may also be predicted where the number of Replikin Counts of a plurality of isolates from a second period and/or a second geographic region that are greater than one standard deviation of the mean is less than the number of Replikin Counts less than one standard deviation of the mean.
  • a decrease, contraction, or failure is predicted if the ratio of the Replikin Count Virus Expansion Index is less than one.
  • Replikin Counts from Replikin Peak Genes may be analyzed from regions (such as all reporting countries) in a given time period (such as a year) for a range of species. Within a country in a year, there may be a range of values over a range of species. The ordinary skilled artisan may select a mean Replikin Count as a control from the range of values, a time, a region, a species, or any combination thereof (such as a time, a region, and a species, e.g., 2004, China, and chicken).
  • Example 7 the mean Replikin Count of all H5N1 isolates from chickens in China in 2004 was selected as an initial control against which Replikin Counts from swans in China in 2004 were compared.
  • a control that shares some similarity with the isolate or group of isolates may be used.
  • a control of all isolates from chicken in China in 2004 may be compared with other isolates from 2004.
  • a control of swans from 2005 in Japan may be compared to future isolates from swans in Japan.
  • the ratio of the percent of isolates having Replikin Counts above mean plus one standard deviation to the percent of isolates having Replikin Counts below the mean minus one standard deviation provides a quantitative index of the viability and expansion of the virus. The index provides a snapshot of current status of the virus population and the propensity for change in that population. If the ratio is greater than one, the RCVE Index predicts an expanding population. If the ratio is less than one, the RCVE Index predicts a contracting or failing virus population.
  • One aspect of the present invention provides methods of preventing or treating outbreaks of influenza virus by predicting an expansion of a strain of influenza virus using a Replikin Count Virus Expansion Index and administering therapies comprising an isolated or synthesized portion of the structure or genome of the influenza virus identified using the RCVE Index to prevent, mitigate, or treat the outbreak of influenza virus.
  • a prediction of an outbreak may be made by (1) determining a mean Replikin Count with standard deviation for a group of isolates of a strain of influenza isolated during a first time period in a first geographic region, (2) determining a Replikin Count of at least one isolate of the same strain of influenza virus from a second time period and/or second geographic region that is different from the first time period and/or is different from the second geographic region, and (3) predicting an expansion of the strain of influenza isolated in said second time period and/or second geographic region if the Replikin Count of the isolate from a second time period and/or second geographic region is greater than one standard deviation from the mean of the Replikin Count of the plurality of isolates isolated in the first time period and in the first geographic region.
  • An outbreak may be prevented, mitigated, or treated by administering a pharmaceutical compound that includes all or some portion of the structure or genome of the at least one isolate of influenza virus.
  • the at least one isolate of influenza from a second time period and/or geographic region may be a plurality of isolates from the second time period and/or second geographic region wherein the Replikin Count of each isolate of the plurality of isolates is compared separately to one standard deviation from the mean. Additionally, an outbreak of influenza may be predicted if the number of Replikin Counts of the plurality of isolates from a second period and/or a second geographic region that is greater than one standard deviation of the mean is greater than the number of Replikin Counts less than one standard deviation of the mean.
  • the portion of the structure or genome may be isolated from an influenza isolate or may be synthesized based on sequences or other structure elucidated from the influenza isolate as well understood by the ordinary skilled artisan.
  • the structure may be a protein or protein fragment that comprises a Replikin peptide or that consists of a Replikin peptide.
  • the structure may comprise or consist of a Replikin Peak Gene or a fragment of a Replikin Peak Gene or may consist of a Replikin peptide identified within a Replikin Peak Gene.
  • the structure may also be a nucleic acid including but not limited to a nucleic acid encoding a Replikin Peak Gene, a Replikin peptide or plurality of Replikin peptides within a Replikin Peak Gene, or a Replikin peptide or plurality or Replikin peptides.
  • a peptide or mixture of peptides may be comprised in an immunogenic compound for influenza and may include at least one of HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1), KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4),
  • HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12).
  • Another aspect of the invention provides methods of predicting an increase in the virulence, morbidity, and/or lethality or an expansion of the population of an isolate of a strain of influenza virus as compared to another isolate or group of isolates of the same or a related strain.
  • Such an increase may be predicted by identifying a cycle of Replikin concentration among a plurality of isolates of influenza and identifying a peak in that cycle.
  • An increase is predicted following the time point or time period when the peak is identified or following a rising portion of the cycle.
  • An increase may likewise be predicted following the time point or time period when a peak is identified in two synchronous cycles wherein a first cycle is the cycle of a strain of influenza and the second cycle is a cycle of a different strain of influenza. The increase is predicted following the time period in which the peaks of the synchronous cycles are identified or in a rising portion identified in both synchronous cycles.
  • a cycle of Replikin concentration or "Replikin cycle" of H9N2 may be seen in Figure 5.
  • a comparison of synchronized cycles of Replikin concentration in H5N1 and H9N2 may be seen in Figure 6.
  • the synchronized cycles in these two influenza strains correspond to and retrospectively predict H5N1 outbreaks in 1997, 2001, 2004, 2007 and the present outbreak in 2008 and 2009.
  • Figure 6 visibly illustrates synchrony between the H9N2 and H5N1 Replikin Cycles.
  • Table 4 mean annual Replikin Count with standard deviation are provided for all amino acid sequences publicly available at www.pubmed.com for H9N2 and H5N1 strains of influenza isolated from 1993 through 2008. The number of poultry flocks reported to have H9N2 infections in Israel are also disclosed for years 2000 through 2004 as a measure of outbreaks of H9N2.
  • Table 4 Synchronous Replikin Cycles in H9N2 and H5N1
  • H9N2 Replikin Counts of Replikin peptides identified as encoded in the pBl region of the influenza genome reached levels twice those found in H5N1.
  • H9N2 Replikin Counts increased in 1996, one year before the H5N1 outbreak in Hong Kong in 1997.
  • Figure 5 illustrates that increasing H9N2 Replikin Counts precede the occurrence of increasing numbers of H9N2 infections in poultry flocks.
  • Figure 5 further demonstrates that Replikin
  • H9N2 Counts in H9N2 began to increase again in 1999, two years before a reported increase of H9N2 outbreaks in poultry in the Middle East, including Israel. As may be seen in Figure 6, following the increase in H9N2, Replikin Counts began to increase in H5N1 in 2000 with infections beginning in 2000 and forward.
  • H9N2 sequences analyzed and reported as mean Replikin Count in Table 4 and in Figures 5 and 6 include all those published on PubMed worldwide. A principal portion of the sequences are from influenza isolated in China and the Middle East.
  • a peptide vaccine of the invention may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in particular virus strains.
  • a vaccine may include a conserved Replikin peptide(s) in combination with a new Replikin(s) peptide or may be based on new Replikin peptide sequences.
  • the Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences, although vaccines based on peptides containing only Replikin sequences are preferred.
  • vaccine compositions of the invention also contain a pharmaceutically acceptable carrier and/or adjuvant.
  • the vaccines of the present invention can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • antiviral drugs such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • the vaccine of the present invention may be administered to any animal capable of producing antibodies in an immune response.
  • the vaccine of the present invention may be administered to a rabbit, a chicken, a shrimp, a pig, or a human.
  • a vaccine of the invention may be directed at a range of strains of a virus or organism or a particular strain of virus or organism.
  • the Replikin peptides of the invention are administered to a subject, in a non-limited embodiment by i.v., intramuscular injection, by mouth, or by spray inhalation, intranasal administration, or intraocular administration.
  • the peptides are administered in order to stimulate the immune system of the subject to produce antibodies to the peptide.
  • the dosage of peptides is in the range of from about 0.01 ⁇ g to about 500 mg, about 0.05 ⁇ g to about 200 mg, or about 0.075 ⁇ g to about 30 mg, from about 0.09 ⁇ g to about 20 mg, from about 0.1 ⁇ g to about 10 mg, from 10 ⁇ g to about 1 mg, and from about 50 ⁇ g to about 500 ⁇ g.
  • isolated Replikin peptides may be used to generate antibodies, which may be used, for example to provide passive immunity in an individual or for diagnostics. See, e.g., U.S. Appln. Ser. No. 11/355,120, filed February 16, 2006 and U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008 (each incorporated herein by reference in their entirety).
  • Replikin sequences is provided in Table 5 below.
  • the mean Replikin Count for each year is provided following the list of accession numbers from isolates in each corresponding year. Standard deviation and significance as compared to the mean Replikin Count of the previous year and of the lowest mean Replikin Count within the data set are also provided along with the mean Replikin Count for each year.
  • Replikin peptides identified in the 1999 isolate reported at accession number CAD49281 are likewise available. See Figure 1 and Table 2. The 1999 isolate is present at the peak of a Replikin cycle, as such, Replikin peptides identified in the isolate reported at CAD49281 may be used as immunogenic compounds. Additionally, the 1998 accession number XPOO 1349534 is identified from an isolate in a rising portion in a Replikin cycle. See Figure 1 and Table 5. Replikin peptides identified in ABU43157, CAD49281, and XPOO 1349534, among others, are likewise useful as immunogenic compounds or vaccines or for diagnosis or treatment of malaria.
  • Figure 3 illustrates cycling of mean annual Replikin Count in West Nile virus in correlation with cycling of West Nile virus morbidity. Cycles are detectable because of repeating conserved virus structures and continuity of the Replikin phenomenon through time.
  • the mean annual Replikin Count of the Envelope Protein of WNV (black), and standard deviation, is compared to the annual number of human cases in the U.S. per CDC reports (gray).
  • 2000 to 2003 The standard deviation of the mean of the Replikin Count of the envelope protein increases markedly from 2000 to 2001 (p ⁇ 0.001).
  • FIG. 4 shows that the annual Replikin Counts (Mean and Standard Deviation (SD)) occurred with two rising portions and a decreasing portion. The first rising portion followed by the first decreasing portion occurred from, 1999-2005 and the second rising portion occurred in 2005-2008. Increases in Replikin Counts provided advance warning signals with p ⁇ 0.001 prior to the 2001-2002 and 2008-2009 severe outbreaks.
  • SD Standard Deviation
  • HKQKIIAPAK SEQ ID NO: 17
  • HKQKIVAPVK SEQ ID NO: 18
  • These sequences have been observed to be conserved in the Replikin cycles illustrated in Figure 4 and, as taught by the invention, are vaccines for predicted outbreaks of foot and mouth disease virus.
  • the two above-listed conserved Replikin peptides have been identified and tracked annually in publicly available sequences in foot and mouth disease virus at www.pubmed.com from 1934 through 2008.
  • the sequence HKQKIIAPAK SEQ ID NO: 17
  • HKQKIVAPVK (SEQ ID NO: 18) is observed also to be conserved in 100% of isolates from 1934 through 2007 with the exception of two substitutions, namely a valine at residue 6 in the peptide and a valine at residue 9 in the peptide.
  • Table 8 provides the accession numbers at www.pubmed.com wherein sequence HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18) were conserved over time. The residue at which the peptide begins in the sequence disclosed in the accession number is noted.
  • ABF 18566 position 43 , ABF 18562 position 43 CAC22209 position 201 , AAL09392 position 153 , ABF 18557 position 43 , ABF 18555 position 43 AAL09391 position 153 , AAK69397 position 153 , ABF 18553 position 43 , ABF 18552 position 43 ABF 18551 position 43 , ABF 18550 position 43 , ABL60850 position 201 , ABL60849 position ABFl 8549 position 43 , ABFl 8548 position 43 , 201 , ABL60848 position 201 , ABL60847 CAC51275 position 201 , CAC51271 position 201 position 201 , ABL60845 position 201 , , CAC51267 position 201 , CAC51264 position ABL60844 position 201 , ABL60843 position 201 , CAC51263 position 201 , CAC51261 201 .
  • ABL60842 position 201 ABL60841 position 201 , CAC51258 position 201 , position 201 , ABL60840 position 201 , CAC51257 position 201 , BAC06475 position 925 ABL60839 position 201 , ABL60838 position , AAG27038 position 153 , AAG27037 position 201 .
  • ABL60837 position 201 ABL60842 position 201 .
  • ACD44915 position 201 ACD 44914 position 201 , ACD44913 position 201 , ACD44912 position 201 , ACD44911 position 201 , ACD44903 position 191 , ACD 44902 position 188 , ACD44898 position 192 , ACD 44897 position 187 , AAR07964 position 153 , ABRl 3026 position 201 , ABRl 3025 position 201 , ABRl 3024 position 201
  • ACD44924 position 200 ACD44923 position ACD44919 position 201 , ACD44916 position 201 200 , ACD44922 position 200 , ABG77560 , ABG77563 position 197 , ABG77564 position 30 position 219 , ABG77557 position 126 .
  • An aspect of the invention is the prediction of an expansion into a geographic region or contraction from a geographic region based on a determination of the progression of mean annual Replikin concentrations in a graph of a cycle or series of cycles of Replikin concentration including observed step-wise cycles. For example, a peak in Replikin concentration in a cycle of Replikin concentration of a plurality of isolates from a given region provides evidence of expansion beyond the geographical area of that region into other contiguous or nearby geographical areas. Furthermore a second, still higher, peak provides even greater evidence of a pathogen that is poised for expansion.
  • a Replikin concentration cycle based on isolates from a particular region demonstrates a prolonged rise in mean annual Replikin Count or a peak following a rise in mean annual Replikin Count
  • the significant rise or peak predicts an expansion of the mortality rate or morbidity rate of that isolate into contiguous or nearby regions that until the significant rise or peak in Replikin Count did not experience the mortality rate or morbidity rate of the particular region.
  • a cycle of Replikin concentration is established in the Sahel region of Africa with two peaks at years 2 and 7. The second peak at year 7 is significantly higher than the first peak at year 2 with a p value of 0.01.
  • the Sahel region between years 0 and 7 has experienced a higher rate of mortality than more southerly regions. Based on the higher peak at year 7, it is predicted that the mortality from malaria will increase in the region contiguous to the south of the Sahel.
  • a plurality of Replikin sequences are isolated from year 7 isolates. Replikins that have been conserved between years 0 and 7 are selected as vaccines for malaria in the Sahel and contiguous regions to the south. Replikins that are new in year 7 are likewise selected as vaccines.
  • a mixture of these Replikin sequences is combined with a pharmaceutically acceptable carrier and/or adjuvant and administered to a subject to produce an immune response to treat and/or protect against malaria predicted to have a higher mortality rate following the dry season in year 8 in the Sahel and in its contiguous regions to the south.
  • the Replikin Count for each individual isolate in a given country in a given year was compared to one standard deviation from the mean Replikin Count for all isolates from chicken in China in that year. Within each country, the number of Replikin Counts greater than one standard deviation of the mean and the number of Replikin Counts less than one standard deviation of the mean were determined. For each country in each year, the percent of Replikin Counts greater than one standard deviation of the mean was then divided by the percent of Replikin Counts less than one standard deviation of the mean to provide a ratio, or Replikin Count Virus Expansion (RCVE) Index. In countries having an RCVE Index of greater than one, an expansion of H5N1 was predicted for the following year or years. In countries having a RCVE Index of less than one, a contraction or viral failure was predicted for the following year or years.
  • RCVE Replikin Count Virus Expansion
  • the RCVE Index for swans in China in 2004 is 12.5/0. Because zero is set as 1 when it is in the denominator, the index returns a ratio of 12.5, which predicts an expanding population. This predicted expansion is seen below in Table 11 in an expanding population in swans in China in 2006.
  • the RCVE Index for swans in Japan in 2007 is 50/0. Because zero is set as 1 when it is in the denominator, the index returns a ratio of 50, which predicts an expanding population. So despite a small sample size, the index predicts expansion, which is seen below in Table 13 in an expanding population in swans in Japan.
  • the RCVE Index for swans in Japan in 2008 is 1.8, which predicts future expansion of influenza in swans in Japan.
  • the RCVE Indices as described above may be practiced by one of ordinary skill in the art as a measure of the current survival and expansion status or contracting/failing status of a population of pathogen engaged in an outbreak.
  • the ordinary skilled artisan may isolate in silico the Replikin Peak Gene, may measure the Replikin Count of the Replikin Peak Gene, and may compare the Replikin Count data of related strains of virus in other geographic regions in the same and previous time periods to understand the severity of the outbreak, the direction of the outbreak, and the attendant risk to neighboring geographic regions.
  • the appreciable advantage to the ordinary skilled artisan is time to develop therapies and to institute public health measures known now or hereafter such as isolation and culling of poultry, vaccination, and other measures.
  • the methods disclosed herein further provide the ordinary skilled artisan with time to manufacture the synthetic Replikin vaccines disclosed herein.
  • a mean Replikin Count with standard deviation is determined.
  • the region having the largest number of isolates or the least variability among Replikin Count in isolates (or both) for each year from 2004 to 2008 is chosen as a control against which other Replikin Counts are analyzed.
  • the Replikin Count for each individual isolate in a given region in a given year is compared to one standard deviation from the mean Replikin Count for all isolates from the control region.
  • the number of Replikin Counts greater than one standard deviation of the mean and the number of Replikin Counts less than one standard deviation of the mean is determined.
  • the Replikin Peak Gene and/or a Replikin peptide (or plurality of Replikin peptides) within the Replikin Peak Gene is selected as an immunogenic compound for diagnostic and/or therapeutic purposes.
  • a vaccine against the expanding population is manufactured comprising the immunogenic compound. The vaccine is administered to mitigate the expanding malarial population.
  • a mean Replikin Count with standard deviation is determined.
  • the region having the largest number of isolates or the least variability among Replikin Count in isolates (or both) for each year from 2000 to 2008 is chosen as a control against which other Replikin Counts are analyzed.
  • the Replikin Count for each individual isolate in a given region in a given year is compared to one standard deviation from the mean Replikin Count for all isolates from the control region.
  • the number of Replikin Counts greater than one standard deviation of the mean and the number of Replikin Counts less than one standard deviation of the mean is determined.
  • RCVE Replikin Count Virus Expansion
  • a Replikin Peak Gene is identified in an isolate having a Replikin Count that is higher than the mean Replikin Count for the region.
  • the Replikin Peak Gene and/or a Replikin peptide (or plurality of Replikin peptides) within the Replikin Peak Gene is selected as an immunogenic compound for diagnostic and/or therapeutic purposes.
  • a vaccine against the expanding population is manufactured comprising the immunogenic compound. The vaccine is administered to mitigate the expanding foot and mouth disease virus population.
  • a synthetic Replikin vaccine containing an approximately equal-parts-by-weight mixture of twelve H5N1 Replikin peptides was tested in chickens against a low pathogenic strain of H5N1 isolated from a black duck in North Carolina, USA.
  • Low-Path H5N1 strains infect migratory birds and impair health and productivity of commercial flocks of U.S. chickens, usually with little mortality in the commercial flocks.
  • These Low-Path H5N1 strains are very closely related in virus structure to their more lethal High-Path H5N1 relatives in Eurasia.
  • a mutation from a Low-Path to a High-Path strain has so far not been observed but mutations of this type over time may be expected by one of skill in the art.
  • the tested vaccine was engineered to block both the entry site of H5N1 virus and the replication site of those H5N1 viruses that manage to enter into host cells.
  • the vaccine is called the TWO-PUNCH vaccine.
  • TWO-PUNCH vaccine As demonstrated below, evidence from the described test of the TWO-PUNCH vaccine in chickens suggests that both mechanisms for which the vaccine was designed were effective: (1) virus entry into inoculated chickens was diminished by immunity from the vaccine and (2) virus replication within infected cells was sufficiently limited by immunity from the vaccine to block excretion of the virus in feces of tested birds.
  • the TWO-PUNCH Replikins Vaccine is based on influenza Replikin peptides shared between influenza strains and conserved for decades within influenza strains.
  • the vaccine was engineered as a mixture of twelve Replikin peptides identified as expressed from the genome of H5N1 virus.
  • Six of the Replikin peptides are synthesized according to sequences isolated from the hemagglutinin protein of H5N1, which is involved in attachment and entry of influenza virus into a cell.
  • Six of the Replikin peptides are synthesized according to sequences isolated from the pBl gene area of H5N1, which has been identified as involved in replication of influenza virus in a host cell.
  • the following six Replikin sequences contained in the vaccine were isolated from the hemagglutinin protein: (1) HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1);
  • HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5);
  • KDVMESMDKEEMEITTH SEQ ID NO: 7
  • the vaccine comprises an approximate equal-parts-by- weight mixture of the twelve peptides.
  • the following peptide amounts were combined to create an initial mixture of the vaccine:
  • HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5) 170.8 mg
  • KDVMESMDKEEMEITTH (SEQ ID NO: 7) 161.9 mg
  • KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9) 217.8 mg
  • HKRTIGKKKQRLNK (SEQ ID NO: 10) 178.0 mg
  • HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK SEQ ID NO: 12233.8 mg
  • the total amount of the mixture was 2237.1 mg.
  • the peptide mixture was then divided into three equal parts for administration of the vaccine on three different days (days 1, 7, and 28). After dissolution with water, the three equal parts were administered to individual birds in two groups of 20 birds each for a total administration at each day of 40 birds.
  • the total amount of active peptide ingredient administered to each bird at the time of administration was about 18.6 mg per bird per administration.
  • the vaccine solution was administered to chickens intranasally at a first administration on day 1 after hatch, intraocularly at a second administration on day 7 after hatch, and via fine spray inhalation at a third administration on day 14 after hatch.
  • Chickens on the first day of life were separated into four groups with twenty chickens per group. The first group was a control group not vaccinated and not challenged with Low-Path H5N1. The second group was vaccinated and not challenged with Low-Path H5N1. The third group was vaccinated and subsequently challenged with Low-Path H5N1. The fourth group was not vaccinated and was challenged with Low-Path H5N1.
  • the synthetic H5N1 Replikins Vaccine was administered intranasally on day 1 after hatch, administered intraocularly on day 7 after hatch, and administered by fine spray inhalation on day 14 after hatch.
  • the groups of challenged chickens were than challenged with Low-Path H5N1 virus on day 28 of the life of the chicken.
  • Serum from selected chickens was analyzed in all groups for antibodies against the H5N1 virus on days 7, 14, and 21 following challenge. PCR for virus fecal excretion was also analyzed for all groups.
  • Unvaccinated control chickens demonstrated both an expected high virus entry (as indicated by a high titer of antibodies against H5N1) and an expected high virus replication (as indicated by high fecal and salival excretion of the virus detected by PCR). In contrast, the vaccinated chickens demonstrated lower virus entry (as indicated by a low titer of antibodies against H5N1 or by the observation of no antibodies against H5N1 in serum) and an absence of fecal or saliva excretion of virus indicating low or no virus replication in the vaccinated chickens.
  • the data in Table 14 below provide the numbers of chickens tested in each of the four groups (Negative Control, Vaccinated, Vaccinated and Challenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (not vaccinated)) on a particular test day and the numbers of chickens in which production of antibodies to H5N1 was detected with a serum titer.
  • Table 15 The data in Table 15 below provide the number of chickens tested for H5N1 virus in their saliva and feces in each of the four groups (Negative Control, Vaccinated, Vaccinated and Challenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (not vaccinated)) on a particular test day and the numbers of chickens in which H5N1 was detected in their feces and saliva based on PCR analysis.
  • Table 15 demonstrates the absence of detectable influenza in the feces and saliva of vaccinated birds. That viral excretion was blocked by this influenza Replikins vaccine is particularly significant because it is generally acknowledged that the maintenance of reservoirs of H5N1 virus in flocks of migratory birds and domestic chickens in both Asia and the U.S. (and the regional spread of H5N1 virus from these reservoirs) is dependent on viral excretions picked up by neighboring chickens and birds. Regardless of the level of lethality of a strain of H5N1 virus, absent excretion of virus, there is expected to be no spread of the virus.
EP20090734389 2008-04-23 2009-04-23 Verfahren und verbindungen zur abschwächung von krankheitsausbrüchen durch replikin-zählzyklen Ceased EP2300493A2 (de)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US12/108,458 US9408902B2 (en) 2007-05-30 2008-04-23 Synthetic replikin peptides against pathogenic infection of invertebrates in aquaculture
PCT/US2008/061336 WO2008156914A2 (en) 2007-05-30 2008-04-23 Synthetic replikin peptides against pathogenic infection of invertebrates in aquaculture
US5401008P 2008-05-16 2008-05-16
US8735408P 2008-08-08 2008-08-08
US14361809P 2009-01-09 2009-01-09
PCT/US2009/041565 WO2009132209A2 (en) 2008-04-23 2009-04-23 Methods and compounds for mitigating pathogenic outbreaks using replikin count cycles

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US9233148B2 (en) 2009-01-09 2016-01-12 Samuel Bogoch Replikin-based compounds for prevention and treatment of influenza and methods of differentiating infectivity and lethality in influenza
EP2734226A2 (de) 2011-07-20 2014-05-28 Samuel Bogoch Gemeinsame peptides von letalen karzinomen und therapeutische zusammensetzungen mit diesen peptiden
SG11201405447QA (en) 2012-03-09 2014-10-30 Samuel Bogoch Therapies, vaccines, and predictive methods for infectious salmon anemia virus

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US7189800B2 (en) * 2001-03-27 2007-03-13 Samuel Bogoch Replikin peptides in rapid replication of glioma cells and in influenza epidemics
WO2005104754A2 (en) * 2004-04-28 2005-11-10 Samuel Bogoch System and method for identifying complex patterns of amino acids
SG160327A1 (en) * 2005-02-16 2010-04-29 Samuel Bogoch Systems and methods for identifying replikin scaffolds and uses of said replikin scaffolds
EP1924282B1 (de) * 2005-08-15 2017-01-11 Altimmune Inc. Immunisierung von avianen durch verabreichung von nicht-repliziert-gerichteten impfstoffen
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