WO2014127155A2 - Procédés d'identification, de prévention et de traitement du virus virulent de la maladie pieds-mains-bouche au moyen de séquences de réplikine - Google Patents

Procédés d'identification, de prévention et de traitement du virus virulent de la maladie pieds-mains-bouche au moyen de séquences de réplikine Download PDF

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Publication number
WO2014127155A2
WO2014127155A2 PCT/US2014/016303 US2014016303W WO2014127155A2 WO 2014127155 A2 WO2014127155 A2 WO 2014127155A2 US 2014016303 W US2014016303 W US 2014016303W WO 2014127155 A2 WO2014127155 A2 WO 2014127155A2
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replikin
hfmdv
peptide
sequence
vaccine
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PCT/US2014/016303
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English (en)
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WO2014127155A3 (fr
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Samuel Bogoch
Elenore S. Bogoch
Samuel Winston Bogoch
Anne-Elenore Bogoch Borsanyi
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Samuel Bogoch
Bogoch Elenore S
Samuel Winston Bogoch
Anne-Elenore Bogoch Borsanyi
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Publication of WO2014127155A2 publication Critical patent/WO2014127155A2/fr
Publication of WO2014127155A3 publication Critical patent/WO2014127155A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • 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
    • C12N2770/32311—Enterovirus
    • C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Hand, foot and mouth disease is a disease in humans generally caused by picornaviridae viruses, which are generally described as non-enveloped, positive- stranded, RNA viruses having an icosahedral capsid.
  • the RNA genome of picornaviridae viruses is known to have a 5' end transcription primer protein used by RNA polymerase.
  • Picornaviruses have a positive sense RNA genome of less than 10,000 kilobases.
  • Viruses that cause HFMD are generally associated with the Enterovirus genus. This genus includes polioviruses, coxsackieviruses, echoviruses, and enteroviruses.
  • Coxsackie A virus The most common strains of picornaviridae viruses causing HFMD are coxsackie A virus and enterovirus 71 (EV-71).
  • Coxsackievirus A16 is the most common cause of hand, foot, and mouth disease in the United States and other coxsackie viruses are also associated with the illness.
  • viruses that cause HFMD are often denoted together as a Hand Foot and Mouth Disease virus family (HFMDV), which includes, and is not limited to, the coxsackie A virus and the enterovirus 71 (EV-71).
  • HFMDV Hand Foot and Mouth Disease virus family
  • EV-71 enterovirus 71
  • Hand Foot and Mouth Disease most commonly affects children, including infants.
  • the disease is generally moderately contagious although virulent forms of HFMD are at times observed.
  • the causative viruses are understood to spread through direct contact with the mucus, saliva, or feces of an infected person.
  • HFMD is often observed in localized epidemics in summer or autumn in places where children are regularly gathered, such as schools and nurseries. Incubation is usually a few days to a week.
  • Complications from infection include meningitis and encephalitis as well as fluid accumulation in the air spaces and parenchyma or bleeding in the lungs. Complications are rare from common infections but have been observed to be considerably less rare in certain virulent outbreaks. Death may result from complications of the infection.
  • HFMD occurs in adults with less frequency than children. Immune- compromised adults are particularly susceptible.
  • the inventors have found a family of conserved small protein sequences that relate to rapid replication in influenza and other viruses.
  • the family of protein sequences is known as Replikins. Rapid replication is characteristic of virulence in viruses where Replikin sequences are identified or where concentrations of Replikin sequences are present. Rapid replication has been associated with the presence of Replikin sequence structures in protein sequences. Replikin sequences have been further associated with viral outbreaks, epidemics and increased rates of host mortality.
  • the inventors provide herein methods of predicting, preventing, treating, and diagnosing Hand Foot and Mouth Disease viruses, including coxsackie A virus and enterovirus 71 (EV-71).
  • the present invention provides methods of determining the relative virulence, replication rate, and/or lethality of an isolate, strain, or population of a virus that causes Hand Foot and Mouth Disease (HFMDV).
  • HFMDV Hand Foot and Mouth Disease
  • the present invention further provides compounds and compositions for prevention and treatment of HFMDV, including immunogenic compositions, blocking compositions, and vaccines against HFMDV, including coxsackie A virus and enterovirus 71 (EV-71).
  • a first aspect of the present invention provides a method of determining the relative virulence of an isolate A of a HFMDV comprising determining a Replikin concentration of at least one amino acid sequence A isolated from an isolate A of an HFMDV and comparing said at least one amino acid sequence A to the Replikin concentration of at least one amino acid sequence B isolated from an isolate B of an HFMDV and determining that isolate A is relatively more virulent than isolate B if the Replikin concentration of said a least one amino acid sequence A is greater than the Replikin concentration of said at least one amino acid sequence B or determining that isolate A is relatively less virulent than isolate B if the Replikin concentration of said at least one amino acid sequence A is less than the Replikin concentration of said at least one amino acid sequence B.
  • said at least one amino acid sequence A is encoded in the same region of the genome of the virus as said at least one amino acid sequence B.
  • sequence A is the amino acid sequence of a protein and sequence B is the amino acid sequence of the same protein as sequence A.
  • sequence A is a Replikin Peak Gene of isolate A and sequence B is a Replikin Peak Gene of isolate B.
  • the Replikin concentration of isolate A reflects the Replikin concentration of the entire genome of isolate A and the Replikin concentration of isolate B reflects the Replikin concentration of the entire genome of isolate B.
  • isolate A is an isolate of coxsackie A virus and isolate B is an isolate of coxsackie A virus.
  • isolate A is an isolate of EV-71 and isolate B is an isolate of EV-71.
  • said at least one sequence A is a plurality of sequences A isolated from a plurality of isolates of population A of a HFMDV and said at least one sequence B is a plurality of sequences B isolated from a plurality of isolates of population B of a HFMDV, wherein a mean Replikin concentration of said plurality of sequences A is compared to a mean Replikin concentration of said plurality of sequences B and virus population A is determined to be relatively more virulent than virus population B if the mean Replikin concentration of said plurality of sequences A is greater than the mean Replikin concentration of said plurality of sequences B or said virus population A is determined to be relatively less virulent than virus population B if the Replikin concentration of said plurality of sequences A is less than the Replikin concentration of said plurality of sequences B.
  • the plurality of sequences A reflects a plurality of whole genome sequences of a plurality of isolates of population A and the plurality of sequences B reflects a plurality of whole genome sequences of a plurality of isolates of population B.
  • said virus population A is a population of a particular strain of a HFMDV isolated within a time period A and said virus population B is a population of the same strain of a HFMDV isolated within a time period B.
  • time period A is a particular calendar year and time period B is a different calendar year.
  • time period A is a particular calendar month and time period B is a different calendar month.
  • said virus population A is a population of a HFMDV isolated within a time period A and within a geographic region A and said virus population B is a population of a HFMDV isolated within a time period B and within a geographic region B.
  • a second non- limiting aspect of the present invention provides an isolated or synthesized Replikin peptide of a HFMDV wherein said peptide consists of 7 to about 50 amino acid residues and comprises (1) at least one lysine residue located 6 to 10 residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues.
  • the isolated or synthesized Replikin peptide has at least one lysine residue on one end of the peptide and at least one lysine residue or at least one histidine residue on the other end of the peptide.
  • the isolated or synthesized Replikin peptide consists of the shortest amino acid sequence of 7 to 50 amino acid residues comprising (1) a first lysine residue located six to ten residues from a second lysine residue, (2) at least one histidine residue; and (3) at least 6% lysine residues.
  • the isolated or synthesized peptide is shared among two or more viruses that cause HFMD.
  • the isolated or synthesized Replikin peptide is shared among EV-71 and coxsackie A virus.
  • a non- limiting embodiment of the second aspect of the present invention provides an isolated or synthesized HFMDV polypeptide comprising a Replikin peptide.
  • the isolated or synthesized polypeptide is an HFMDV protein or protein fragment comprising a Replikin peptide sequence.
  • Another non- limiting embodiment of the second aspect of the present invention provides a peptide or polypeptide consisting essentially of an HFMDV Replikin peptide.
  • the HFMDV Replikin peptide is a Replikin peptide from a coxsackie A virus.
  • the coxsackie A virus is a coxsackie A16 virus.
  • the HFMDV Replikin peptide is a Replikin peptide from an EV-71 virus.
  • the HFMDV Replikin peptide is a synthesized peptide derived from a sequence of a HFMDV such as, but not limited to, a coxsackie A virus or an EV-71 virus.
  • the Replikin peptide sequence is shared among two or more viruses that cause HFMD.
  • the Replikin peptide sequence is shared among EV-71 and coxsackie A virus.
  • a non- limiting embodiment of the second aspect of the present invention provides an immunogenic composition comprising at least one HFMDV Replikin peptide sequence.
  • the immunogenic composition comprises a pharmaceutically-acceptable carrier, excipient, and/or adjuvant.
  • the immunogenic composition comprises a Replikin peptide sequence shared among two or more viruses that cause HFMD.
  • a non- limiting embodiment of the second aspect of the present invention provides a vaccine against HFMDV comprising at least one HFMDV Replikin sequence.
  • the vaccine comprises an isolated or synthesized HFMDV polypeptide comprising an HFMDV Replikin peptide.
  • the isolated or synthesized polypeptide is an HFMDV protein or protein fragment comprising a Replikin peptide.
  • the vaccine comprises a peptide or polypeptide consisting essentially of an HFMDV Replikin peptide or plurality of HFMDV Replikin peptides.
  • the vaccine comprises at least one polypeptide or peptide comprising at least one of SEQ ID NO(s): 1-22. In a further embodiment, the vaccine comprises at least one polypeptide or peptide consisting essentially of at least one of SEQ ID NO(s): 1-22. In a further embodiment, the vaccine comprises at least one polypeptide or peptide consisting of at least one of SEQ ID NO(s): 1-22. In a non- limiting embodiment, the vaccine comprises at least one Replikin sequence shared among at least two viruses that cause HFMD. In another non- limiting embodiment, the Replikin sequence is shared among EV-71 and Coxsackie virus. In a non- limiting embodiment, the Replikin sequence is SEQ ID NO(s): 2, 3, and/or 7.
  • a non-limiting embodiment of the second aspect of the present invention provides an immunogenic and/or blocking compound or composition comprising at least one protein, protein fragment, polypeptide, or peptide of any one of the above-listed proteins, protein fragments, polypeptides, or peptides including and not limited to at least one protein, protein fragment, polypeptide, or peptide comprising at least one Replikin peptide sequence identified in an HFMDV or at least one homologue of said at least one Replikin peptide identified in an HFMDV or at least one functional fragment of at least one Replikin peptide sequence identified in HFMDV.
  • the immunogenic and/or blocking compound or composition comprises at least one peptide sequence of SEQ ID NO(s): 1-22. In a further non-limiting embodiment, the immunogenic and/or blocking compound or composition comprises at least one peptide consisting essentially of any one of SEQ ID NO(s): 1-22. In further non-limiting embodiment, the immunogenic and/or blocking compound or composition comprises at least one peptide consisting of any one of SEQ ID NO(s): 1-22 or at least one functional fragment of any one of SEQ ID NO(s): 1-22. In a non- limiting embodiment, the compound is a blocking compound. In a non-limiting embodiment, the compound blocks infection of a host cell by an HFMDV. In a non-limiting embodiment, the compound blocks replication of an HFMDV in a host cell. In a non-limiting embodiment, the compound blocks excretion of an HFMDV from a host.
  • a non- limiting embodiment of the second aspect of the invention provides a nucleic acid sequence that is antisense to a nucleic acid that encodes for any Replikin peptide, homologue, or functional fragment thereof present in or identified in an HFMDV isolate.
  • This may include at least one of SEQ ID NO(s): 1-22 or a small interfering nucleic acid sequence that interferes with a nucleic acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes any Replikin peptide homologue or functional Replikin fragment of HFMDV including, for example, any one of SEQ ID NO(s): 1-22 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any one of SEQ ID NO(s): 1-22 or any functional fragment thereof.
  • a non- limiting embodiment of the second aspect of the present invention provides a method of making a preventive or therapeutic HFMDV vaccine comprising identifying at least one Replikin sequence or at least one functional fragment of the at least one Replikin sequence in at least one protein, polypeptide, or peptide of HFMDV wherein the at least one Replikin sequence comprises 7 to 50 amino acid residues and comprises (1) at least one lysine residue six to ten amino acid residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues, and making the virus vaccine comprising the at least one protein, polypeptide, or peptide of HFMDV.
  • the at least one protein, polypeptide, or peptide is isolated or synthesized and combined with a pharmaceutically- acceptable carrier, excipient, and/or adjuvant to make the HFMDV vaccine.
  • the at least one Replikin sequence is conserved for at least two consecutive years in isolates of HFMDV.
  • the at least one Replikin sequence consists of 7 to 50 amino acid residues.
  • the at least one Replikin sequence has at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence.
  • the at least one Replikin sequence is the shortest sequence comprising the elements of the Replikin sequence.
  • the Replikin sequence is shared among at least two viruses that cause HFMD.
  • Another non-limiting embodiment of the second aspect of the present invention provides a method of making a HFMDV vaccine comprising identifying at least one Replikin sequence or at least one functional fragment of at least one Replikin sequence in an amino acid sequence of a HFMDV, wherein said at least one Replikin sequence comprises 7 to 50 amino acid residues and comprises (1) at least one lysine residue six to ten amino acid residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues, and making the HFMDV vaccine comprising the at least one Replikin sequence.
  • the at least one Replikin sequence consists of 7 to 50 amino acids.
  • the at least one Replikin sequence has at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence.
  • the Replikin sequence is the shortest sequence comprising the elements of the Replikin.
  • Another non-limiting embodiment of the second aspect of the present invention provides a method of preventing, mitigating, or treating HFMDV infection or an outbreak of HFMDV infection comprising administering at least one compound comprising an HFMDV Replikin peptide to a subject.
  • the compound may comprise a protein, protein fragment, other polypeptide, or peptide comprising an HFMDV Replikin peptide sequence.
  • Another non- limiting embodiment of the second aspect of the present invention provides a compound comprising an HFMDV Replikin peptide or functional fragment thereof for use in the treatment of HFMD.
  • the compound is a protein, protein fragment, polypeptide, or peptide.
  • a non- limiting embodiment of the second aspect of the present invention provides a method of stimulating the immune system of a subject comprising administering at least one HFMDV Replikin peptide or at least one polypeptide comprising said at least one HFMDV Replikin peptide to a subject to stimulate the immune system.
  • Another non- limiting embodiment of the second aspect of the present invention provides an isolated antibody that specifically binds at least a portion of an HFMDV Replikin peptide.
  • a non- limiting embodiment of the second aspect of the present invention provides an antagonist of the replication of a HFMDV wherein said antagonist interacts with at least a portion of an HFMDV Replikin peptide sequence.
  • the antagonist binds at least a portion of an HFMDV Replikin peptide sequence.
  • the antagonist is an antibody.
  • a third non- limiting aspect of the present invention provides an isolated or synthesized Replikin Peak Gene of an HFMDV wherein said Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein, or protein fragment of the virion of the virus.
  • the Replikin Peak Gene is the portion of a protein or protein fragment consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to all other proteins or protein fragments in the virion of the virus.
  • a non- limiting embodiment of the third aspect of the present invention provides an immunogenic composition comprising an isolated or synthesized Replikin Peak Gene.
  • the immunogenic composition comprises a Replikin sequence identified within a Replikin Peak Gene.
  • a non-limiting embodiment of the third aspect of the present invention provides an isolated or synthesized HFMDV peptide or polypeptide comprising an HFMDV Replikin Peak Gene.
  • the isolated or synthesized peptide or polypeptide is an HFMDV protein or protein fragment comprising a Replikin Peak Gene.
  • the peptide or polypeptide consists essentially of an HFMDV Replikin Peak Gene.
  • a non- limiting embodiment of the third aspect of the present invention provides an immunogenic composition comprising at least one HFMDV Replikin peptide.
  • the immunogenic composition comprises a pharmaceutically- acceptable carrier, excipient, and/or adjuvant.
  • a non- limiting embodiment of the third aspect of the present invention provides a method of stimulating the immune system of a subject comprising administering at least one HFMDV Replikin Peak Gene or at least one polypeptide comprising said HFMDV Replikin Peak Gene to the subject to stimulate the immune system.
  • a non- limiting embodiment of the third aspect of the present invention provides a vaccine against HFMDV comprising at least one HFMDV Replikin Peak Gene.
  • the vaccine comprises an isolated or synthesized HFMDV polypeptide comprising an HFMDV Replikin Peak Gene.
  • the isolated or synthesized polypeptide is an HFMDV protein or protein fragment comprising a Replikin Peak Gene.
  • the vaccine comprises a peptide or polypeptide consisting essentially of an HFMDV Replikin Peak Gene or plurality of HFMDV Replikin Peak Genes.
  • Another non-limiting embodiment of the third aspect of the present invention provides an isolated antibody or other antagonist that specifically binds at least a portion of an HFMDV Replikin Peak Gene.
  • a non- limiting embodiment of the third aspect of the present invention provides an antagonist of the replication of HFMDV wherein said antagonist interacts with at least a portion of an HFMDV Replikin Peak Gene.
  • the antagonist binds at least a portion of an HFMDV Replikin Peak Gene.
  • the antagonist is an antibody.
  • a fourth non- limiting aspect of the invention provides a method of preventing, mitigating, or treating an outbreak of HFMDV comprising
  • predicting an expansion of a strain of HFMDV comprising (1) determining a mean Replikin concentration and a standard deviation of said mean Replikin concentration for a plurality of isolates of a strain of HFMDV for a first time period in a first geographic region, (2) determining a Replikin concentration of at least one isolate of the same or a related strain of HFMDV 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 concentration of said at least one isolate is greater than one standard deviation of the mean of the Replikin concentration of the plurality of isolates isolated in said first time period and in said first geographic region; and
  • 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.
  • 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 sequence.
  • said protein or protein fragment is a Replikin peptide.
  • said protein or protein fragment consists essentially of a Replikin peptide or a Replikin Peak Gene.
  • 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 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.
  • a fifth non- limiting aspect of the present invention provides a method of predicting an expansion of a strain of HFMDV comprising
  • Figure 1 illustrates annual mean Replikin concentration of all protein sequences (available to applicants at www.pubmed.com from all available regions) of isolates of viruses reported to cause Hand Foot and Mouth Disease (HFMDV).
  • HFMDV Hand Foot and Mouth Disease
  • FIG 1 wider, gray bars with gradient shading represent annual mean Replikin concentration and thinner, darker, solid gray bars above the annual mean Replikin concentration represent standard deviation from the annual mean.
  • Table 1 contains the data reflected in Figure 1.
  • Replikin concentrations were determined for all genomic sequences available to applicants at ww . pubmed . com for isolates of HFMDV from all available regions globally.
  • Annual increase in genomic Replikin concentration in HFMDV as illustrated in Figure 1 was observed to be followed in the next year by increases in human outbreaks of HFMDV and increases in human mortality from HFMDV infections. See Table 2 and Figures 2 and 3. Applicants determined that Replikin concentration correlated with and predicted rises in disease incidence and resulting human mortality.
  • Figure 2 illustrates total recorded morbidity in humans of HFMDV for 2008 through February 9, 2013 as reported through various sources gathered at Wikipedia and from the World Health Organization (WHO). See Wikipedia.org Hand, foot and mouth disease and http://www.wpro.who.int emerging diseases/HFMD/en/iridex.html.
  • the data for 2010 reflect data only from January through June 2010.
  • the data for 2011 reflect data only from January through October 2011.
  • the data for 2012 reflect data only from January through July 2012.
  • the data for 2012-2013 reflect data only from January 1, 2012 through February 9, 2013.
  • the data are provided in tabular form in Table 2.
  • Figure 3 illustrates total recorded deaths in humans from HFMDV infections for 2008 through February 9, 2013, as reported through various sources gathered at Wikipedia and from the World Health Organization (WHO). See Wikipedia.org Hand, foot and mouth disease and http://www.wpro.who.int emerging diseases/HFMD/en/iridex.html.
  • the data for 2010 reflect data only from January through June 2010.
  • the data for 2011 reflect data only from January through October 2011.
  • the data for 2012 reflect data only from January through July 2012.
  • the data for 2012-2013 reflect data only from January 1, 2012 through
  • a "Replikin sequence” is an amino acid sequence of 7 to about 50 amino acids comprising (1) a first lysine residue located six to ten residues from a second lysine residue, (2) at least one histidine residue; and (3) at least 6% lysine residues.
  • a Replikin sequence may have a lysine residue on one end of the sequence and a lysine residue or histidine residue on the other end of the sequence.
  • a Replikin sequence is the shortest amino acid sequence of 7 to 50 amino acid residues comprising (1) a first lysine residue located six to ten residues from a second lysine residue, (2) at least one histidine residue; and (3) at least 6% lysine residues.
  • the Replikin sequence may comprise any number of lysine residues and any number of histidine residues so long as any two lysine residues and any one histidine residue reflect the requirements of the Replikin sequence.
  • a Replikin sequence counted as part of the Replikin concentration of a sequence of amino acid residues may comprise overlapping Replikin sequences.
  • Replikin sequence can also refer to a nucleic acid sequence encoding an amino acid sequence having 7 to about 50 amino acids comprising:
  • amino acid sequence may have a lysine residue on one end of the sequence and a lysine residue or histidine residue on the other end of the sequence or may be the shortest amino acid sequence having any two lysine residues and any one histidine residue reflecting the requirements of the Replikin sequence.
  • Replikin CountTM 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 amino acid sequence or in a nucleic acid that encodes at least one Replikin peptide sequence.
  • a Hand Foot and Mouth Disease virus or HFMDV includes any virus known now or hereafter to cause Hand Foot and Mouth Disease (HFMD).
  • HFMDV includes, and it not limited to, viruses of the picornaviridae family including, and not limited to, viruses of the Enterovirus genus, such as polioviruses, coxsackieviruses, echoviruses, and enteroviruses.
  • 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.
  • a protein or peptide may be 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 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.
  • 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, 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.
  • Isolated or related words may also mean: identified within such a Replikin sequence identified within a larger polypeptide.
  • a Replikin sequence may be isolated in silico from any medium, including from a database such as the databases at www.pubmed.com.
  • “Functional derivatives" of the Replikins as described herein are fragments, variants, analogs, or chemical derivatives of Replikin sequences that retain at least a portion of the immunological cross reactivity with an antibody specific for the Replikin sequence.
  • a fragment of the Replikin peptide refers to any subset of the molecule.
  • a functional derivative fragment or "functional fragment” of the Replikin peptide or a Replikin Peak Gene is any subset of the molecule that retains at least a portion of immunological cross reactivity with an antibody specific for the Replikin peptide or Replikin Peak Gene.
  • Variant peptides may be made by direct chemical synthesis, for example, using methods well known in the art.
  • An analog of a Replikin to a non-natural protein substantially similar to either the entire protein or a fragment thereof.
  • Chemical derivatives of a Replikin contain additional chemical moieties not normally a part of the peptide or peptide fragment.
  • sequence identity As used herein, “homologous” or “homology” or “sequence identity” are used to indicate that an amino acid sequence or nucleic acid sequence exhibits substantial structural or functional equivalence with another sequence. Any structural or functional differences between sequences having sequence identity or homology will be de minimus; that is, they will not affect the ability of the sequence to function as indicated in the desired application. Structural differences are considered de minimus if there is a significant amount of sequence overlap or similarity between two or more different sequences or if the different sequences exhibit similar physical characteristics even if the sequences differ in length or structure.
  • Such characteristics include, for example, the ability to hybridize under defined conditions, or in the case of proteins, immunological cross-reactivity, similar enzymatic activity, etc. The ordinary skilled practitioner can readily determine each of these characteristics by art- known methods.
  • the sequences are aligned for optimal comparison purposes ⁇ e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or more of the length of a reference sequence is aligned for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences as compared to the total length of the sequence identified as a reference sequence.
  • 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.
  • a "Replikin CountTM Virus Expansion Index” or "RCVE Index” or a “Replikin CountTM Expansion Index” or “RCE Index” is the number of Replikin CountsTM 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 CountTM 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 CountTM 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.
  • 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 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.
  • a "Replikin Peak Gene” 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.
  • Replikin Peak Gene 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 as the Replikin Peak Gene. More than one RPG may be identified within a gene, gene segment, protein, or protein fragment.
  • 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 7 to about 50 amino acids comprising:
  • an RPG may include the protein or protein fragment that contains an identified RPG.
  • a Replikin concentration 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.
  • continuous Replikin sequences means a series of two or more Replikin sequences that are overlapped and/or are directly covalently linked.
  • expansion of a pathogen or a population of pathogen and “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 HFMDV, etc.) and/or an expansion of the population of a pathogen (e.g. , strain of P. falciparum, a strain of HFMDV, 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 HFMDV, etc.
  • an increase or decrease in "virulence” includes an increase or decrease in virulence, morbidity, lethality, host mortality, rate of replication, rate of distribution, and/or expansion of a pathogen, such as an HFMDV.
  • 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.
  • One non-limiting aspect of the present invention is, therefore, a method of forecasting rise in incidence, virulence, and morbidity in HFMDV in humans.
  • SEQ ID NO(s): 2, 3, and 7 have to date been observed to be shared among EV-71 and Coxsackie virus. Because these sequences are shared by different viral pathogens that together are the predominant agent known to cause Hand Foot and Mouth Disease and because these sequences are Replikin sequence and, therefore, known to be involved in rapid replication and virulence in these viruses, SEQ ID NO(s): 2, 3, and 7 represent a non- limiting aspect of the invention that are an excellent targets for control of Hand Foot and Mouth Disease (HFMD) across HFMD causing viruses such as EV-71 and Coxsackie virus as well as other viruses that cause HFMD.
  • HFMD Hand Foot and Mouth Disease
  • any shared sequence including any of SEQ ID NO(s): 2, 3, and 7 (as well as any other sequences shared among causative agents in Hand Foot and Mouth disease) as an isolated or synthesized peptide in a therapeutic composition
  • inclusion of a protein, protein fragment, other polypeptide or peptide comprising any shared sequence (such as SEQ ID NO(s): 2, 3, or 7) in a therapeutic composition is likewise a non-limiting aspect of the invention.
  • One element of controlling Hand Foot and Mouth Disease is to target sequences involved in rapid replication and shared across the various causative agents.
  • a therapeutic composition comprising SEQ ID NO(s): 2, 3, or 7 or any combination of these sequences or other shared sequences as a target for control of the causal agents of HFMD is likewise a non- limiting aspect of the invention.
  • SEQ ID NO(s): 2, 3, and 7 have presently been observed to be shared among EV-71 and Coxsackie virus as Hand Foot and Mouth Disease causative pathogens. It is further observed that SEQ ID NO: 2 has so far first been observed in EV-71 isolates from 1999. SEQ ID NO: 3 has so far first been observed in EV-71 isolates from 2000. SEQ ID NO: 7 has so far first been observed in EV-71 isolates from 1966. Surprisingly, SEQ ID NO(s) 2, 3, and 7 have all so far first been observed in isolates from 2008. The movement of these sequences from EV-71 into Coxsackie virus around 2008 is postulated, wherein shared Replikin sequences may be a causative agent (or accompanying a causative agent) in manifestation of Hand Foot and Mouth Disease.
  • the vaccine comprises at least one sequence of SEQ ID NO(s): 1-22.
  • the vaccine may further comprise more than one sequence of SEQ ID NO(s): 1-22 and may comprise each sequence of SEQ ID NO(s): 1-22.
  • the vaccine may further comprise a functional fragment of any of SEQ ID NO(s): 1-22.
  • the vaccine may further comprise a polypeptide, protein fragment, or protein comprising at least one of SEQ ID NO(s): 1-22 or a functional fragment of SEQ ID NO(s): 1-22.
  • the vaccine may comprise a pharmaceutically-acceptable carrier and/or adjuvant including sterile water or other carrier and may be administered by any method known to one of skill in the art including spray to the eyes or nose or parenteral administration.
  • the vaccine may comprise equal parts by weight of each of SEQ ID NO(s): 1-22.
  • a vaccine comprising a homologue of any one of SEQ ID NO(s): 1-22 may be directed at outbreaks of HFMDV.
  • Vaccines may be administered by any mode that contacts immune-responding cells of a host or that provides blocking of the virus at entry, replication, or excretion.
  • a vaccine may be administered orally, via protected encapsulated oral administration, nasally, via respiratory administration, and any form of mucosal
  • the vaccines provided in this application are directed against conserved mechanisms of rapid replication in HFMDV. As a result, previous failures of vaccine attempts are overcome by direct targeting of rapid replication.
  • One aspect of the present invention provides methods of determining the relative virulence, replication rate, and/or lethality of an isolate, strain, or population of a Hand Foot and Mouth Disease virus (HFMDV). Another aspect of the present invention also provides compounds for preventing, treating, and diagnosing HFMDV. A further aspect of the present invention provides methods of identifying Replikin sequences and Replikin Peak Gene sequences to be comprised in therapies and diagnostics for HFMDV. An aspect of the present invention also provides methods of predicting outbreaks of HFMDV.
  • the examples herein are illustrative of the uses of Replikin sequences and Replikin Peak Gene sequences in the invention and do not limit the scope of the invention described herein. One of ordinary skill in the art will understand the full scope of utilities of the Replikin sequences and Replikin Peak Gene sequences described herein based on the description and examples provided herein.
  • An aspect of the present invention provides methods of determining the relative virulence of an isolate, plurality of isolates, or population of an HFMDV.
  • Relative virulence in an HFMDV includes increased or decreased morbidity, lethality, host mortality or viral lethality, rate of replication, rate of distribution, expansion of a pathogen and/or any metric known to one of skill in the art concerning the virulence of a virus.
  • An embodiment of the first aspect of the present invention provides a method of determining the relative virulence of an isolate A of a HFMDV by determining the Replikin concentration of at least one amino acid sequence isolated from isolate A of a HFMDV and comparing the Replikin
  • isolate A is determined to be relatively less virulent than isolate B.
  • the at least one amino acid sequence A may be encoded in the same region of the genome of the virus as said at least one amino acid sequence B.
  • the at least one amino acid sequence A and the at least one amino acid sequence B may be isolated from the same protein of a HFMDV.
  • the sequence A and sequence B may also be isolated from the same region of the same protein.
  • Sequence A and B may also be the amino acid sequence of the same protein in isolate A and isolate B, respectively.
  • Sequence A may also be a Replikin Peak Gene of isolate A and sequence B may be a Replikin Peak Gene of isolate B.
  • At least one sequence A may also be plurality of sequences A isolated from a plurality of isolates of HFMDV wherein a mean Replikin concentration of said plurality of sequences A is compared to the Replikin concentration of said at least one amino acid sequence B.
  • At least one sequence B may also be a plurality of sequences B isolated from a plurality of isolates of HFMDV where a mean Replikin concentration of the plurality of sequences B is compared to the Replikin concentration of the at least one amino acid sequence A.
  • a mean Replikin concentration of a plurality of sequences A may also be compared to a mean Replikin concentration of a plurality of sequences B.
  • a plurality of sequences A may also be isolated from a plurality of isolates of virus population A and a plurality of sequences B may be isolated from a plurality of isolates of virus population B where virus population A is determined to be relatively more virulent than virus population B if the mean Replikin concentration of the plurality of sequences A is greater than the mean Replikin concentration of the plurality of sequences B or virus population A may be determined to be relatively less virulent than virus population B if the Replikin concentration of the plurality of sequences A is less than the Replikin concentration of the plurality of sequences B.
  • Virus population A may be a population of a strain A of a HFMDV and virus population B may be a population of a strain B of a HFMDV where strain A is any particular strain of a HFMDV and strain B is any particular strain of a HFMDV that is different from strain A.
  • Strain A may differ from strain B based on physical or genetic differences between strain A and strain B or may differ based on the time or region in which strain A was isolated as compared to the time or region in which strain B was isolated. Strain A may also differ from strain B based on any difference understood by one of skill in the art.
  • virus population A may be a population of a particular strain of HFMDV isolated within a time period A and virus population B may be a population of the same strain of HFMDV isolated within a time period B.
  • Time period A may be a particular month and time period B a different month.
  • Time period A may be a particular year and time period B a different year.
  • Time periods A and B may also represent different calendar years.
  • Another embodiment of the first aspect of the present invention provides a method of determining an increased probability of an outbreak of HFMDV within about six months to about three years following an increase in Replikin concentration in at least one isolate of HFMDV comprising identifying said increase in the concentration of Replikin sequences in at least one first isolate of a HFMDV as compared to at least one other isolate of a HFMDV wherein said at least one first isolate is isolated at a later time point than said at least one other isolate is isolated, and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of HFMDV within about six months to about three years following the increase in the concentration of Replikin sequences.
  • said at least one first isolate is isolated at least one month later than said at least one other isolate. In a further embodiment, said at least one first isolate is isolated at least six months later than said at least one other isolate. In a further embodiment, said at least one first isolate is isolated at least one year or more later than said at least one other isolate. In one non- limiting embodiment, the increase in Replikin concentration in a population of a HFMDV predicts an increase in virulence in the virus in about one year.
  • the invention provides a method of determining an increased probability of an outbreak of HFMDV comprising:
  • the outbreak of HFMDV is predicted within about six months to about five years. In a further embodiment of the invention, the outbreak of HFMDV is predicted within about one year to about three years. In a further embodiment, the outbreak of HFMDV is predicted within about one year.
  • a non- limiting aspect of the present invention provides an isolated or synthesized Replikin peptide of HFMDV.
  • An HFMDV Replikin peptide consists of 7 to about 50 amino acid residues and comprises (1) at least one lysine residue located 6 to 10 residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues.
  • An isolated or synthesized Replikin peptide may also have at least one lysine residue on one end of the peptide and at least one lysine residue or at least one histidine residue on the other end of the peptide.
  • An isolated or synthesized Replikin peptide may have at least one lysine residue on one end of the peptide and at least one lysine residue on the other end of the peptide or may have at least one lysine residue on one end of the peptide and at least one histidine residue on the other end of the peptide.
  • Replikin peptide may contain as few as two lysine residues and may contain all lysines with the exception of one histidine.
  • the isolated or synthesized Replikin peptide may contain as few as one histidine residue and may contain many histidine residues so long as the peptide contains at least 6% lysines.
  • An isolated or synthesized Replikin peptide sequence may be the shortest sequence that comprises the elements of the Replikin peptide.
  • a non- limiting embodiment provides a homologue of a Replikin peptide sequence identified in an isolate of an HFMDV.
  • the homologue may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more homologous with the Replikin sequence.
  • a homologue of a Replikin peptide sequence may have the same lysine residues and histidine residue required to define the Replikin peptide sequence where the residue positions between the required lysine and histidine residues may have differing amino acids.
  • a non- limiting embodiment also provides an isolated HFMDV polypeptide comprising an HFMDV Replikin peptide or a synthesized polypeptide comprising an HFMDV Replikin peptide.
  • the synthesized polypeptide may be derived from a polypeptide of HFMDV as understood by one of skill in the art.
  • the synthesized polypeptide may be homologous with an HFMDV polypeptide.
  • the synthesized or isolated polypeptide may be an HFMDV protein or protein fragment comprising a Replikin peptide.
  • a synthesized or isolated peptide or polypeptide may consist essentially of an HFMDV Replikin peptide.
  • an isolated or synthesized peptide or polypeptide may comprise a plurality of Replikin peptides or a mixture of at least one Replikin peptide and at least one Replikin Peak Gene.
  • An isolated or synthesized peptide or polypeptide may likewise consist essentially of a plurality of Replikin peptides or a mixture of at least one Replikin peptide and at least one Replikin Peak Gene.
  • An isolated or synthesized peptide or polypeptide may also consist of a plurality of Replikin peptides or a mixture of at least one Replikin peptide and at least one Replikin Peak Gene.
  • a non- limiting embodiment of an aspect of the present invention provides a composition comprising at least one HFMDV Replikin peptide.
  • the composition may comprise a pharmaceutically-acceptable carrier and/or adjuvant.
  • Another non-limiting embodiment of the aspect of the present invention provides an immunogenic composition comprising at least one HFMDV Replikin peptide.
  • the immunogenic composition may comprise a pharmaceutically-acceptable carrier and/or adjuvant and/or excipient.
  • a non- limiting embodiment provides a vaccine against HFMDV.
  • a vaccine may comprise at least one HFMDV Replikin sequence.
  • a vaccine may also comprise an isolated or synthesized HFMDV polypeptide comprising an HFMDV Replikin peptide.
  • the isolated or synthesized polypeptide may be an HFMDV protein or protein fragment comprising a Replikin peptide.
  • a vaccine may comprise a peptide or polypeptide consisting essentially of an HFMDV Replikin peptide or plurality of HFMDV Replikin peptides or may comprise a mixture of at least one Replikin peptide and at least one Replikin Peak Gene.
  • the vaccine may comprise at least one polypeptide or peptide comprising any one of SEQ ID NO(s): 1-22, at least one polypeptide or peptide consisting essentially of any one of SEQ ID NO(s): 1-22, or at least one polypeptide or peptide consisting of any one of SEQ ID NO(s): 1-22.
  • a non-limiting embodiment of the aspect of the present invention provides a method of making a preventive or therapeutic HFMDV vaccine comprising identifying at least one Replikin sequence or at least one functional derivative fragment of the at least one Replikin sequence in at least one protein, polypeptide, or peptide of HFMDV where the at least one Replikin sequence comprises 7 to 50 amino acid residues and comprises (1) at least one lysine residue six to ten amino acid residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues, and making the virus vaccine comprising the at least one protein, polypeptide, or peptide of HFMDV.
  • the at least one protein, polypeptide, or peptide in which said at least one Replikin sequence or said at least one functional fragment of said at least one Replikin sequence is isolated or synthesized and further combined with a pharmaceutically acceptable carrier and/or adjuvant to make the HFMDV vaccine.
  • the at least one Replikin sequence may be conserved in HFMDV and further may be conserved for at least two consecutive years.
  • the at least one Replikin sequence may consist of 7 to 50 amino acid residues.
  • the at least one Replikin sequence may have at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence or may be the shortest sequence comprising the elements of the Replikin sequence.
  • Another non-limiting embodiment provides a vaccine comprising a protein or protein fragment comprising a Replikin sequence.
  • a vaccine comprising a protein fragment of up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acid residues on either or both termini of at least one Replikin sequence.
  • Another non- limiting embodiment provides a vaccine comprising a homologue of a Replikin sequence identified in an HFMDV that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more homologous with the Replikin sequence.
  • Another non-limiting embodiment of the aspect of the present invention provides a method of preventing, mitigating, or treating HFMDV infection or an outbreak of HFMDV infection comprising administering at least one compound comprising an HFMDV Replikin peptide to a subject.
  • the compound may comprise a protein, protein fragment, other polypeptide, or peptide comprising an HFMDV Replikin peptide.
  • the compound may comprise a protein, protein fragment, other polypeptide, or peptide consisting essentially of an HFMDV Replikin peptide.
  • a non- limiting embodiment of the aspect of the present invention provides a method of stimulating the immune system of a subject comprising administering at least one HFMDV Replikin peptide to stimulate the immune system.
  • the immune system is stimulated against HFMDV.
  • the immune system is stimulated to provide protection of the subject against infection of HFMDV.
  • the subject is a human.
  • Another non-limiting embodiment of the aspect of the present invention provides an antibody that binds at least a portion of an HFMDV Replikin peptide.
  • the antibody specifically binds at least a portion of an HFMDV Replikin peptide.
  • the antibody specifically binds to a portion of an HFMDV Replikin peptide with at least two-times more affinity than it binds to another HFMDV epitope.
  • the antibody specifically binds with at least 10-times more affinity than it binds to another HFMDV epitope.
  • the antibody is an isolated antibody.
  • the antibody is a single chain antibody, a chimeric antibody, or an antibody fragment.
  • a non- limiting embodiment of the aspect of the present invention provides an antagonist of the replication of HFMDV wherein said antagonist interacts with at least a portion of an HFMDV Replikin peptide.
  • the antagonist binds at least a portion of an HFMDV Replikin peptide.
  • a non- limiting embodiment of an aspect of the present invention provides a method of antagonizing the replication of HFMDV comprising administering to a subject an agent capable of binding at least a portion of an HFMDV Replikin peptide.
  • the agent binds at least a portion of an HFMDV Replikin peptide comprised within a protein, protein fragment, other polypeptide or peptide.
  • the agent binds a portion of the HFMDV Replikin peptide and a portion of the protein, protein fragment, other polypeptide, or peptide comprising the HFMDV Replikin peptide.
  • the agent is an antibody.
  • the antibody is a chimerized antibody, a single chain antibody, or an antibody fragment.
  • Another non-limiting aspect of the present invention provides an isolated or synthesized peptide that is an immunogenic fragment of an HFMDV Replikin sequence.
  • Imunogenic fragments of an HFMDV Replikin sequence are fragments that provide at least a portion of cross-reactivity with an antibody or antibody fragment against the HFMDV Replikin sequence.
  • One embodiment of the present invention provides an isolated or synthesized immunogenic fragment of at least one HFMDV Replikin sequence as a target for control of replication of the virus.
  • One embodiment provides the isolated or synthesized immunogenic fragment as an antigen for stimulating the immune system of a subject, including but not limited to a human, to produce antibodies against the fragment.
  • Another embodiment of the invention provides the isolate or synthesized immunogenic fragment as a vaccine or as a component of a vaccine in combination with an acceptable pharmaceutical carrier or in combination with other proteins, peptides, immunogenic substances, and/or adjuvant(s).
  • Another non-limiting aspect of the present invention provides an isolated or synthesized Replikin Peak Gene of HFMDV.
  • a non- limiting embodiment provides an isolated or synthesized Replikin Peak Gene for diagnosis, prevention or treatment of an outbreak of HFMDV prepared by the method comprising:
  • a Replikin sequence from the Replikin Peak Gene or a peptide consisting essentially of the Replikin sequence from the Replikin Peak Gene or a fragment of the Replikin Peak Gene may be isolated or synthesized for diagnostic, preventive, or therapeutic purposes.
  • Another non-limiting embodiment provides an immunogenic composition comprising an isolated or synthesized Replikin Peak Gene or comprising a Replikin sequence identified within a Replikin Peak Gene.
  • a non-limiting embodiment of this aspect of the present invention also provides an isolated or synthesized HFMDV polypeptide comprising an HFMDV Replikin Peak Gene.
  • the isolated or synthesized polypeptide may be an HFMDV protein or protein fragment comprising a Replikin Peak Gene.
  • the peptide or polypeptide may consist essentially of an HFMDV Replikin Peak Gene.
  • a non- limiting embodiment of this aspect of the present invention provides a composition comprising at least one HFMDV Replikin Peak Gene.
  • the composition may also comprise a pharmaceutically- acceptable carrier, excipient, and/or adjuvant.
  • a non- limiting embodiment of this aspect of the present invention may provide an immunogenic compound comprising at least one HFMDV Replikin peptide and may comprise a pharmaceutically-acceptable carrier, excipient, and/or adjuvant.
  • a non- limiting embodiment of this aspect of the present invention provides a method of stimulating the immune system of a subject comprising administering at least one HFMDV Replikin Peak Gene to stimulate the immune system.
  • the immune system is stimulated with a Replikin sequence identified within the Replikin Peak Gene.
  • the immune system is stimulated against HFMDV.
  • the immune system is stimulated to provide protection of the subject against infection of HFMDV.
  • a non- limiting embodiment of this aspect of the present invention provides a vaccine against HFMDV.
  • the vaccine may comprise at least one isolated or synthesized HFMDV Replikin Peak Gene or may comprise an isolated or synthesized HFMDV polypeptide comprising an HFMDV Replikin Peak Gene.
  • the isolated or synthesized polypeptide may be an HFMDV protein or protein fragment comprising a Replikin Peak Gene or may consist essentially of an HFMDV Replikin Peak Gene or plurality of HFMDV Replikin Peak Genes.
  • a non- limiting embodiment of the aspect of the present invention provides a method of making a preventive or therapeutic HFMDV vaccine.
  • the method may comprise identifying at least one Replikin Peak Gene sequence or at least one functional derivative fragment of the at least one Replikin Peak Gene sequence in at least one protein, polypeptide, or peptide of HFMDV and making the virus vaccine comprising the at least one protein, polypeptide, or peptide of HFMDV.
  • the protein, polypeptide, or peptide in which said at least one Replikin Peak Gene sequence or said at least one functional derivative fragment of said at least one Replikin Peak Gene sequence may be isolated or synthesized and combined with a pharmaceutically-acceptable carrier, excipient, and/or adjuvant to make the HFMDV vaccine.
  • the at least one Replikin Peak Gene sequence may be conserved in HFMDV and may be conserved for at least two consecutive years.
  • the Replikin Peak Gene sequence may have at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence or may be the shortest sequence comprising the elements of a Replikin sequence.
  • Another non- limiting embodiment provides a method of preventing, mitigating, or treating HFMDV infection or an outbreak of HFMDV infection comprising administering to a subject at least one compound comprising an HFMDV Replikin peptide.
  • the compound may comprise a protein, protein fragment, other polypeptide, or peptide comprising an HFMDV Replikin peptide or may comprise a protein, protein fragment, other polypeptide, or peptide consisting essentially of an HFMDV Replikin peptide.
  • Another non- limiting embodiment provides an antibody that binds at least a portion of an HFMDV Replikin Peak Gene.
  • the antibody may be isolated from other biological or chemical contaminants or may be isolated from a subject.
  • the antibody may specifically bind at least a portion of an HFMDV Replikin Peak Gene and may bind with at least two-times more affinity than it binds another HFMDV epitope or may bind with at least 10-times more affinity than it binds to another HFMDV epitope.
  • An antibody may be a single chain antibody, a chimeric antibody, an antibody fragment, or any other portion or chimera of the antibody or any agent that behaves like an antibody.
  • a non- limiting embodiment provides an antagonist of the replication of HFMDV where the antagonist may interact with at least a portion of an HFMDV Replikin Peak Gene.
  • the antagonist may bind at least a portion of an HFMDV Replikin Peak Gene.
  • the antagonist may be an antibody.
  • a non-limiting embodiment provides a method of antagonizing the replication of HFMDV comprising administering to a subject an agent capable of binding at least a portion of an HFMDV Replikin Peak Gene.
  • the agent binds at least a portion of an HFMDV Replikin Peak Gene comprised within a protein, protein fragment, other polypeptide or peptide.
  • the agent binds a portion of the HFMDV Replikin Peak Gene and a portion of the protein, protein fragment, other polypeptide, or peptide comprising the HFMDV Replikin peptide.
  • Another non- limiting aspect of the present invention provides a method of preventing, mitigating, or treating an outbreak of HFMDV predicted to have an expansion of population.
  • the method comprises predicting an expansion of a population of HFMDV and administering a compound comprising an isolated or synthesized portion of the structure or genome of the HFMDV predicted to expand.
  • the method comprises:
  • a compound comprising an isolated or synthesized portion of the structure or genome of the HFMDV predicted to expand to mitigate, prevent, or treat the predicted outbreak of the pathogen.
  • a further embodiment provides a method of preventing, mitigating, or treating an outbreak of HFMDV 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 HFMDV as compared to another isolate or plurality of isolates of the same or a related strain of HFMDV comprising: (1) identifying a first cycle in the Replikin concentration of a plurality of isolates of said first strain of HFMDV, (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
  • 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.
  • the identified peak in the cycle of Replikin concentration may also have a higher Replikin concentration than a chronologically earlier peak in the cycle of Replikin concentration.
  • the identified peak in the cycle of Replikin concentration may be
  • a p value may be less than 0.01 or may be less than 0.001.
  • the isolated or synthesized portion of the structure or genome of the at least one isolate of HFMDV may be 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 HFMDV or may be 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 isolated or synthesized portion may consist essentially of a Replikin peptide or a Replikin Peak Gene.
  • Another non- limiting embodiment provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified as conserved in an isolate of HFMDV from among a plurality of isolates of HFMDV, 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.
  • Any of the above-listed or herein identified Replikin peptides may be comprised in an immunogenic compound of the invention.
  • a further non-limiting embodiment 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 HFMDV or an increase in virulence, morbidity, and/or mortality of HFMDV.
  • 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 HFMDV 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 computer system including a processor coupled to a network and a memory coupled to the processor, the memory containing a plurality of instructions to perform a method of predicting an increase in an HFMDV and/or a method of identifying and/or counting Replikin peptide sequences in an HFMDV.
  • a signal is provided containing a plurality of instructions to perform the method of predicting the lethality of HFMDV and/or the method of identifying and/or counting.
  • a computer-readable medium, a computer system, and a signal provide methods of predicting an expansion of a strain of an HFMDV or an increase in virulence, morbidity, and/or mortality of HFMDV employing regression analysis and/or statistical analysis.
  • a computer-readable medium, a computer system, and a signal provide methods of identifying and/or counting Replikin sequences in an HFMDV.
  • Changes in HFMDV virulence and mortality may be predicted by identifying a peak within an identified cycle in the Replikin concentration of isolates of a plurality of HFMDV and predicting an increase in the virulence, morbidity, and/or mortality of HFMDV 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.
  • Another non- limiting aspect provides a method of diagnosing HFMDV comprising identifying an HFMDV Replikin sequence or HFMDV Replikin Peak Gene sequence in an animal, including human, infected by HFMDV.
  • the blood or tissue of human or other animal may be screened for an HFMDV Replikin sequence or an HFMDV Replikin Peak Gene sequence.
  • an antibody or other binding agent may be used to screen for an HFMDV Replikin sequence or HFMDV Replikin Peak Gene sequence.
  • the blood or tissue of human or other animal may be screened for DNA or RNA encoding an HFMDV Replikin sequence or an HFMDV Replikin Peak Gene sequence.
  • Replikin or Replikin Peak Gene nucleotide sequences may be used in hybridization assays of biopsied tissue or blood, e.g., Southern or Northern analysis, including in situ hybridization assays, to diagnose the presence of HFMDV in a tissue sample or an environmental sample.
  • a further non- limiting embodiment contemplates kits containing antibodies specific for particular Replikins that are present in a particular pathogen of interest, or containing nucleic acid molecules (sense or antisense) that hybridize specifically to a particular Replikin, and optionally, various buffers and/or reagents needed for diagnosis.
  • the present invention also provides a method of predicting an expansion of a strain of pathogen such as HFMDV by (1) determining a mean Replikin concentration and a standard deviation of the mean Replikin concentration for a plurality of isolates of a strain of pathogen for a first time period in a first geographic region; (2) determining a Replikin concentration 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 concentration of the at least one isolate is greater than one standard deviation of the mean of the Replikin concentration 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 concentration 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 concentrations 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 concentrations 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 concentrations that are greater than one standard deviation of the mean divided by the number of Replikin concentrations that are less than one standard deviation of the mean.
  • the ratio is called a Replikin CountTM Expansion Index (RCE Index).
  • RCE Index Replikin CountTM Expansion Index
  • Another way to determine the RCE Index is to divide the percent of Replikin CountsTM 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 CountsTM 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 CountsTM in strains of pathogen over time.
  • the mean Replikin concentration 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 concentration 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 reflect a highest number of isolates reported in a year or in several years in a geographic area.
  • 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.
  • An aspect of the invention 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, vaccines that block virus entry, replication, and/or excretion, and vaccines that provide a protective effect.
  • 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.
  • 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 peptide vaccine may comprise a Replikin peptide or plurality of Replikin peptides, it may consist essentially of a Replikin peptide or a plurality of Replikin peptides, or may consist of a Replikin peptide or plurality of Replikin peptides.
  • 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.
  • a vaccine may likewise comprise a protein or protein fragment that comprises a Replikin peptide. The protein or protein fragment may be identified by identifying the Replikin peptide or a homologue of a Replikin peptide.
  • Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences.
  • Vaccine composition may comprise a pharmaceutically acceptable carrier and/or adjuvant.
  • Replikin peptides for use in a virus or pathogen vaccine are those Replikins observed to "re- emerge" after an absence from the amino acid sequence for one or more years.
  • the vaccines of an aspect 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 a particular strain of virus.
  • the Replikin peptides of the invention alone or in various combinations 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 or polypeptides comprising 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.
  • the skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.
  • Blocking agents may comprise any Replikins sequence identified in an HFMDV, any homologue of any Replikin sequence identified in HFMDV, or any functional fragment of any Replikin sequence identified in HFMDV.
  • Replikin sequences have been shown to block replication of viruses. See, e.g., Example 2 of US20090041795 published 12 February 2009 from U.S. Appln. Ser. No. 12/108,458, filed April 23, 2008, and Example 10 of US20090269367 published 29 October 2009 from U.S. Appln. Ser. No. 12/429,044, filed April 23, 2009.
  • This blocking mechanism allows for therapies that provide acute blocking against infection (within hours or a few days) and then, in addition, may provide an immune response against infection (within weeks, such as an initial response within about two weeks and more mature responses within about four weeks).
  • Replikin sequences, homologues, or functional fragments thereof interfere with the replication process within cells and the infection process between cells and provide a mechanism of stopping spread of the virus between hosts. This mechanism provides an acute response as soon as infection is observed in a population.
  • a blocking agent may comprise at least one protein, protein fragment, or peptide comprising a Replikin sequence, homologue, or functional fragment of a Replikin sequence of an HFMDV. It may consist essentially or at least one Replikin sequence, homologue, or functional fragment thereof. It may consist of at least one Replikin sequence, homologue, or functional fragment thereof. It may comprise a plurality of peptides each consisting of or consisting essentially of at least one Replikin sequence, homologue, or functional fragment thereof. It may comprise a mixture of many peptides consisting of or consisting essentially of at least one Replikin sequence, homologue, or functional fragment thereof.
  • One non- limiting aspect of the invention provides a nucleic acid sequence that is antisense to a nucleic acid that encodes for any Replikin peptide, homologue, or functional fragment thereof present in or identified in an HFMDV isolate.
  • This may include at least one of SEQ ID NO(s): 1-22 or a small interfering nucleic acid sequence that interferes with a nucleic acid sequence that is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes any Replikin peptide of HFMDV including, for example, any one of SEQ ID NO(s): 1-22 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any one of SEQ ID NO(s): 1-22 or any functional fragment thereof.
  • oligoribonucleotide sequences that include antisense RNA and DNA molecules and ribozymes that function to inhibit the translation of Replikin-containing mRNA.
  • Both antisense RNA and DNA molecules and ribozymes may be prepared by any method known in the art.
  • the antisense molecules can be incorporated into a wide variety of vectors for delivery to a subject. The skilled practitioner can readily determine the best route of delivery, although generally intravenous or intramuscular delivery is routine. The dosage amount is also readily ascertainable.
  • An aspect of the invention further provides antisense nucleic acid molecules that are complementary to a nucleic acid of the invention, wherein the antisense nucleic acid molecule is complementary to a nucleotide sequence encoding a peptide of the invention.
  • the nucleic acid sequence may be anti-sense to a nucleic acid sequence
  • compositions comprising RNAi- inducing entities used to inhibit or reduce HFMDV infection or replication including small interfering RNA, which is a class of about 10 to about 50 and often about 20 to about 25 nucleotide-long double- stranded RNA molecules. siRNA is involved in the RNA
  • compositions of the invention may comprise a single siRNA species targeted to a target transcript or may comprise a plurality of different siRNA species targeting one or more target transcripts.
  • the invention provides a small interfering nucleic acid sequence that is about 10 to about 50 nucleic acids in length and is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that encodes for any portion of an HFMDV Replikin peptide including, for example, any portion of SEQ ID NO(s): 1-22 or is 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more homologous with a nucleic acid that is antisense to a nucleic acid that encodes for any portion of a Replikin peptide, including, for example, a portion of one of SEQ ID NO(s): 1-22.
  • the nucleic acid sequence is about 15 to about 30 nucleic acids. In a further non- limiting embodiment, the nucleic acid sequence is about 20 to about 25 nucleic acids. In a further non-limiting embodiment, the nucleic acid sequence is about 21 nucleic acids.
  • isolated Replikin peptides may be used to generate antibodies, which may be used, for example for diagnostic purposes, to identify protein or protein fragments of interest for development of vaccines and other therapies, or, for example, to provide passive immunity in an subject.
  • Various procedures known in the art may be used for the production of antibodies to Replikin sequences.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Antibodies that are linked to a cytotoxic agent may also be generated.
  • Antibodies may also be administered in combination with an antiviral agent.
  • combinations of antibodies to different Replikins may be administered as an antibody cocktail.
  • An antibody of the invention may bind to a Replikin peptide or a Replikin Peak Gene. It may bind to a protein or protein fragment comprising a Replikin peptide or a Replikin Peak Gene. It may also bind to a portion of a Replikin peptide or a portion of a Replikin Peak Gene or a portion of a protein, protein fragment, polypeptide, or peptide comprising a Replikin peptide or Replikin Peak Gene.
  • An antibody that specifically binds to a portion of a Replikin peptide or a portion of a Replikin Peak Gene generally binds to an epitope on the Replikin peptide or an epitope that is at least partially on the Replikin peptide or to an epitope on the Replikin Peak Gene or an epitope that is at least partially on the Replikin Peak Gene when the antibody or fragment of the antibody binds to the epitope more readily than it would bind to a random, unrelated epitope.
  • Monoclonal antibodies to Replikins may be prepared by using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, ⁇ Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81 :6851- 6855) or other techniques may be used. Alternatively, techniques described for the production of single chain antibodies (U.S. 4,946,778) can be adapted to produce Replikin- specific single chain antibodies.
  • Antibodies to any peptides observed to be present in an emerging or re- emerging strain of virus and combinations of such antibodies are useful in the treatment and/or prevention of viral infection, including Replikin peptides, Replikin Peak Gene peptides, and Replikin sequences isolated within Replikin Peak Gene peptides.
  • Antibody fragments that contain binding sites for a Replikin may be generated by known techniques.
  • fragments include but are not limited to F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be generated (Huse et al., 1989, Science,
  • immune serum containing antibodies to one or more Replikins obtained from an individual exposed to one or more Replikins may be used to induce passive immunity in another individual or animal.
  • Immune serum may be administered via i.v. to a subject in need of treatment.
  • Passive immunity also can be achieved by injecting a recipient with preformed antibodies to one or more Replikins.
  • Passive immunization may be used to provide immediate protection to individuals who have been exposed to an infectious organism.
  • Administration of immune serum or preformed antibodies is routine and the skilled practitioner can readily ascertain the amount of serum or antibodies needed to achieve the desired effect.
  • One of the reasons that vaccines directed towards a particular protein antigen of a disease causing agent have not been fully effective in providing protection against the disease is that the best antibodies have not been produced, that is - it is likely that the antibodies to the Replikins have not been produced.
  • a vaccine may be formulated with a pharmaceutically acceptable excipient, carrier, or adjuvant.
  • a pharmaceutically acceptable carrier or excipient is water.
  • Excipients, carriers, or adjuvants may include, but are not limited to, excipients, carriers and adjuvants known to those of skill in the art now or hereafter.
  • compositions of an aspect of the invention may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (topical), transmucosal or any other routes.
  • parenteral e.g., intravenous
  • intradermal subcutaneous, oral, nasal, bronchial
  • ophthalmic e.g., transdermal (topical), transmucosal or any other routes.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • Solutions or suspensions used for intranasal, intraocular, spray inhalation, parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water (for dermal, nasal, or ocular application, spraying, or injection), saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use typically include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition should be sterile and should be fluid to the extent that easy syringability exists.
  • the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • Administration of the vaccine via any method may produce an immune response in the animal or human, it may further produce an antibody response in the animal or human.
  • the vaccine may produce a protective effect in the animal or human.
  • the vaccine of the present invention may be administered to a rabbit, a chicken, a shrimp, a pig, a ferret, a human, or any animal capable of an immune resposne. Because of the universal nature of Replikin sequences, a vaccine of the invention may be directed at a range of strains of HFMDV.
  • Replikin sequences for formulation in a vaccine were identified in isolates of virulent strains of HFMDV. The following twenty-two sequences were identified as excellent targets for controlling rapid replication and virulence in the virus. Any one or more of the following sequences may be comprised in a vaccine.
  • KHYPVSFSK (SEQ ID NO: 4)
  • HAQPGKTIWVEHK (SEQ ID NO: 6)
  • HFC KFQPLYATEAK (SEQ ID NO: 7)
  • HLIGSEGAVEKILKNLVK (SEQ ID NO: 9)
  • HLIGS DGAVEKILKNLIK (SEQ ID NO: 10)
  • HDVFEGNKEPAVLHSK (SEQ ID NO: 16)
  • a vaccine was designed comprising peptides of each of the sequences in equal parts by weight in sterile water.
  • the vaccine is administered in humans or animals via mucosal administration (protected encapsulated oral or respiratory administration) or parenteral injection.
  • the vaccine provides a block of the pathogen or an immune stimulant against the pathogen or both, thereby providing protection against HFMDV.
  • SEQ ID NO: 2 is shared by different viral pathogens that together are dominant agents known to cause Hand Foot and Mouth Disease and because SEQ ID NO: 2 is a Replikin sequence and known to be involved in rapid replication and virulence in these viruses, SEQ ID NO: 2 is an aspect of the invention that is a target for control of Hand Foot and Mouth Disease (HFMD) across HFMD causing viruses such as E V-71 and Coxsackie virus as well as other viruses that cause HFMD.
  • HFMD Hand Foot and Mouth Disease
  • SEQ ID NO: 2 is an isolated or synthesized peptide, or inclusion of a protein, protein fragment, other polypeptide or peptide comprising SEQ ID NO: 2, in a therapeutic composition, such as an immunogenic composition and/or vaccine, is a non- limiting aspect of the invention.
  • a therapeutic composition comprising SEQ ID NO: 2 as a target for control of the causal agents of HFMD in combination with other shared sequences as targets for control is likewise a non-limiting aspect of the invention.
  • SEQ ID NO(s): 2, 3, and 7 have presently been observed to be shared among EV-71 and Coxsackie virus as Hand Foot and Mouth Disease causative pathogens. It is further observed that SEQ ID NO: 2 has so far first been observed in EV-71 isolates from 1999. SEQ ID NO: 3 has so far first been observed in EV-71 isolates from 2000. SEQ ID NO: 7 has so far first been observed in EV-71 isolates from 1966. Surprisingly, SEQ ID NO(s) 2, 3, and 7 have all so far first been observed in isolates from 2008. The movement of these sequences from EV-71 into Coxsackie virus around 2008 is postulated, wherein shared Replikin sequences may be a causative agent (or accompanying a causative agent) in manifestation of Hand Foot and Mouth Disease.
  • Coxsackie virus isolates were queried at www.pudmed.com for the sequence KEPAVLHSK (SEQ ID NO: 3). Accession numbers of isolates having the sequence are identified in Table 3 below along with the year in which each isolate was reported to have been identified. The position in the reported sequence at which SEQ ID NO: 3 begins is also noted. SEQ ID NO: 3 appeared first in PubMed in 2000 in EV-71 isolates and in 2008 in Coxsackie virus isolates. Table 4 - Sharing of KEPAVLHSK (SEP ID NO: 3) within Genome of Isolates of EV-71 and Coxsackie Virus
  • a position 38 AFL65915 position position 221 , AFM85269 position 221 , 1769 .
  • ADE80721 position 1769, AFM85274 position 221 AM85272 ADE80719 position 1769, ADE80715 position 22 1 , AFM85270 position 221 , position 3769, ADE807 i position 1769 , AFM85268 position 22 1 , ADE80709 position 1769 , ADE80722 3N6M_A position 38, 3N6N_A position position 1769 , ADE80710 position 1769 38, 3N6N_ position 38, AFP57465
  • SEQ ID NO: 3 is shared by different viral pathogens that together are dominant agents known to cause Hand Foot and Mouth Disease and because SEQ ID NO: 3 is a Replikin sequence and known to be involved in rapid replication and virulence in these viruses, SEQ ID NO: 3 is an aspect of the invention that is a target for control of Hand Foot and Mouth Disease (HFMD) across HFMD causing viruses such as EV-71 and Coxsackie virus as well as other viruses that cause HFMD.
  • HFMD Hand Foot and Mouth Disease
  • SEQ ID NO: 3 is an isolated or synthesized peptide, or inclusion of a protein, protein fragment, other polypeptide or peptide comprising SEQ ID NO: 3, in a therapeutic composition, such as an immunogenic composition and/or vaccine, is a non- limiting aspect of the invention.
  • a therapeutic composition comprising SEQ ID NO: 3 as a target for control of the causal agents of HFMD in combination with other shared sequences as targets for control is likewise a non-limiting aspect of the invention.
  • HFCRKFQPLYATEAK (SEQ ID NO: 7). Accession numbers of isolates having the sequence are identified in Table 5 below along with the year in which each isolate was reported to have been identified. The position in the reported sequence at which SEQ ID NO: 7 begins is also noted. SEQ ID NO: 7 appeared first in PubMed in 1966 in EV-71 isolates and in 2008 in Coxsackie virus isolates.
  • SEQ ID NO: 7 is shared by different viral pathogens that together are a dominant agents known to cause Hand Foot and Mouth Disease and because SEQ ID NO: 7 is a Replikin sequence and known to be involved in rapid replication and virulence in these viruses, SEQ ID NO: 7 is an aspect of the invention as a target for control of Hand Foot and Mouth Disease (HFMD) across HFMD causing viruses such as EV-71 and Coxsackie virus as well as other viruses that cause HFMD.
  • HFMD Hand Foot and Mouth Disease
  • SEQ ID NO: 7 is an isolated or synthesized peptide, or inclusion of a protein, protein fragment, other polypeptide or peptide comprising SEQ ID NO: 7, in a therapeutic composition, such as an immunogenic composition and/or vaccine, is a non- limiting aspect of the invention.
  • a therapeutic composition comprising SEQ ID NO: 7 as a target for control of the causal agents of HFMD in combination with other shared sequences as targets for control is likewise a non-limiting aspect of the invention.
  • ADR70702 position 70 ADR70696 position 70 ADR70690 position 70 position 70 ,
  • ADR70678 position 70 ADR70672 position 70 ADR70666 position 70 position 70 ,
  • ADR70654 position 70 ADR70648 position 70 ADR70642 position 70 position 70 ,
  • ADR70630 position 70 ADR70624 position 70 ADR70618 position 70 position 70 ,
  • ADR70606 position 70 ADR70600 position 70 ADR70594 position 70 position 70 ,
  • ADR70582 position 70 ADR70576 position 70 ADR70570 position 70 position 70 ,
  • ADR70558 position 70 ADR70552 position 70 ADR70546 position 70 position 70 ,
  • ADR70534 position 70 ADR70528 position 70 ADR70522 position 70 position 70 ,
  • ADR70510 position 70 ADR70504 position 70 ADR70498 position 70 position 70 ,
  • ADR70480 position 70 ADR70474 position 70 ADR70468 position 70 position 70 ,
  • ADR70456 position 70 ADR70450 position 70 ADR70723 position 70 position 70 ,
  • ADR70711 position 70 ADR70705 position 70 ADR70699 position 70 position 70 ,
  • ADR70687 position 70 ADR70681 position 70 ADR70675 position 70 position 70 ,
  • ADR70663 position 70 ADR70657 position 70 ADR70651 position 70 position 70 ,
  • ADR70639 position 70 ADR70633 position 70 ADR70627 position 70 position 70 ,
  • ADR70615 position 70 ADR70609 position 70 ADR70603 position 70 position 70 ,
  • ADR70591 position 70 ADR70585 position 70 ADR70579 position 70 position 70 ,
  • ADR70567 position 70 ADR70561 position 70 ADR70555 position 70 position 70 ,
  • ADR70543 position 70 ADR70537 position 70 ADR70531 position 70 position 70 ,
  • ADR70519 position 70 ADR70513 position 70 ADR70507 position 70 position 70 ,
  • ADR70495 position 70 ADR70489 position 70 ADR70483 position 70 position 70 ,
  • ADR70471 position 70 ADR70465 position 70 ADR70459 position 70 position 70 .
  • AAD52643 position 932 AAD22046 position 932 , AAD44710 position 932 , AAD22045 position 932
  • AAL29451 position 63 AAL29449 position 63 , AAL29447 position 63 , AAL29445 position 63 , AAK74071 position 63 , AAL29450 position 63 , AAL29448 position 63 , AAL29446 position 63 , AAK30618 position 932 , AAK13008 position 932 .
  • ADR70441 position 70 ADR70435 position 70 , ADR70429 position 70 , AC089619 position
  • ACD50101 position 932 ABN11270 position 932

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés permettant de prévoir la virulence de virus de la maladie pieds-mains-bouche et d'épidémies desdits virus, consistant à comparer les concentrations en réplikine dans différents isolats de virus et à identifier les isolats les plus virulents comme étant ceux présentant les concentrations en réplikine les plus élevées. La présente invention concerne en outre des applications diagnostiques, préventives et thérapeutiques de peptides de réplikine identifiés dans des virus de la maladie pieds-mains-bouche, notamment des peptides de réplikine identifiés comme étant partagés entre des virus responsables de la maladie pieds-mains-bouche.
PCT/US2014/016303 2013-02-15 2014-02-13 Procédés d'identification, de prévention et de traitement du virus virulent de la maladie pieds-mains-bouche au moyen de séquences de réplikine WO2014127155A2 (fr)

Applications Claiming Priority (2)

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US201361765106P 2013-02-15 2013-02-15
US61/765,106 2013-02-15

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WO2014127155A2 true WO2014127155A2 (fr) 2014-08-21
WO2014127155A3 WO2014127155A3 (fr) 2014-12-04

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020151677A1 (en) * 2001-03-27 2002-10-17 Samuel Bogoch Replikins and methods of identifying replikin-containing sequences
US20050123550A1 (en) * 2003-05-12 2005-06-09 Laurent Philippe E. Molecules enhancing dermal delivery of influenza vaccines
WO2012108840A1 (fr) * 2011-02-08 2012-08-16 Temasek Life Sciences Laboratory Limited Nouvelle cassette d'expression pour une présentation en surface efficace des protéines antigéniques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020151677A1 (en) * 2001-03-27 2002-10-17 Samuel Bogoch Replikins and methods of identifying replikin-containing sequences
US20050123550A1 (en) * 2003-05-12 2005-06-09 Laurent Philippe E. Molecules enhancing dermal delivery of influenza vaccines
WO2012108840A1 (fr) * 2011-02-08 2012-08-16 Temasek Life Sciences Laboratory Limited Nouvelle cassette d'expression pour une présentation en surface efficace des protéines antigéniques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHANG, LY ET AL.: 'Viral Virulence Factors: EV71 Genetics Of 3C And Its Correlation With Clinical Outcome: 3C Protein' PARTIAL [ENTEROVIRUS A71 & DATABASE GENBANK 26 July 2006 Database accession no. ABG78216 *

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