WO2006088962A2 - Replikin peptides and uses thereof - Google Patents

Replikin peptides and uses thereof Download PDF

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Publication number
WO2006088962A2
WO2006088962A2 PCT/US2006/005343 US2006005343W WO2006088962A2 WO 2006088962 A2 WO2006088962 A2 WO 2006088962A2 US 2006005343 W US2006005343 W US 2006005343W WO 2006088962 A2 WO2006088962 A2 WO 2006088962A2
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Prior art keywords
replikin
lysine
virus
peptide
seq
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PCT/US2006/005343
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French (fr)
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WO2006088962A3 (en
WO2006088962A9 (en
Inventor
Samuel Bogoch
Elenore S. Bogoch
Samuel Winston Bogoch
Anne-Elenore Bogoch Borsanyi
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Samuel Bogoch
Bogoch Elenore S
Samuel Winston Bogoch
Bogoch Anne-Elenore Elizabeth
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Priority claimed from PCT/US2005/014443 external-priority patent/WO2005104754A2/en
Application filed by Samuel Bogoch, Bogoch Elenore S, Samuel Winston Bogoch, Bogoch Anne-Elenore Elizabeth filed Critical Samuel Bogoch
Priority to AU2006214332A priority Critical patent/AU2006214332B2/en
Priority to NZ560415A priority patent/NZ560415A/en
Priority to EP06735145A priority patent/EP1859063A4/en
Priority to JP2007555371A priority patent/JP2008539164A/en
Priority to CA002598381A priority patent/CA2598381A1/en
Publication of WO2006088962A2 publication Critical patent/WO2006088962A2/en
Publication of WO2006088962A9 publication Critical patent/WO2006088962A9/en
Priority to IL185308A priority patent/IL185308A0/en
Publication of WO2006088962A3 publication Critical patent/WO2006088962A3/en

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • This invention relates generally to two newly discovered classes of peptides that share structural characteristics and the use of bioinformatics to search databases of amino acids, nucleic acids and other biological information to identify shared structural characteristics.
  • Replikins are a newly discovered class of peptides that share structural characteristics and have been correlated with rapid replication of viruses and organisms.
  • Replikin Scaffolds are a sub-set of the class of Replikin peptides.
  • Exoskeleton Scaffolds are another newly discovered class of peptides that share structural characteristics and have been correlated with a decrease in replication.
  • Rapid replication is characteristic of virulence in certain bacteria, viruses and malignancies, but no chemistry common to rapid replication in different organisms has been described.
  • the inventors have found a family of conserved small protein sequences related to rapid replication, Replikins. Such Replikins offer new targets for developing effective detection methods and therapies. There is a need in the art for methods of identifying patterns of amino acids such as Replikins. Bioinformatic Identification of Amino Acid Sequences
  • PROSITE pattern description may be assembled according to the following rules:
  • Ambiguities are indicated by listing the acceptable amino acids for a given position, between square parentheses '[ ]'. For example: [ALT] would stand for Alanine or Leucine or Threonine. (4) Ambiguities are also indicated by listing between a pair of curly brackets ' ⁇ ⁇ ' the amino acids that are not accepted at a given position. For example: ⁇ AM ⁇ stands for any amino acid except Alanine and Methionine.
  • Each element in a pattern is separated from its neighbor by a '-'.
  • Repetition of an element of the pattern can be indicated by following that element with a numerical value or a numerical range between parentheses.
  • x(3) corresponds to x-x-x
  • x(2,4) corresponds to x-x or x-x-x or x-x-x-x.
  • (7) When a pattern is restricted to either the N- or C-terminal of a sequence, that pattern either starts with a ' ⁇ ' symbol or respectively ends with a '>' symbol.
  • PROSITE patterns include:
  • PA [AC]-x-V-x(4)- ⁇ ED ⁇ This pattern is translated as: [Alanine or Cysteine] -any- Valine -any-any-any-any- ⁇ any but Glutamic Acid or Aspartic Acid ⁇
  • amino acid sequence patterns In the context of describing amino acid sequence patterns, a simplified set of regular expression capabilities is typically employed. Amino acid sequence patterns defined by these simple regular expression rules end up looking quite similar to PROSITE patterns, both in appearance and in result. A regular expression description for an amino acid sequence may be created according to the following rules:
  • [LIVM] means that any one of the amino acids L, I, V, or M can be in that position.
  • ⁇ CF ⁇ means C and F should not be in that particular position.
  • exclusion capability can be specified with a " ⁇ " character.
  • ⁇ G would represent all amino acids except Glycine
  • [ ⁇ ILMV] would represents all amino acids except I, L, M, and V.
  • n is a number
  • x(3) is the same as “xxx”.
  • x(l,4) represents “x” or "xx” or “xxx” or "xxxx”.
  • BLAST Basic Local Alignment Search Tool
  • NCBI Accession numbers NCBI Accession numbers
  • GenBank sequence numbers NCBI Accession numbers
  • PROSITE patterns An example sequence in FASTA format is: >gi
  • BLAST sequence comparison algorithms that are used to search sequence databases for regions of local alignments in order to detect relationships among sequences which share regions of similarity.
  • the BLAST tools are limited in terms of the structure of amino acid sequences that can be discovered and located. For example, BLAST is not capable of searching for a sequence that has "at least one lysine residue located six to ten amino acid residues from a second lysine residue," as required by a Replikin pattern, for example.
  • BLAST capable of searching for amino acid sequences that contain a specified percentage or concentration of a particular amino acid, such as a sequence that has "at least 6% lysine residues.”
  • Need for Replikin Search Tools [00013] As can be seen from its definition, a Replikin pattern description cannot be represented as a single linear sequence of amino acids. Thus, PROSITE patterns and regular expressions, both of which are well suited to describing ordered strings obtained by following logical set-constructive operations such as negation, union and concatenation, are inadequate for describing Replikin patterns. [00014] In contrast to linear sequences of amino acids, a Replikin pattern is characterized by attributes of amino acids that transcend simple contiguous ordering.
  • the present invention provides a method for identifying nucleotide or amino acid sequences that include a Replikin sequence.
  • the method is referred to herein as a 3 -point-recognition method.
  • peptides comprising from 7 to about 50 amino acids including (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues and having replication, transformation, or redox functions may be identified.
  • An aspect of the present invention provides a method of identifying a Replikin Scaffold in a virus or organism comprising identifying a series of Replikin Scaffold peptides comprising about 16 to about 30 amino acids comprising (1) a terminal lysine and a lysine immediately adjacent to said terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to said terminal histidine, (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines.
  • An aspect of the invention may provide a method of identifying a Replikin Scaffold peptide in a virus or organism comprising about 16 to about 30 amino acids comprising (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine, (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines.
  • An aspect of the invention may also provide a method of making a preventive or therapeutic virus vaccine comprising identifying a Replikin Scaffold comprising about 16 to about 30 amino acids and synthesizing said Replikin Scaffold as a preventive or therapeutic virus vaccine wherein said Replikin Scaffold further comprises: (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine; (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines.
  • the Replikin Scaffold may contain influenza virus peptide Replikins.
  • a Replikin Scaffold may further comprise a group of Replikins comprising : (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine; (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines.
  • An aspect of the invention may provide a method of identifying an
  • Exoskeleton Scaffold wherein a Replikin Scaffold is identified in a first strain of virus or organism and the Exoskeleton Scaffold is identified in a later-arising strain of said virus or organism wherein said Exoskeleton Scaffold comprises an amino acid sequence comprising the same number of amino acids as the Replikin Scaffold and further comprising (1) two terminal lysines, (2) two terminal histidines, and (3) no lysine within about 6 to about 10 amino acids from another lysine.
  • an isolated or synthesized influenza virus peptide is provided with from 7 to about 50 amino acids, at least one lysine residue located six to ten residues from a second lysine residue, at least one histidine residue and at least 6% lysine residues.
  • the peptide comprises a terminal lysine.
  • the peptide is present in an emerging strain of influenza virus such as the influenza virus strain H5N1.
  • an isolated or synthesized influenza virus peptide comprising the H5N1 peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHH.
  • an isolated or synthesized influenza virus peptide having about 16 to about 30 amino acids; a terminal lysine and a lysine immediately adjacent to the terminal lysine; a terminal histidine and a histidine immediately adjacent to the terminal histidine; a lysine within about 6 to about 10 amino acids from another lysine; and at least 6% lysines.
  • a preventive or therapeutic virus vaccine having at least one isolated or synthesized peptide of influenza virus with at least one lysine residue located six to ten residues from a second lysine residue; at least one histidine residue; and at least 6% lysine residues.
  • the isolated or synthesized peptide is present in an emerging strain of influenza virus or is present in an H5N1 strain of influenza virus.
  • a preventive or therapeutic virus vaccine comprises the peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHH having alternatively a synthetic UTOPE tail, an adjuvant, or a combination thereof.
  • the preventive or therapeutic virus vaccine comprises a pharmaceutically acceptable carrier.
  • the preventive or therapeutic virus vaccine comprises the peptide
  • a method of stimulating the immune system of a subject to produce antibodies to influenza virus comprising administering an effective amount of at least one isolated or synthesized influenza virus Replikin peptide comprising from 7 to about 50 amino acids comprising (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
  • the administered Replikin peptide may further comprise a pharmaceutically acceptable carrier and/or adjuvant and prevent or treat an influenza infection.
  • the method of stimulating the immune system may further comprise an isolated or synthesized influenza virus peptide present in an emerging virus or present in an H5N1 strain of influenza virus.
  • the method may further comprise administration of the peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHKKLH.
  • An aspect of the invention may also provide a method comprising: applying a plurality of criteria to data representing protein sequences; based on the criteria, identifying an arbitrary sub-sequence within the protein sequences; and outputting the identified sub-sequence to a data file; wherein the criteria include: a set ⁇ a ⁇ of amino acids to be included in the sub-sequence; a set ⁇ b ⁇ of amino acids to be excluded from the sub-sequence; and a minimum and a maximum permissible gap between members of sets ⁇ a ⁇ and ⁇ b ⁇ .
  • the protein sequences may be obtained via a network.
  • An aspect of the invention may further comprise a machine-readable medium storing computer-executable instructions to perform such a method.
  • An aspect of the invention may further provide a method comprising applying a plurality of criteria to data representing protein sequences; based on the criteria, identifying a sub-sequence within the protein sequences, the identified subsequence having a predetermined allowed range of distance between lysine amino acids thereof, and a predetermined allowed range of distance between a histidine amino acid and a farthest Lysine acid thereof; and outputting an identified sub-sequence to a data file.
  • the protein sequences may be obtained via a network.
  • a machine-readable medium storing computer-executable instructions may perform such a method.
  • Figure 2 is a graph depicting the percentage of malignin per milligram total membrane protein during anaerobic replication of glioblastoma cells.
  • Figure 3 is a bar graph showing amount of antimalignin antibody produced in response to exposure to the recognin 16-mer.
  • Figure 4A is a photograph of a blood smear taken with ordinary and fluorescent light.
  • Figure 4B is a photograph of a blood smear taken with ordinary and fluorescent light illustrating the presence of two leukemia cells.
  • Figure 4C is a photograph of a dense layer of glioma cells in the presence of antimalignin antibody.
  • Figure 4D and Figure 4E are photographs of the layer of cells in Figure 4C taken at 30 and 45 minutes following addition of antimalignin antibody.
  • Figure 4F is a bar graph showing the inhibition of growth of small cell lung carcinoma cells in vitro by antimalignin antibody.
  • Figure 5 is a plot of the amount of antimalignin antibody present in the serum of patients with benign or malignant breast disease pre-and post surgery.
  • Figure 6 is a box diagram depicting an aspect of the invention wherein a computer is used to carry out the 3-point-recognition method of identifying Replikin sequences.
  • Figure 7 is a graph showing the concentration of Replikins observed in hemagglutinin of influenza B and influenza A strain, HlNl, on a year by year basis from 1940 through 2001.
  • Figure 8 is a graph of the Replikin concentration observed in hemagglutinin of influenza A strains, H2N2 and H3N2, as well as an emerging strain defined by its constituent Replikins, designated H3N2(R), on a year by year basis from 1950 to 2001.
  • Figure 9 is a graph depicting the Replikin count per year for several virus strains, including the coronavirus nucleocapsid Replikin, from 1917 to 2002.
  • Figure 10 is a chart depicting the mean Replikin count per year for nucleocapsid coronavirus isolates.
  • Figure 11 is a chart depicting the Replikin count per year for H5N1 Hemagglutinins.
  • Figure 12 is a conversion table that enables amino acids to be encoded as single alphabetic characters according to a standard supplied by the International Union of Pure and Applied Chemistry (IUPAC).
  • IUPAC International Union of Pure and Applied Chemistry
  • Figure 13 is a printout of a human cancer protein obtained by searching a protein database maintained by the National Center for Biotechnology Information
  • Figure 14 is a conversion table illustrating a correspondence between nucleic acid base triplets and amino acids.
  • Figure 15 is a graph illustrating a rapid increase in the concentration of Replikin patterns in the hemagglutinin protein of the H5N1 strain of influenza prior to the outbreak of three "Bird Flu” epidemics.
  • Figure 15 illustrates that increasing replikin concentration ('Replikin Count') of hemagglutinin protein of H5N1 preceded three 'Bird Flu' Epidemics.
  • the decline in 1999 occurred with the massive culling of poultry in response to the El epidemic in Hong Kong.
  • Figure 16 is a table illustrating selected examples of Replikin patterns that have been found in various organisms.
  • Figure 17 is a high-level block diagram of a computer system incorporating a system and method for identifying Replikin patterns in amino acid sequences, in accordance with an aspect of the present invention.
  • Figure 18 is a simple flow chart illustrating a general method for locating a
  • Figure 19 is a flow chart illustrating a generalized method for locating a plurality of Replikin-like patterns in a sequence of amino acids, according to an aspect of the present invention.
  • Figure 20 is a source code listing containing a procedure for discovering Replikin patterns in a sequence of amino acids, in accordance with an aspect of the present invention.
  • Figure 21 is a table illustrating Replikin Scaffolds occurring in substantially fixed amino acid positions in different proteins.
  • Figure 22 is a simplified block diagram of a computer system platform useful with the present invention.
  • peptide or "protein” refers to a compound of two or more amino acids in which the carboxyl group of one is united with an amino group of another, forming a peptide bond.
  • peptide is also used to denote the amino acid sequence encoding such a compound.
  • 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 Replikin peptide or Replikin protein is an amino acid sequence having 7 to about 50 amino acids comprising:
  • a Replikin sequence is the amino acid sequence encoding such a peptide or protein.
  • an "earlier-arising” virus or organism 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.
  • a “later-arising” virus or organism is a specimen of a virus or organism collected from a natural source of the virus or organism on a date subsequent to the date on which another specimen of the virus or organism was collected.
  • emerging strain refers to a strain of a virus, bacterium, fungus, or other organism identified as having an increased increasing concentration of Replikin sequences in one or more of its protein sequences relative to the concentration of Replikins in other strains of such organism.
  • the increase or increasing concentration of Replikins occurs over a period of at least about six months, and preferably over a period of at least about one year, most preferably over a period of at least about three years or more, for example, in influenza virus, but may be a much shorter period of time for bacteria and other organisms.
  • mutation refers to change in this structure and properties of an organism caused by substitution of amino acids. In contrast, the term
  • replikin count refers to the number of replikins per 100 amino acids in a protein or organism. A higher replikin count in a first strain of virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier- or later-arising strain of the virus or organism having a lower replikin count.
  • Replikin Scaffold refers to a series of conserved Replikin peptides wherein each of said Replikin peptide sequences comprises about 16 to about 30 amino acids and further comprises: (1) a terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine; (3) a lysine within 6 to 10 amino acid residues from another lysine; and (4) about 6% lysine.
  • “Replikin Scaffold” peptides may comprise an additional lysine immediately adjacent to the terminal lysine.
  • Replikin Scaffold also refers to an individual member or a plurality of members of a series of a “Replikin Scaffold.” Identification of Replikins
  • identification of a new family of small peptides related to the phenomenon of rapid replication provides targets for detection of pathogens in a sample and developing therapies, including vaccine development.
  • knowledge of and identification of this family of peptides enables development of effective therapies and vaccines for any organism that harbors Replikins.
  • Identification of this family of peptides also provides for the detection of viruses and virus vaccine development.
  • identification of this family of peptides provides for the detection of influenza virus and provides new targets for influenza treatment and vaccines including treatment and vaccines for influenza H5N1. Further examples provided by the identification of this family of peptides include the detection of infectious disease Replikins, cancer immune Replikins and structural protein Replikins.
  • Rapid replication is characteristic of virulence in certain bacteria, viruses and malignancies, but no chemistry common to rapid replication in different organisms has been described.
  • Replikins a family of conserved small protein sequences related to rapid replication, which we have named Replikins. Such Replikins offer new targets for developing effective detection methods and therapies.
  • the first Replikin found was the glioma Replikin, which was identified in brain glioblastoma multiforme (glioma) cell protein, called malignin.
  • glioma glycoprotein 1OB has a 50% reduction in carbohydrate residues when compared to the normal 1OB. This reduction is associated with virus entry in other instances, and so may be evidence of the attachment of virus for the delivery of virus Replikins to the 1OB of glial cells as a step in the transformation to the malignant state.
  • a varying structure provides an inconstant target, which is a good strategy for avoiding attackers, such as antibodies that have been generated specifically against the prior structure and thus are ineffective against the modified form.
  • This strategy is used by influenza virus, for example, so that a previous vaccine may be quite ineffective against the current virulent virus.
  • the Replikin structures are conserved and thus provide new constant targets for treatment.
  • Certain structures too closely related to survival functions apparently cannot change constantly. Because an essential component of the Replikin structure is histidine (h), which is know for its frequent binding to metal groups in redox enzymes and probable source of energy needed for replication, and since this histidine structure remains constant, this structure remains all the more attractive a target for destruction or stimulation.
  • h histidine
  • Replikin concentration and composition provide new quantitative methods to detect and control the process of replication, which is central to the survival and dominance of each biological population.
  • Examples of peptide sequences of cancer Replikins or as containing a Replikin, i.e., a homologue of the glioma peptide, kagvaflhkk, may be found in such cancers of, but not limited to, the lung, brain, liver, soft-tissue, salivary gland, nasopharynx, esophagus, stomach, colon, rectum, gallbladder, breast, prostate, uterus, cervix, bladder, eye, forms of melanoma, lymphoma, leukemia, and kidney.
  • Replikins provide for: 1) detection of pathogens by qualitative and quantitative determinations of Replikins; 2) treatment and control of a broad range of diseases in which rapid replication is a key factor by targeting native Replikins and by using synthetic Replikins as vaccines; and 3) fostering increased growth rates of algal and plant foods.
  • the first Replikin sequence to be identified was the cancer cell Replikin found in a brain cancer protein, malignin, which was demonstrated to be enriched tenfold during rapid anaerobic replication of glioblastoma multiforme (glioma) cells.
  • Malignin is a 10KDa portion of the 250 KDa glycoprotein 1OB, which was isolated in vivo and in vitro from membranes of glioblastoma multiforme (glioma) cells.
  • 16-mer peptide sequence YKAGVAFLHKKNDIDE obtained from malignin by hydrolysis and mass spectrometry
  • the present invention provides a method for identifying nucleotide or amino acid sequences that include a Replikin sequence.
  • the method is referred to herein as a 3-point-recognition method.
  • the three point recognition method comprises: a peptide from 7 to about 50 amino acids including (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues. (Replikin).
  • These peptides or proteins constitute a new class of peptides in species including algae, yeast, fungi, amoebae, bacteria, plant, virus and cancer proteins having replication, transformation, or redox functions.
  • the present invention further provides a method for identifying nucleotide or amino acid sequences that include a Replikin sequence comprising from 7 to about 50 amino acids including (1) at least one first lysine located at either terminus of the isolated or synthesized peptide, (2) a second lysine located six to ten residues from the first lysine residue; (3) at least one histidine; and (4) at least 6% lysines.
  • the isolated or synthesized peptides are influenza virus peptides.
  • the isolated or synthesized peptides are H5N1 influenza virus peptides. Table 2
  • an amino acid sequence as a Replikin or as containing a Replikin i.e., a homologue of the glioma peptide, kagvaflhkk, requires that the three following requirements be met.
  • the sequences have three elements: (1) at least one lysine residue located six to ten residues from another lysine residue; (2) at least one histidine residue; and (3) a composition of at least 6% lysine within an amino acid sequence of 7 to about 50 residues.
  • An exemplary non-limiting Replikin comprises a terminal lysine.
  • viruses or viral peptides, as, but not limited to, adenovirus, lentivirus, a-virus, retrovirus, andeno-associated virus, human immunodeficiency virus, hepatitis virus, influenza virus, maize streak virus, herpes virus, bovine herpes virus, feline immunodeficiency virus, foot and mouth disease virus, small pox virus, rous sarcoma virus, neuroblastoma RAS viral oncogene, polyamavirus, Sindbis, human papilloma virus, myelomonocytic tumor virus, murine acute leukemia, T-cell lymphotropic virus, and tomato leaf curl virus.
  • adenovirus lentivirus
  • a-virus retrovirus
  • Eno-associated virus human immunodeficiency virus
  • human immunodeficiency virus hepatitis virus
  • influenza virus hepatitis virus
  • maize streak virus herpes virus
  • bovine herpes virus feline immunodeficiency virus
  • homologues of the amino acid sequence kagvafhkk are present in known classes of coronavirus, which are members of a family of enveloped viruses that replicate in the cytoplasm of host cells. Additionally, the homologue of the amino acid sequence kagvatlhkk is present in the recently identified class of coronavirus responsible for severe acute respiratory syndrome, or SARS. The replikin is located in the nucleocapsid whole protein sequence of the SARS coronavirus. In addition, the location of the replikins is present in other members of the coronavirus class and, more specifically, are also present in the nucleocapsid protein sequences from these coronaviruses.
  • Replikins are present in such bacteria as, but not limited to, Acetobacter, Achromobacter, Actinomyces, Aerobacter, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Chainia, Clostridium, Corynebacterium, Erwinia, Escheria, Lebsiella, Lactobacillus, Haemophilus, Flavobacterium, Methylomonas, Micrococcus, Mycobacterium, Micronomspora, Mycoplasma, Neisseria, Nocardia, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Staphylococcus, Streptocossus, Streptomyces, Streptosporangium, Strepto-virticillium, Vibrio peptide, and Xanthomas.
  • Replikins are present in such fungi as, but not limited to, Penicillium, Diseula, Ophiostoma novo-ulim, Mycophycophta, Phytophthora infestans, Absidia, Aspergillus, Candida, Cephalosporium, Fusarium, Hansenula, Mucor, Paecilomyces, Pichia, Rhizopus, Torulopsis, Trichoderma, and Erysiphe.
  • Replikins are present in such yeast as, but not limited to, Saccharomyces, Cryptococcus, including Cryptococcusneoformas, Schizo-saccharomyces, and Oryza.
  • Replikins are present in algae such as, but not limited to, Caldophera, Isolepisprolifera, Chondrus, Gracilaria, Gelidium, Caulerpa, Laurencia, Cladophexa, Sargassum, Penicillos, Halimeda, Laminaria, Fucus, Ascophyllum, Undari, Rhodymenia, Macrocystis, Eucheuma, Ahnfeltia, and Pteroclasia.
  • Replikins are present in amoeba such as, but not limited to, Entamoeba (including Entamoeba invadens), Amoebidae, Acanthamoeba and Naegleria.
  • Replikins are present in plants such as, but not limited to, Arabidopsis, wheat, rice, and maize. Auxiliary Specifications
  • auxiliary specifications to the basic "3-point-recognition” requirements may be added: (a) on a structural basis, such as the common occurrence of adjacent di- and polylysines in cancer cell proteins (e.g., transforming protein P21B(K-RAS 2B), lung, Table 2, SEQ ID NO: 89), and other adjacent di-amino acids in TOLL-like receptors, or b) on a functional basis, such as exhibiting ATPase, tyrosine kinase or redox activity as seen in Table 2.
  • a structural basis such as the common occurrence of adjacent di- and polylysines in cancer cell proteins (e.g., transforming protein P21B(K-RAS 2B), lung, Table 2, SEQ ID NO: 89), and other adjacent di-amino acids in TOLL-like receptors
  • b) on a functional basis such as exhibiting ATPase, tyrosine kinase or redox activity as seen in Table 2.
  • “Functional derivatives" of the Replikins as described herein are fragments, variants, analogs, or chemical derivatives of the Replikins, which retain at least a portion of the immunological cross reactivity with an antibody specific for the Replikin.
  • a fragment of the Replikin peptide refers to any subset of the molecule.
  • 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.
  • the concentration of Replikins in influenza viruses was examined by visually scanning the hemagglutinin amino acid sequences published in the National Library of Medicine "PubMed" data base for influenza strains isolated world wide from human and animal reservoirs year by year over the past century, i.e., 1900 to 2001. These Replikin concentrations (number of Replikins per 100 amino acids, mean +/- SD) were then plotted for each strain.
  • the concentration of Replikins was found to directly relate to the occurrence of influenza pandemics and epidemics.
  • the concentration of Replikins found in influenza B hemagglutinin and influenza A strain, HlNl is shown in Figure 7, and the concentration of Replikins found in the two other common influenza virus A strains, H2N2 and H3N2 is shown in Figure 8 (H2N2, H3N2).
  • the data in Figure 8 also demonstrate an emerging new strain of influenza virus as defined by its constituent Replikins (H3N2(R)).
  • Each influenza A strain has been responsible for one pandemic: in 1918, 1957, and 1968, respectively.
  • the data in Figures 7 and 8 show that at least one
  • kshfanlk (SEQ ID NO: 104) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 kshfanlkgtk (SEQ ID NO: 105) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 kshfanlkgtktrgklcpk (SEQ ID NO: 106) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hekygglnk (SEQ ID NO: 107) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hekygglnksk (SEQ ID NO: 108) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hekygglnksk (SEQ ID NO: 108) 1940,43
  • hsdneiqmvklygdsk (SEQ ID NO: 116) hsdneiqdkmvklygdskpqk (SEQ ID NO: 117) kyggl ⁇ kskpyytgeh (SEQ ID NO: 122) kcmgtipsakasilhevk (SEQ ID NO: 125) 1943, 75,76,77, 93 klygdskpqkftssa ⁇ gvtth (SEQ ID NO: 130) 1943, 75,76,77, 93,97, OO hsdnetqmaklygdskpqk (SEQ ID NO: 131) 1943, 75,76,77, 93 hfanlkgtqtrgk (SEQ ID NO: 132) 1959 hfanlkgtktrgk (SEQ ID NO: 114) 1976, 89,90, 99,00,01 hfanlkgtktrgklcpk (SEQ ID
  • Influenza B has not been responsible for any human pandemic.
  • hp(v/i)tigecpkyv(r/k)(s/t)(t/a)k (SEQ ID NO: 135) 1918,25.28.30.31.35.47.48.51.52.55.56.57.59.63.77.79.80.81.85.87,88,89,91.92,95.96.97.98.99,0O hdsnvknly(e/g)kv(k/r)(n/s)ql(k/r)nnak (SEQ ID NO: 136) 1918, 28,30,31 77,79,80, 88, 91, 95, 98 hdsnvknly(e/g)kv(k/r)(n/s)qlk (SEQ ID NO: 137) 1918.
  • khfekvkilpk (SEQ ID NO: 230) 1957,58,59,60,61,64,65,68, 78,83,84,91 khllssvkhfekvk (SEQ ID NO: 231) 1957.58.59.60.61. 83,84,91 ha(k/q/m)(d/n)ilekthngk (SEQ ID NO: 232) 1957.58.59.60.61.64.65.68.
  • hpltigecpkyvksek (SEQ DD NO: 248) 1957, 60, 65 kcqtplgaikttlpfh (SEQ ID NO: 249) 1957, 65 hhsndqgsgyaadkestqka(f/i)dgitnkvnsviek-
  • Influenza H2N2 was responsible for the human pandemic (global distribution) of 1957.
  • Influenza H3N2 was responsible for the human pandemic (global distribution) of 1968.
  • HlNl Replikin concentration also declined between 1997 and 2000, and the presence of HlNl strains decreased in isolates obtained during these years.
  • H2N2 Replikins recovery from a 35 year decline has not occurred ( Figure 8), and this correlates with the absence of H2N2 from recent isolates.
  • H3N2 the Replikin concentration of many isolates fell to zero during the period from 1996 to 2000, but other H3N2 isolates showed a significant, sharp increase in Replikin concentration. This indicates the emergence of a substrain of H3N2, which is designated herein as H3N2(R).
  • Figures 7 and 8 demonstrate that frequently, a one to three year stepwise increase is observed before Replikin concentration reaches a peak. This stepwise increase proceeds the occurrence of an epidemic, which occurs concurrently with the Replikin peak. Thus, the stepwise increase in concentration of a particular strain is a signal that particular strain is the most likely candidate to cause an epidemic or pandemic.
  • each year one or more of the original Replikin structures is conserved, while at the same time, new Replikins emerge.
  • new Replikins emerge.
  • influenza virus B hemagglutinin five Replikins were constantly conserved between 1919 and 2001, whereas 26 Replikins came and went during the same period (some recurred after several years absence).
  • the disappearance and re-emergence years later of a particular Replikin structure suggests that the Replikins return from another virus host pool rather than through de novo mutation.
  • influenza virus hemagglutinin protein sequence for the presence and concentration of Replikins provides a predictor of influenza pandemics and/or epidemics, as well as a target for influenza vaccine formulation. It is worth noting again with reference to this data, previously, no strain-specific chemical structures were known with which to predict the strains that would predominate in coming influenza seasons, nor to devise annual mixtures of whole-virus strains for vaccines.
  • FIG. 10 depicts the automated Replikin analysis of nucleocapsid coronavirus proteins for which the protein sequence is available on isolates collected from 1962 to 2003. Each individual protein is represented by an accession number and is analyzed for the presence of Replikins. The Replikin Count (number of Replikins per 100 amino acid) is automatically calculated as part of the automated Replikin analysis.
  • SARS and H3N2-Fujian influenza virus replikins traced back to a 1918 pandemic replikin The origin of the SARS virus is as yet unknown. We report evidence that certain SARS virus peptides can be traced back through homologous peptides in several strains of influenza virus isolates from 2002 to a sequence in the strain of the 1918 influenza pandemic responsible for the deaths of over 20 million people. [000109] By quantitative analysis of primary protein sequences of influenza virus and other microorganisms recorded through the last century we have found a new class of peptide structures rich in lysines and histidine, related to the phenomenon of rapid replication itself and to epidemics, rather than to the type of organism (eg. Table 1) and named them Replikins.
  • HIV TAT protein Smallpox virus [jreejhicjk] 8 B.anthracis, HATPase jjatvljkj 8 Ebola virus polymerase
  • the Goose Replikin Since the Goose Replikin has at least an 85 year history involving most or all of the A-strains of influenza and SARS, it and its components are conserved vaccine candidates for pan-strain protection. Condensed short SARS replikins, 7 to 21 amino acids long, enriched in % lysine and histidine compared to the Goose Replikin, occurred in association with the higher mortality rate of SARS (10-55%) when compared to that (2.5%) of the Goose Replikin, 29 amino acids long. Short replikins here mixed with long replikins in SARS may be responsible for high mortality. This is also the case for replikins of other organisms such as the ebola and smallpox viruses and anthrax bacteria (Table 7a).
  • Short synthetic vaccines besides being much more rapidly produced (days rather than months), and far less expensive, should avoid the side effects attendant on the contamination and the immunological interference engendered by multiple epitopes of thousands of undesired proteins in current whole virus vaccines in general. In any case for influenza, current whole virus vaccines are ineffective in more than half of the elderly. But would short replikins be sufficiently immunogenic?
  • the short glioma replikin 'kagvaflhkk' proved to be a successful basis for a synthetic anti-glioblastoma multiforme and anti-bronchogenic carcinoma vaccine. It produced anti-malignin antibody, which is cytotoxic to cancer cells at picograms/cell and relates quantitatively to the survival of cancer patients.
  • anti-malignin antibody which is cytotoxic to cancer cells at picograms/cell and relates quantitatively to the survival of cancer patients.
  • we synthesized four SARS short replikins found in nucleocapsid, spike, and envelope proteins. We found that these synthetic short SARS replikins when injected into rabbits also produced abundant specific antibody.
  • the 21 amino acid SARS nucleocapsid replikin antibody binds at dilutions greater than 1 in 204,800. Because of previous unsuccessful attempts by others to achieve with various small peptides a strong immune response without the unwanted side effects obtained with a whole protein or the thousands of proteins or nucleic acids as in smallpox vaccine, the ability of small synthetic replikin antigens to achieve strong immune responses is significant for the efficacy of these SARS vaccines. [000116]
  • Replikin structure in influenza and SARS viruses was examined the relationship of Replikin structure in influenza and SARS viruses to increased mortality, with results as shown in Table 7.
  • the first 2003 human SARS Replikin in Table 7 shows certain sequence homologies to the influenza virus goose 1917 and human 1918 Replikins through an intermediary structure of influenza H 1N2 in 2002 (e.g., see Replikin "k” in positions 1, 18 and 19).
  • the 1917 Goose Replikin sequence is seen in Table 7 to have been largely conserved despite many substitutions in amino acids which are not crucial to the definition of Replikins through 1999 (substitutions are show in italics).
  • the original 29 amino acid 1917 Replikin sequence was then found to have been almost exactly restored to its structure of 1917-1918 in the 2001 H1N2 Replikin.
  • the 2002 H1N2 influenza Replikin has been shortened from 29 to 28 amino acids and the "shift to the left" of amino acids kevl(i/v)wg (v/i)hh is clearly evident.
  • one Replikin was further shortened (or compacted) to the 21 amino acid Replikin of the first listed 2003 human SARS virus.
  • the % k of the 2003 SARS Replikin is now 38.1 % (8/21) in comparison to 20.7 % of the Goose Replikin and the 1918 Human Pandemic Replikin.
  • three SARS Replikins were found to be further shortened (or compacted) to 19, 11 and 9 amino acid long sequences, respectively.
  • the % k is 44.4% (4/9).
  • SARS mortality rate in humans rose to 10% in the young and 55.5 % in the elderly compared to the 2.5 % mortality in the 1918 influenza pandemic.
  • the amino acid sequences are shown in Table 7 to emphasize the degree of homology and conservation for 85 years (1917-2002) of the influenza Replikin, for which evidence has first been observed in the 1917 Goose Replikin. No such conservation has ever been observed before.
  • Table 7 also illustrates that the Replikins in the 2003 human SARS virus, in addition to having homologies to the influenza Replikins which first appeared as the 1917 Goose Replikin and the 1918 Human Pandemic influenza Replikin, show certain sequence homologies to both the coronavirus avian bronchitis virus Replikin (e.g. "k” in positions 1 and 2, end in “h") and to the coronavirus acute diarrhea virus Replikin (e.g. "k” in positions 1 and 11, "h” at the end of the Replikin).
  • KSHFANLKGTK SEQ ID NO: 105
  • KSHFANLKGTKTRGKLCPK SEQ ID NO: 106
  • HAKAIGNCPIWVK (SEQ ID NO: 110)
  • HAKAIGNCPIWVVKKTPLKLANGTK (SEQ ID NO : 111 )
  • HAKAIGNCPIWVKTPLKLANGTKYRPPAKLLK (SEQ ID NO: 113) [000121] Tables 3 and 4 indicate that there appears to be much greater stability of the Replikin structures in influenza B hemagglutinins compared with HlNl Replikins. Influenza B has not been responsible for any pandemic, and it appears not to have an animal or avian reservoirs. (Stuart-Harris et al., Edward Arnold Ltd., London (1985)). Replikins in Influenza over Time
  • Influenza H2N2 Replikins Influenza H2N2 was responsible for the human pandemic of 1957. Three of the 20 Replikins identified in that strain for 1957 were conserved in each of the H2N2 isolates available for examination on PubMed until 1995 (Table 5).
  • Influenza H3N2 was responsible for the human pandemic of 1968. Five Replikins which appeared in 1968 disappeared after 1977, but reappeared in the 1990s (Table 6). The only Replikin structure which persisted for 22 years was hcd(g/q)f(q/r)nekwdlf(v/i)er(s/t)k, which appeared first in 1977 and persisted through 1998.
  • H5N1 Influenza Conservation of Replikin Scaffold [000130] There is concern that the current outbreak of high mortality H5N1 "bird flu" in several countries may represent the first phase of an overdue influenza pandemic. A recent report suggests that in the first probable person-to-person transmission of H5N1, "sequencing of the viral genes identified no change in the receptor-binding site of hemagglutinin or other key features of the virus. The sequences of all eight viral gene segments clustered closely with other H5N1 sequences from recent avian isolates in Thailand.” Phylogenetic analysis suggested that from the absence of evidence of "reassortment with human influenza viruses" that H5N1 is not a new variant. However, we now report three recent changes in a specific H5N1 protein sequence at sites which had not been changed in the last two H5N1 epidemics and in fact had been conserved since 1959.
  • a Replikin Scaffold comprises a series of conserved peptides comprising a sequence of about 16 to about 30 amino acids and further comprising ( 1 ) a terminal lysine ;
  • a Replikin Scaffold may further comprise a an additional lysine immediately adjacent to the terminal lysine.
  • "Replikin Scaffold” peptides may comprise an additional lysine immediately adjacent to the terminal lysine.
  • “Replikin Scaffold” peptide also refers to an individual member or a plurality of members of a series of a "Replikin Scaffold.”
  • a non-limiting and preferred target for synthetic influenza vaccines may be a Replikin Scaffold in influenza virus further comprising a sequence of about 29 amino acids and a lysine immediately adjacent to the terminal lysine.
  • a non-preferred target for synthetic influenza may be an Exoskeleton Scaffold in a first strain of influenza virus comprising a first peptide of about 29 amino acids and
  • FIG. 15 illustrates a rapid increase in the concentration of Replikins per 100 amino acids just prior to epidemics in 1997 (indicated as El), 2001 (indicated as E2) and 2004 (indicated as E3).
  • Table 8 above, provides further support for the role of replikins in epidemics and pandemics in humans and birds.
  • Table 8 the history of the Goose Replikin and its homologues are tracked from 1917 to the present outbreak of avian H5N1 virus.
  • Table 8 demonstrates conservation of the "scaffold" homology of the Goose Replikin in virulent strains of influenza.
  • Table 8 illustrates the history, by year or smaller time period, of the existence in the protein structure of the Goose Replikin and its homologues in other influenza Replikins. Table 8 further illustrates the history of amino acid substitutions in those homologues and the conservation of certain amino acids of the Replikin structure which are essential to the definition of a Replikin and the function of rapid replication supplied by Replikins. [000141] A review of Table 8 illustrates that if random substitution of amino acids were to occur in virulent strains of influenza from 1917 through the present, certain framework amino acids of the Goose Replikin would not be conserved from year to year in strains in which epidemics occurred.
  • An aspect of the present invention is a combination of replikin structure and function to track the pathogenicity or rate of replication of a virus, epidemic or pandemic or to predict the occurrence of epidemics or pandemics.
  • An example of this combination is the ability of the Replikin algorithm of the invention to be used to count increases in Replikin counts in influenza strains such as the strain of 1918 and the current H5N 1 strain of H5N 1.
  • the Replikin Count of the 1918 influenza pandemic and the current outbreak of "Bird Flu" demonstrate the predictive capacity of this exemplary aspect in accordance with and made possible by the invention.
  • FMDV isolates were visually examined for the presence of both the entire Replikin and each of the component Replikin amino acid residues observed in a particular Replikin. [000151] Rather than being subject to extensive substitution over time as occurs in neighboring amino acids, the amino acids which comprise the Replikin structure are substituted little or not at all, that is the Replikin structure is conserved.
  • the Replikin (SEQ ID NO: 3) "hkqkivapvk" was found to be conserved in 78% of the 236 isolates reported in PubMed, and each amino acid was found to be conserved in individual isolates as follows: his, 95.6%; lys, 91.8%; gin 92.3%; lys, 84.1%; ile, 90.7%; val, 91.8%; ala, 97.3%; pro, 96.2%; ala, 75.4%; and lys, 88.4%.
  • the high rate of conservation suggests structural and functional stability of the Replikin structure and provides constant targets for treatment.
  • sequence conservation was found in different isolates of HIV for its Replikins, such as (SEQ ID NO: 5) “kcfncgkegh” or (SEQ ID NO: 6) "kvylawvpahk” in HIV Type 1 and (SEQ ID NO: 7) "kcwncgkegh” in HIV Type 2 (Table 2). Further examples of sequence conservation were found in the HIV tat proteins, such as (SEQ ID NO: 613) "hclvckqkkglgisygrkk,” wherein the key lysine and histidine amino acids are conserved. (See Table 9).
  • Tat trans-activator proteins
  • TAR trans-activating response sequence
  • anti-Replikin antibodies also support Replikin class unity.
  • An anti-Replikin antibody response has been quantified by immunoadsorption of serum antimalignin antibody to immobilized malignin (see Methods in U.S. Patent No. 5,866,690).
  • the abundant production of antimalignin antibody by administration to rabbits of the synthetic version of the 16-mer peptide whose sequence was derived from malignin, absent carbohydrate or other groups, has established rigorously that this peptide alone is an epitope, that is, provides a sufficient basis for this immune response (Figure 3).
  • the 16-mer peptide produced both IgM and IgG forms of the antibody.
  • Antimalignin antibody was found to be increased in concentration in serum in 37% of 79 cases in the U.S.
  • transformed cells may be one form of safe haven for the virus: prolonging cell life and avoiding virus eviction, so that the virus remains inaccessible to anti-viral treatment.
  • peptide vaccines based on the particular influenza virus Replikin or group of Replikins observed to be most concentrated over a given time period provide protection against the particular strain of influenza most likely to cause an outbreak in a given influenza season, e.g., an emerging strain or re-emerging strain
  • analysis of the influenza virus hemagglutinin amino acid sequence on a yearly or bi-yearly basis provides data which are useful in formulating a specifically targeted influenza vaccine for that year. It is understood that such analysis may be conducted on a region-by-region basis or at any desired time period, so that strains emerging in different areas throughout the world can be detected and specifically targeted vaccines for each region can be formulated.
  • Influenza Vaccines, Treatments and Therapeutics [000163] Currently, vaccine formulations for influenza are changed twice yearly at international WHO and CDC meetings. Vaccine formulations are based on serological evidence of the most current preponderance of influenza virus strain in a given region of the world. However, prior to the present invention there has been no correlation of influenza virus strain specific amino acid sequence changes with occurrence of influenza epidemics or pandemics.
  • influenza virus pandemics and epidemics provide the first specific quantitative early chemical correlates of influenza pandemics and epidemics and provides for production and timely administration of influenza vaccines tailored specifically to treat the prevalent emerging or re-emerging strain of influenza virus in a particular region of the world.
  • influenza virus pandemics and epidemics can be predicted.
  • influenza peptide vaccine of the invention may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in influenza virus strains.
  • the peptide vaccine is based on Replikin sequence(s) shown to be increasing in concentration over a given time period and conserved for at least that period of time.
  • a vaccine may include a conserved Replikin peptide(s) in combination with a new Replikin(s) peptide or may be based on new Replikin peptide sequences.
  • the Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences, although vaccines based on peptides containing only Replikin sequences are preferred.
  • vaccine compositions of the invention also contain a pharmaceutically acceptable carrier and/or adjuvant.
  • influenza vaccines of the present invention can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • antiviral drugs such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • influenza vaccine of the present invention may be administered to any animal capable of producing antibodies in an immune response.
  • influenza vaccine of the present invention may be administered to a rabbit, a chicken, a pig or a human.
  • an influenza vaccine of the invention may be directed at a range of strains of influenza or a specific strain of influenza.
  • an influenza vaccine may be directed to an immune response against animal or human strain of influenza including influenza B, (A)HlNl, (A)H2N2 and (A)H3N2, or any human variant of the virus that may arise hereafter, as well as strains of influenza predominantly in animals such as the current avian H5N1.
  • An influenza vaccine may further be directed to a particular replikin amino acid sequence in any portion of an influenza protein.
  • an influenza vaccine may comprise a Replikin Scaffold of the H5N1 virus such as KKNSTYPTIKRSYNNTNQEDLLVLWGIHH.
  • an influenza vaccine may comprise a UTOPE such as KKKKH or KKKKHKKKKKH.
  • a vaccine may comprise the addition of an adjuvant such as the well known key limpet hemocyanin having amino acid sequence KLH.
  • an influenza vaccine may comprise a Replikin Scaffold of influenza H5N1 further comprising two UTOPES and an adjuvent sequence such as KKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK-KLH (Vaccine V120304U2).
  • An aspect of the present invention may comprise the Replikin Scaffold previously constructed and shown in Table 8 as one of the Bird Flu Replikins labelled"2004 H5N1 Vietnam, highly pathogenic.”
  • Table 8 the Replikin Scaffold previously constructed and shown in Table 8 as one of the Bird Flu Replikins labelled"2004 H5N1 Vietnam, highly pathogenic.
  • Plasmodium farciparum malaria antigen located at the merozoite surface and/or within the parasitophorous vacuole revealed that this organism, like influenza virus, also contains numerous Replikins.
  • this organism like influenza virus, also contains numerous Replikins.
  • Plasmodium falciparum contains several partial Replikins.
  • Another difference seen in Plasmodium falciparum is a frequent repetition of individual Replikin structures within a single protein, which was not observed with influenza virus. Repetition may occur by (a) sharing of lysine residues between Replikins, and (b) by repetition of a portion of a Replikin sequence within another Replikin sequence.
  • isolated Replikin peptides may be used to generate antibodies, which may be used, for example to provide passive immunity in an individual. Passive immunity to the strain of influenza identified by the method of the invention to be the most likely cause of future influenza infections may be obtained by administering antibodies to Replikin sequences of the identified strain of influenza virus to patients in need. Similarly, passive immunity to malaria may be obtained by administering antibodies to Plasmodium falciparum Replikin(s). [000175] 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 an 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. Furthermore, combinations of antibodies to different Replikins may be administered as an antibody cocktail.
  • 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.
  • Particularly useful antibodies of the invention are those that specifically bind to Replikin sequences contained in peptides and/or polypeptides of influenza virus.
  • antibodies to any of peptides observed to be present in an emerging or re-emerging strain of influenza virus and combinations of such antibodies are useful in the treatment and/or prevention of influenza.
  • antibodies to any Replikins present on malaria antigens and combinations of such antibodies are useful in the prevention and treatment of malaria.
  • Antibody fragments which contain binding sites for a Replikin may be generated by known techniques.
  • such fragments include but are not limited to F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be generated (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Antimalignin does not increase with benign proliferation, but specifically increases only with malignant transformation and replication in breast in vivo and returns from elevated to normal values upon elimination of malignant cells (Figure 5). Antimalignin antibody concentration has been shown to relate quantitatively to the survival of cancer patients, that is, the more antibody, the longer the survival. Taken together, these results suggest that anti-Replikin antibodies may be a part of a mechanism of control of cell transformation and replication.
  • Augmentation of this immune response may be useful in the control of replication, either actively with synthetic Replikins as vaccines, or passively by the administration of anti-Replikin antibodies, or by the introduction of non-immune based organic agents, such as for example, carbohydrates, lipids and the like, which are similarly designed to target the Replikin specifically.
  • non-immune based organic agents such as for example, carbohydrates, lipids and the like, which are similarly designed to target the Replikin specifically.
  • 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.
  • Synthetic Replikin vaccines based on Replikins such as the glioma Replikin (SEQ ID NO: 1) "kagvaflhkk” or the hepatitis C Replikin (SEQ ID NO: 18) "hyppkpgcivpak", or HIV Replikins such as (SEQ ID NO: 5) “kcfncgkegh” or (SEQ ID NO: 6) "kvylawvpahk” or preferably, an influenza vaccine based on conserved and/or emerging or re-emerging Replikin(s) over a given time period may be used to augment antibody concentration in order to lyse the respective virus infected cells and release virus extracellularly where chemical treatment can then be effective.
  • Replikins such as the glioma Replikin (SEQ ID NO: 1) "kagvaflhkk” or the hepatitis C Replikin (SEQ ID NO: 18) "hyppkpgcivpak", or HIV Replikins such as (SEQ
  • a malaria vaccine based on Replikins observed in Plasmodium falciparum malaria antigens on the merozoite surface or within the parasitophorous vacuole, for example, can be used to generate cytotoxic antibodies to malaria.
  • Table 7 shows the relation of shortening or compacting of Replikin sequences to mortality rate caused by the organisms which contain these Replikins, to as short as seven amino acids. This correlation has been found by us to be a general phenomenon regardless of the type of organism. We have also found that there may be a progression over time to the shortened Replikin structure, as in influenza and SARS viruses. [000184] There is abundant evidence that there are constant evolutionary and competitive pressures for the emergence of constantly increasing "efficacy" of each infectious organism.
  • Replikin permitted by the algorithm which defines a Replikin, that is six amino acids (two ks six to ten amino acids apart), with the highest possible % k ( see deduced Replikin "kkkklik", which contains 83.3% k, 5/6, and one obligatory "h”).
  • so-to-speak taken what appears to be, or might be, the next evolutionary step, not apparently as yet taken by the organisms themselves, and devised the resultant deduced Replikins to use as general vaccines.
  • UVAX Ultraviolet X
  • deduced synthetic vaccines may be used as sole vaccines or as adjuvants when administered with more specific Replikin vaccines oi • other vaccines.
  • Recognin and/or Replikin peptides may be administered to a subject to induce the immune system of the subject to produce anti-Replikin antibodies.
  • a 0.5 to about 2 mg dosage, preferably a 1 mg dosage of each peptide is administered to the subject to induce an immune response. Subsequent dosages may be administered if desired.
  • the Replikin sequence structure is associated with the function of replication.
  • the Replikins of this invention are used for targeting sequences that contain Replikins for the purpose of diagnostic identification, promoting replication, or inhibiting or attacking replication, for example, the structure-function relationship of the Replikin is fundamental.
  • the larger protein sequence may be known in the art as having a "replication associated function," vaccines using the larger protein often have failed or proven ineffective.
  • the larger protein sequence invariably has one or more epitopes (independent antigenic sequences that can induce specific antibody formation); Replikin structures usually comprise one of these potential epitopes.
  • the presence of other epitopes within the larger protein may interfere with adequate formation of antibodies to the Replikin, by "flooding" the immune system with irrelevant antigenic stimuli that may preempt the Replikin antigens, See, e.g., Webster, R.G., J. Immunol, 97(2):177-183 (1966); and Webster et al, J.
  • decoy epitopes may bind anti-Replikin antibodies, but may keep the antibodies away from histidine-bound respiratory enzymes. Treatment may therefore be most efficacious in two stages: 1) proteases to hydro lyze decoys, then; 2) anti-Replikin antibodies or other anti-Replikin agents.
  • an antibody that specifically recognizes a Replikin sequence has a better chance of inactivating or destroying the cell in which the Replikin is located, as seen with anti-malignin antibody, which is perhaps the most cytotoxic anti-cancer antibody yet described, being active at picograms per cell.
  • anti-malignin antibody which is perhaps the most cytotoxic anti-cancer antibody yet described, being active at picograms per cell.
  • either epitopes other than Replikins present in the larger protein fragments may interfere according to the phenomenon of antigenic primacy referred to above, and/or because the hydrolysis of larger protein sequences into smaller sequences for processing to produce antibodies results in loss of integrity of any Replikin structure that is present, e.g., the Replikin is cut in two and/or the histidine residue is lost in the hydrolytic processing.
  • the present studies suggest that for an effective vaccine to be produced, the Replikin sequences, and no other epitope, should be used as the vaccine.
  • a vaccine of the invention can be generated using any one of the Replikin peptides identified by the three-point recognition system.
  • Particularly preferred peptides - for example - an influenza vaccine include peptides that have been demonstrated to be conserved over a period of one or more years, preferably about three years or more, and/or which are present in a strain of influenza virus shown to have the highest increase in concentration of Replikins relative to Replikin concentration in other influenza virus strains, e.g., an emerging strain.
  • the increase in Replikin concentration preferably occurs over a period of at least about six months to one year, preferably at least about two years or more, and most preferably about three years or more.
  • the preferred Replikin peptides for use in an influenza virus vaccine are those Replikins observed to "re- emerge" after an absence from the hemagglutinin amino acid sequence for one or more years.
  • the Replikin peptides of the invention are administered to a subject, preferably by i.v. or intramuscular injection, 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 0.1 ⁇ g to about 10 mg, preferably about 10 ⁇ g to about 1 mg, and most preferably about 50 ⁇ g to about 500 ug. The skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.
  • Various adjuvants may be used to enhance the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, key limpet hemocyanin, dintrophenol, and potentially useful human adjuvants such as BCG and Corynebacterium parvum.
  • UTOPEs can be used as adjuvants to other Replikin vaccines and to non-Replikin vaccines.
  • Replikin DNA or RNA may have a number of uses for the diagnosis of diseases resulting from infection with a virus, bacterium or other Replikin encoding agent.
  • Replikin 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 a particular organism in a tissue sample or an environmental sample, for example.
  • 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.
  • oligoribonucleotide sequences that include antisense RNA and DNA molecules and ribozymes that function to inhibit the translation of Replikin- or recognin-containing mRNA.
  • 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 i.v. or i.m. delivery is routine. The dosage amount is also readily ascertainable.
  • Particularly preferred antisense nucleic acid molecules are those that are complementary to a Replikin sequence contained in a mRNA encoding, for example, an influenza virus polypeptide, wherein the Replikin sequence comprises from 7 to about 50 amino acids including (1) at least one 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.
  • antisense nucleic acid molecules that are complementary to a Replikin present in the coding strand of the gene or to the mRNA encoding the influenza virus hemagglutinin protein, wherein the antisense nucleic acid molecule is complementary to a nucleotide sequence encoding a Replikin that has been demonstrated to be conserved over a period of six months to one or more years and/or which are present in a strain of influenza virus shown to have an increase in concentration of Replikins relative to Replikin concentration in other influenza virus strains.
  • the increase in Replikin concentration preferably occurs over a period of at least six months, preferably about one year, most preferably about two or three years or more.
  • antisense nucleic acid molecules that are complementary to mRNA those that are complementary to a mRNA encoding bacterial Replikins comprising a Replikin sequence of from 7 to about 50 amino acids including (1) at least one 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. More preferred are antisense nucleic acid molecules that are complementary to the coding strand of the gene or to the mRNA encoding a protein of the bacteria.
  • a method of preventing or treating ⁇ a virus infection comprising administering to a patient in need thereof a preventive or therapeutic virus vaccine comprising at least one isolated Replikin present in a protein of an emerging strain of the virus and a pharmaceutically acceptable carrier and/or adjuvant.
  • the isolated or synthesized peptides are influenza virus peptides.
  • the isolated or synthesized peptides are H5N1 influenza virus peptides.
  • the present invention also provides a method of making a preventive or therapeutic virus vaccine comprising: (1) identifying a strain of a virus as an emerging strain,
  • step (3) synthesizing peptides having the amino acid sequence of the at least one Replikin sequence selected in step (2), and (4) combining a therapeutically effective amount of the peptides of step
  • the isolated Replikin is from influenza virus.
  • the isolated Replikin is from an influenza H5N1 virus.
  • the invention is directed to a method of identifying an emerging strain of a virus for diagnostic, preventive or therapeutic purposes comprising:
  • a process for stimulating the immune system of a subject to produce antibodies that bind specifically to a Replikin sequence comprising administering to the subject an effective amount of a dosage of a composition comprising at least one Replikin peptide.
  • a further aspect of the present invention comprises at least one peptide that is present in an emerging strain of the organism if such new strain emerges.
  • Another aspect of the present invention comprises at least one peptide that is present in influenza H5N1.
  • the present invention also provides antibodies that bind specifically to a Replikin, as defined herein, as well as antibody cocktails containing a plurality of antibodies that specifically bind to Replikins.
  • Another aspect of the present invention provides compositions comprising an antibody or antibodies that specifically bind to a Replikin and a pharmaceutically acceptable carrier.
  • the present application also provides isolated, or separated from nucleocapsid proteins, amongst others, recombinant, or synthesized peptides or other methods containing a viral Replikin sequence.
  • a process for stimulating the immune system of a subject to produce antibodies that bind specifically to a viral Replikin sequence comprising administering to the subject an effective amount of a dosage of a composition comprising at least one Replikin peptide.
  • Another aspect of the present invention comprises at least one peptide that is present in an emerging strain of the virus if such new strain emerges.
  • the present invention also provides antibodies that bind specifically to a viral Replikin, as defined herein, as well as antibody cocktails containing a plurality of antibodies that specifically bind to viral Replikins.
  • Another aspect of the present invention provides compositions comprising an antibody or antibodies that specifically bind to a viral Replikin and a pharmaceutically acceptable carrier.
  • the present invention also provides therapeutic compositions comprising one or more of isolated Replikin virus peptides and a pharmaceutically acceptable carrier.
  • an antisense nucleic acid molecule complementary to a virus Replikin mRNA sequence, said Replikin mRNA sequence denoting from 7 to about 50 amino acids comprising:
  • a method of simulating the immune system of a subject to produce antibodies to viruses comprising: administering an effective amount of at least one virus Replikin peptide.
  • a virus peptide for inclusion in a preventive or therapeutic virus vaccine comprising:
  • step (4) selecting at least one Replikin sequence present in the strain of the virus peptide identified in step (4) as a peptide for inclusion in the virus vaccine.
  • influenza virus peptides comprising a Replikin sequence.
  • a process for stimulating the immune system of a subject to produce antibodies that bind specifically to an influenza virus Replikin sequence comprising administering to the subject an effective amount of dosage of a composition comprising at least one influenza virus Replikin peptide.
  • a further aspect of the present invention comprises at least one Replikin peptide that is present in an emerging strain of influenza virus.
  • Yet another aspect of the present invention comprises a composition comprising at least one influenza H5N1 Replikin peptide.
  • the present invention also provides antibodies that bind specifically to an influenza virus Replikin, as defined herein, as well as antibody cocktails containing a plurality of antibodies that specifically bind to influenza virus Replikins.
  • compositions comprising an antibody or antibodies that specifically bind to an influenza Replikin and a pharmaceutically acceptable carrier.
  • the present invention also provides therapeutic compositions comprising one or more of isolated influenza virus peptides having from 7 to about 50 amino acids comprising:
  • an antisense nucleic acid molecule complementary to an influenza virus hemagglutinin Replikin mRNA sequence, said Replikin mRNA sequence denoting from 7 to about 50 amino acids comprising:
  • a method of simulating the immune system of a subject to produce antibodies to influenza virus comprising administering an effective amount of at least one influenza virus Replikin peptide having from 7 to about 50 amino acids comprising: (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
  • an influenza virus peptide for inclusion in a preventive or therapeutic influenza virus vaccine comprising:
  • step (4) selecting at least one Replikin sequence present in the strain of influenza virus peptide identified in step (4) as a peptide for inclusion in an influenza virus vaccine.
  • the present invention also provides a method of making a preventive or therapeutic influenza virus vaccine comprising:
  • the invention is directed to a method of identifying an emerging strain of influenza virus for diagnostic, preventive or therapeutic purposes comprising:
  • a preventive or therapeutic influenza virus vaccine comprising at least one isolated Replikin present in the hemagglutinin protein of an emerging strain of influenza virus and a pharmaceutically acceptable carrier and/or adjuvant.
  • Also provided by the present invention is a method of preventing or treating influenza virus infection comprising administering to a patient in need thereof a preventive or therapeutic vaccine comprising at least one isolated Replikin present in the hemagglutinin protein of an emerging strain of influenza virus and a pharmaceutically acceptable carrier and/or adjuvant.
  • Embodiments of the present invention are directed to a system and method for identifying and/or locating complex patterns in an amino acid sequence such as Replikin patterns, Replikin Scaffold structures, Exoskeleton Scaffold structures and other complex patterns in amino acid and nucleic acid sequences.
  • techniques are provided to facilitate queries of protein databases. For protein descriptions received in response to the queries, aspects of the present invention may include a scan of the received protein descriptions to identify and locate Replikin patterns.
  • a Replikin pattern is a sequence of from 7 to about 50 amino acids that include the following three (3) characteristics, each of which may be recognized as an aspect of the present invention: (1) the sequence has at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) the sequence has at least one histidine residue; and (3) at least 6% of the amino acids in the sequence are lysine residues.
  • Another aspect of the present invention may identify and/or locate a complex amino acid sequence having specified length constraints, which further includes any combination of the following characteristics: (1) a first amino acid residue located more than N positions and less than M positions away from a second amino acid residue; (2) a third amino acid residue located anywhere in the sequence; and (3) at least R percent of a fourth amino acid residue.
  • the present invention may count occurrences of the identified amino acid sequences and may report the counted occurrences, either as raw absolute values or as ratios of the number of identified amino acid sequences per N amino acids in the protein.
  • Still another aspect of the present invention may analyze the evolution of identified amino acid sequence patterns in variants of a given protein over time, and may also analyze the similarities and differences between instances of identified amino acid sequence patterns across a plurality of different proteins over time. As a result of the analysis, yet another aspect of the present invention may identify potential amino acid scaffolding structures that appear to be preserved over time and across different proteins, as component elements of the identified amino acid sequence patterns mutate and/or evolve. [000229] Embodiments of the present invention will be described with reference to the accompanying drawings, wherein like parts are designated by like reference numerals throughout, and wherein the leftmost digit of each reference number refers to the drawing number of the figure in which the referenced part first appears. [000230] FIG.
  • FIG. 17 is a high-level block diagram of a computer system incorporating a system and method for identifying Replikin patterns in amino acid sequences, in accordance with an aspect of the present invention.
  • computer workstation 610 may be a computer having a processor and a memory configured to permit a researcher to search protein databases and to scan protein descriptions for selected amino acid patterns.
  • computer workstation 610 may include protein and amino acid research system 630, which may receive instructions from a user/researcher to conduct protein searching and amino acid scanning operations.
  • protein and amino acid research system 630 may further include amino acid sequence scanner 640 that scans and searches retrieved protein and amino acid sequences for specific patterns of amino acids, including Replikin patterns.
  • Protein and amino acid research system 630 may communicate with network interface 620 to obtain protein sequences and amino acid sequences from resources on network 660, which may include the Internet. Alternatively, protein and amino acid research system 630 may obtain protein sequences and amino acid sequences from a local protein database 650. In addition, protein and amino acid research system 630 may obtain protein sequences and amino acid sequences directly from other input means, such as keyboard input. Protein and amino acid research system 630 may also communicate with network interface 620 to transmit results to other computers on network 660. Automated Scanning for Replikin Patterns
  • Embodiments of the present invention may include a generalized method and system for identifying complex patterns of amino acids within proteins.
  • the user may direct aspects of the invention to search for a variety of complex patterns of amino acids.
  • the present invention provides a method for identifying nucleotide or amino acid sequences that include a Replikin pattern.
  • FIG. 18 is a simple flow chart illustrating a general method for locating a Replikin pattern in a sequence of amino acids, according to an aspect of the present invention.
  • the method 700 may begin after a sequence of amino acids has been obtained.
  • the sequence of amino acids may be represented by alphabetic characters according to the code supplied in FIG. 12.
  • the string contains from 7 to about 50 amino acids
  • the string contains at least one lysine residue located 6 to 10 positions from a second lysine residue
  • the string contains at least one histidine residue
  • the string contains at least 6% lysine residues.
  • a given sequence of amino acids may contain many subsequences or strings that match the Replikin pattern. Additionally, Replikin patterns may overlap each other. Thus, to locate and identify all possible Replikin patterns in a sequence of amino acids, method 700 may be invoked iteratively for each subsequence of amino acids contained within the original sequence of amino acids. [000235] When method 700 is invoked iteratively to identify and locate all possible Replikin patterns in an amino acid sequence, an aspect of the present invention may count the number of resulting Replikin patterns. A Replikin count may be reported as an absolute number.
  • aspects of the invention may also determine a ratio of the number of Replikins per N amino acids in the sequence. For example, an aspect of the present invention may determine that a given protein contains a ratio of 6 Replikins for every 100 amino acids. Replikin ratios have been shown by laboratory experiment and by epidemiological evidence to correlate directly to the rate that a given protein replicates. Rapid replication of proteins may be an indication of disease. For example, the presence of relatively high ratios of Replikin patterns has been correlated to epidemics of influenza. Similarly, an increase in the count of Replikin patterns observed in a protein over time may also be an indication of future disease caused by the organism from which the protein was obtained (see, e.g., FIG. 15). Thus, the ability to detect and count Replikin patterns within sequences of amino acids is a significant advantage of the present invention.
  • aspects of the present invention may utilize method 700 to identify and locate other complex patterns of amino acids, which exhibit characteristics similar to Replikin patterns. That is, although some aspects of the present invention may specify exact values for: (1) distances between amino acids, (2) acceptable lengths of recognized amino acid sequences, and (3) the percentage or concentration of specific amino acids, these exact values may also be expressed as variables. Thus a researcher may employ an aspect of the present invention to identify sequences of amino acids in a protein that have the following characteristics:
  • the sequence contains at least one lysine residue located kmin to kmax amino acid residues from a second lysine residue; (3) the sequence contains at least one histidine residue;
  • FIG. 19 is a flow chart illustrating a generalized method 800 for locating a plurality of Replikin-like patterns in a given sequence of amino acids, according to an aspect of the present invention.
  • the method 800 begins by locating a first lysine residue in the given sequence (810). Then, the method 800 may determine whether a second lysine residue resides within kmin to kmax positions of the first lysine residue (820). As indicated in FIG. 19, kmin and kmax define the limits on the distance between the first and second lysine residues. For a typical Replikin pattern, kmin will equal 6 and kmax will equal 10.
  • method 800 may examine every histidine residue that resides within rmax positions of both the first and second lysine residues (830).
  • rmax will usually be set to equal 50.
  • method 800 will construct the shortest string of amino acid residues that includes the first lysine residue, the second lysine residue, and the identified histidine residue (840).
  • method 800 will determine whether the length of that shortest string is within the desired range - that is, whether it contains at least rmin amino acid residues and no more than rmax amino acid residues (850). Finally, if the identified string of amino acids also contains at least kpercent of lysine residues (860), the string will be identified as matching the desired Replikin-like pattern (870).
  • method 800 may identify several Replikin-like patterns from a single given amino acid sequence. This may happen because method 800 may examine more than one histidine residue that resides within rmax positions of the two identified lysine residues. Each identified histidine residue may, in combination with the two lysine residues, match the desired Replikin-like pattern.
  • FIG. 20 is a source code listing containing a procedure for discovering all Replikin patterns present in a given sequence of amino acids, in accordance with an aspect of the present invention.
  • the "match" procedure shown in FIG. 20 is programmed in an interpreted shell language called “TcI” and recognizes Replikins in a straightforward fashion.
  • TcI interpreted shell language
  • the "Tool Command Language” or TcI is a simple interpreted scripting language that has its roots in the Unix command shells, but which has additional capabilities that are well-suited to network communication, Internet functionality and the rapid development of graphical user interfaces.
  • the present invention may include a search engine to access and interact with amino acid and protein databases, either locally or over a network such as the Internet, to retrieve protein definitions.
  • protein and amino acid research system 630 may accept protein search criteria from a user, and may then access a plurality of on-line amino acid and protein database search engines to retrieve protein definitions that match the supplied search criteria.
  • Protein database search criteria may comprise any text string that may form a valid search term in any of the on-line protein or amino acid search engines. Typically, these search criteria relate to text that may be found in the printout that describes each specific protein.
  • aspects of the present invention may forward this text string to a plurality of Internet protein and amino acid search engines, each of which may then return any protein descriptions found in their databases that contained the terms "influenza type A.”
  • a plurality of Internet protein and amino acid search engines each of which may then return any protein descriptions found in their databases that contained the terms "influenza type A.”
  • amino acid sequence scanner 640 each of the returned protein descriptions may be scanned for the presence of Replikin patterns.
  • Additional aspects of the present invention may permit a user to select or de-select a plurality of Internet protein search engines and to customize the search criteria and protein retrieval capabilities of the present invention for each of the selected on-line protein search engines.
  • aspects of the invention may also permit a user to access a local protein database 650 or to supply a specific protein definition directly, for example, by supplying a local file name containing the protein definition, or by other methods known in the art for supplying parameters to computer software.
  • Another aspect of the present invention may include a search engine to access and interact with amino acid and protein databases on the Internet to retrieve protein definitions or amino acid sequence definitions. After accepting protein or amino acid sequence search criteria from a user, the present invention may access a plurality of amino acid and protein database search engines, through on-line access, to retrieve protein definitions or amino acid sequence definitions that match the supplied search criteria.
  • Initial existing protein search criteria based on existing definitions may comprise any text string that may form a valid search term in any of the on-line protein or amino acid search engines.
  • these search criteria relate to text that may be found in the printout that describes each specific protein. For example, if the user supplied the search criteria "influenza type A," the present invention would forward this text string to the plurality of Internet protein and amino acid search engines, each of which would then return any protein definitions in their databases that contained the terms "influenza type A.”
  • a non-limiting aspect of the present invention comprising a protein search engine entitled “Genome Explorer” is included in Appendix A.
  • the TcI procedure named “GenomalEnquirer” may control the macro level operation of the protein search engine (see “proc GenomalEnquirer ⁇ database term additionalCriteria ⁇ ).”
  • GenomalEnquirer a series of specific on-line protein search engines may be accessed and queried using the user-supplied protein search terms and additional criteria.
  • Additional aspects of the invention may permit a user to select or de-select a plurality of Internet protein search engines and to customize the search criteria and protein retrieval capabilities of the present invention for each of the selected on-line protein search engines.
  • aspects of the invention may also permit a user to access local protein databases or to supply a specific protein definition directly, for example, by supplying a local file name containing the protein definition, or by other methods known in the art for supplying parameters to computer software.
  • Embodiments of the present invention may be employed not only to identify and locate Replikin patterns in amino acid sequences. Embodiments may also be used to discover and analyze similarities in the structure of Replikin patterns occurring in different proteins, or to analyze different Replikin patterns occurring in the same protein over time.
  • FIG. 21 for example, is a table illustrating a Replikin Scaffold or "fixed scaffold" structure that was preserved in a "Bird Flu” influenza virus over an 87 year period from 1917 to 2004.
  • Embodiments of the present invention may assemble a number of discovered Replikin patterns in proteins, including Replikin patterns discovered in variants of the same protein. Along with each Replikin pattern, aspects of the present invention may also associate a date when each protein was first identified.
  • an aspect of the present invention may include sorting and displaying a plurality of selected Replikin patterns according to content, date or other criteria, in order to reveal substantially fixed amino acid structures that have been preserved in Replikin patterns over time and which may be present in different proteins as well as variants of the same protein. Further, when directed by a researcher, an aspect of the invention may employ known methods of pattern analysis to compare a plurality of selected Replikin patterns in order to identify such fixed amino acid structures automatically. As an example, in FIG.
  • the illustrated Replikin patterns appear to demonstrate - in this case - a relatively fixed scaffold structure of (usually) 29 amino acids that begins with a pair of lysine residues (kk) at the amino terminal, ends with a pair of histidine residues (hh) at the carboxyl terminal, and contains a lysine residue in either position 8, 10 or 11.
  • This conservation of scaffold structure over decades permits synthetic vaccines to be prepared rapidly and inexpensively.
  • a researcher may select elements of that scaffolding structure that are conserved over time and which are also present in a current variant of a protein. A vaccine may then be prepared based on the selected elements from the scaffolding structure.
  • identification of Replikins also provides for the detection of other pathogens, such as malaria, anthrax and small pox virus, in addition to enabling the development of therapies and vaccines that target Replikin structures. Additional examples provided by the identification of Replikins include the detection of infectious disease Replikins, cancer immune Replikins and structural protein Replikins.
  • Embodiments of the present invention enable important Replikin patterns of amino acids to be recognized, located and analyzed in manners that are not found in the prior art.
  • researchers have been limited in by existing techniques for describing sequences of amino acids. Indeed, limitations of the prior art have in some ways dampened research in this field, since heretofore it has not been possible to specify sequences of amino acids that comprise non-linear attributes.
  • descriptions of amino acid sequences were limited to linear sequences containing, at most, repetitive substrings and logical constraints on substring content.
  • Embodiments of the present invention enable a new class of amino acid sequences to be discovered, located and analyzed using tools not found in the prior art.
  • This new class of amino acids is characterized by attributes such as specific amino acid concentration and distance relationships between specific amino acids. These attributes transcend simple contiguous ordering and thus are not easily described, discovered or located by existing methods known in the art. [000251] For example, rather than examining strict amino acid sequence matches (homologies) as is done by other widely used programs such as BLAST, the present inventors have discovered a unique quantitative "language" related to rapid replication which defines a new class of amino acid grouping. Novel computer programs described herein detect instances of this new language. [000252] These programs include functionality to search electronic data for amino acid sub-sequences meeting predetermined criteria. The data, which may be obtained online, may include data defining a specified group of protein sequences.
  • the criteria may include: i) the occurrence within a protein sequence of two amino acids, in this case Lysine(K) and histidine(H) in specific concentrations in the sequence ii) the spacing of one of these (K) to a second K in the sequence, and iii) the concentration of one or more amino acids (e.g. K) in a percentage greater than a defined percentage.
  • the programs include the capability to identify Replikin sub-sequences in genome sequences.
  • One source of the genome sequences may be published genome sequences obtained from online, electronic databases, using search criteria provided by a user.
  • the databases may be NCBI (National Center for Biotechnology Information) or LANL (Los Alamos National Laboratory) databases.
  • the programs further include the capability to search for arbitrary sub-sequences (i.e., not only Replikin sub-sequences), based on user- supplied criteria.
  • Genome Explorer may generate a user interface to prompt a user for search terms.
  • Genome Explorer may apply the search terms to online databases, such as NCBI or LANL databases, to obtain raw sequence data. Additional data may be further obtained, such as article names, protein source, strain, serotype and year of discovery for all the raw sequences which match the search terms.
  • Genome Explorer may further apply additional search criteria to identify Replikin sub-sequences within the raw sequences.
  • the search criteria can be specified by the user in such a way as to implement relatively strict, or relatively relaxed definitions of what can be included in the set of matching sub-sequences to be reported by Genome Explorer.
  • Genome Explorer may compile ongoing statistics and display a progress bar in a user interface. When Genome Explorer completes its processing, it may save resulting statistics in a data file.
  • the data file may be an HTML file that can be opened in any word processor for inspection of results.
  • a program herein entitled "Dr. Peptide” search criteria may be applied to identify sub-sequences other than Replikin sub-sequences. With Dr. Peptide it is possible to search for, e.g., all instances of the sequence hlk hlk, separated by not more than 15 amino acids, in publicly available genome databases.
  • Dr. Peptide may include much the same functionality as Genome Explorer. For example, like Genome Explorer, Dr. Peptide may, via a user interface, prompt a user for search terms and apply the search terms to online databases, such as NCBI or LANL databases, to obtain raw sequence data. Additional data may be further obtained, such as article names, protein source, strain, serotype and year of discovery for all the raw sequences which match the search terms. Once the raw data has been acquired, Dr. Peptide may further process the data to identify arbitrary sub-sequences and present its output in a data file, for example in the form of HTML pages that can be opened in any word processor.
  • a data file for example in the form of HTML pages that can be opened in any word processor.
  • an "initial server inquiry" refers to search criteria to be applied to one or more network elements, such as server computers, storing electronic data representing protein sequences.
  • the network elements may be included in private networks or, for example, the Internet.
  • the data may be in the form of a "protein page,” i.e., a quantum of data representing protein sequences.
  • the character "k” represents a lysine amino acid, and the character “h” represents a histidine amino acid.
  • Search parameters include:
  • search parameters are identical, reuse cached protein pages as data input. If the search parameters are not identical (cached protein pages are not relevant),
  • Extract found sequence data (seq-data.ncbieaa). Extract article names (descr ⁇ article.title ⁇ name).
  • Extract protein source (source. org.taxname).
  • Parse HTML page for strain, definition, source, year, serotype, and raw nucleotide sequence Convert nucleotides to amino acids by mapping every three nucleotides in sequence to the corresponding amino acid. Save parsed value for this protein.
  • update user interface as to progress via progress bar, and: For each sequence data found on the page, Scan the amino acid sequence data for each sub-sequence matching
  • Genome Explorer implements a method including applying a plurality of criteria to data representing protein sequences, and based on the criteria, identifying a sub-sequence within the protein sequences, the identified sub-sequence having a predetermined allowed range of distance between Lysine amino acids thereof, and a predetermined allowed range of distance between a histidine amino acid and a farthest Lysine acid thereof.
  • An identified sub-sequence may be output to a data file.
  • the functionality of the herein aspects may be provided on various computer platforms executing program instructions. One such platform 1100 is illustrated in the simplified block diagram of FIG. 22.
  • the platform 1100 is shown as being populated by a processor 1160, which communicates with a number of peripheral devices via a bus subsystem 1150. These peripheral devices typically include a memory subsystem 1110, a network interface subsystem 1170, and an input/output (I/O) unit 1180.
  • the processor 1160 may be any of a plurality of conventional processing systems, including microprocessors, digital signal processors and field programmable logic arrays. In some applications, it may be advantageous to provide multiple processors (not shown) in the platform 1100.
  • the processor(s) 1160 execute program instructions stored in the memory subsystem 1110.
  • the memory subsystem 1110 may include any combination of conventional memory circuits, including electrical, magnetic or optical memory systems. As shown in FIG.
  • the memory system may include read only memories 1120, random access memories 1130 and bulk storage 1140.
  • Memory subsystem 1110 not only stores program instructions representing the various methods described herein but also may store the data items on which these methods operate.
  • Network interface subsystem 1170 may provide an interface to outside networks, including an interface to communications network 1190 comprising, for example, the Internet. I/O unit 1180 would permit communication with external devices, which are not shown.
  • nucleocapsid protein of the shrimp white spot virus has an exceptionally high Replikin Count as compared to all other viruses and organisms surveyed for replikins up to the present time (with the exception of malaria).
  • Falciparum is a trypanosome, both spend an essential part of their reproductive cycles in red blood cells, an unusual host cell whether in shrimp (white spot virus) or man (malaria), both are fulminating rapidly replicating diseases with high mortality rates of their hosts, and both appear to use the same methods of increasing their high Replikin Counts to such record highs, namely, Replikin Repeats and Replikin Overlap.
  • Table 10 illustrates intramolecular Replikin Repeats and Replikin Overlap in shrimp white spot syndrome virus (WSSV) nucleocapsid protein (VP35) gene with a Replikin Count (number of replikins per 100 amino acids) of 103.8 (497 total replikins per 479 amino acids).
  • WSSV shrimp white spot syndrome virus
  • VP35 nucleocapsid protein
  • a further aspect of the present invention comprises a protein search engine directed to recognizing generalized amino acid and nucleic acid patterns online databases.
  • Appendix D is an exemplary protein search engine directed to recognizing complex amino acid patterns such as Scaffold Exoskeletons. Appendix D is entitled “Dr. Peptide.”
  • Appendix D is an exemplary non-limiting aspect of the present invention and is designed to recognize generalized amino acid patterns in addition to the Replikin pattern.
  • an "initial server inquiry" refers to search criteria to be applied to one or more network elements, such as server computers, storing electronic data representing protein sequences.
  • the network elements may be included in private networks or, for example, the Internet.
  • the data may be in the form of a "protein page,” i.e., a quantum of data representing protein sequences .
  • Extract protein source (source.org.taxname).
  • the match patterns are a sequence of alternative steps:
  • amino acid in the amino acid sequence data is in a set of specific amino acids as defined in user parameter (2) above.
  • amino acid in the amino acid sequence data is not in the set of specific amino acids defined in user parameter (3) above.
  • An amino acid in the amino acid sequence data has a spacing gap of m to n amino acids from another amino acid in the amino acid sequence data as defined in user parameter (4) above.
  • the initial sub-sequence set is all possible terminal sequences, or "tails" of the sequence data at the first pattern step,
  • next amino acid of the sub-sequence is also in the set of amino acids.
  • the sub-sequence is a matched sub-sequence. Ignore sequences with no matches. Accept the sequence with the most matches. If a sequence has been accepted, Catalog each sub-sequence by the year it was discovered. For each additional criteria, Check the additional criteria against other parsed fields. If it does not match, do not accept the page. If the page was accepted, Add it as a passed page.
  • Dr. Peptide implements a method including applying a plurality of criteria to data representing protein sequences, and based on the criteria, identifying arbitrary subsequences within the protein sequences.
  • An identified sub-sequence may be output to a data file.
  • the criteria may include: a set ⁇ a ⁇ of amino acids to be included in the sub-sequence; a set of amino acids to be excluded from the sub-sequence; and a minimum and a maximum permissible gap between members of sets ⁇ a ⁇ and ⁇ b ⁇ .
  • a non-limiting and exemplary aspect of the invention employs the complex amino acid analysis aspect of the invention to analyze Replikin Scaffold sequences in earlier strains of influenza that have degenerated into non-rep likin sequences but maintained the scaffold structure of the Replikin Scaffold.
  • the exemplary and non-limiting software program in Appendix D to recognize generalized amino acid patterns, the inventors first discovered by visual scanning of protein sequences (now by Dr. Peptide software) that what was in earlier-arising specimens of a particular influenza species a Replikin Scaffold, was in later specimens changed as follows:
  • Replikin Scaffolds as 1) targets for anti-rapid replication agents such as antibodies or small inhibitory RNAs and 2) the basis of anti- viral vaccines.
  • Software according to aspects of the present invention may comprise logic to obtain and analyze protein sequences to identify sequences having characteristics 1, 2, 3 and 4 above. For example, Scaffold Exoskeletons can now be detected and counted in any protein sequence by the exemplary software in Appendix D.
  • Another non-limiting aspect in accordance with the present invention is a method of identifying a Replikin Scaffold comprising indentifying a series of peptides comprising about 17 to about 30 amino acids and further comprising
  • the method of identifying a Replikin Scaffold may comprise identifying a single or plurality of individual members of the series of a Replikin Scaffold.
  • the method of identifying a Replikin Scaffold further comprises the identification of a second lysine immediately adjacent to the terminal lysine.
  • Software according to aspects of the present invention may comprise logic to obtain and analyze protein sequences to identify sequences using steps 1, 2, 3 and 4 above.
  • TcI (the "Tool Command Language,” pronounced “tickle") is a simple interpreted scripting language that has its roots in the Unix command shells, but which has additional capabilities that are well-suited to network communication, Internet functionality and the rapid development of graphical user interfaces. TcI was created by John K. Ousterhout at the University of California at Berkeley in 1988. Originally conceived as a reusable, embeddable language core for various software tools, it is now widely used in applications including web scripting, test automation, network and system management, and in a variety of other fields. [000280] In aspects, Genome Explorer and Dr. Peptide may be coded in Tcl/Tk, a scripting programming language that includes powerful facilities for internet access, user interface design, and string manipulation.
  • Tcl/Tk has been ported to nearly all available computer architectures and is familiar to those skilled in the art, programs written in Tcl/Tk can be run on nearly any operating system.
  • Source code for specific implementations of Genome Explorer and Dr. Peptide are provided in Appendices A and D. The specific implementations are provided by way of illustration and example only, and the present invention is not in any way limited to the specific implementations illustrated.
  • a sequence is screened for subsequences containing three or more desired structural characteristics.
  • the subsequence of 7 to about 50 covalently linked units should contain (1) at least one first amino acid, carbohydrate or lipid residue located seven to ten residues from a second of the first amino acid, carbohydrate or lipid residue; (2) encoding at least one second amino acid, lipid or carbohydrate residue; and (3) at least 6% of the first amino acid, carbohydrate or lipid residue.
  • the subsequence of about 21 to about 150 nucleotides should contain (1) at least one codon encoding a first amino acid located within eighteen to thirty nucleotides from a second codon encoding the first amino acid residue; (2) at least one second amino acid residue; and (3) encodes at least 6% of said first amino acid residue.
  • the following algae were collected from Bermuda water sites and either extracted on the same day or frozen at -20 degrees C and extracted the next day.
  • the algae were homogenized in a cold room (at 0 to 5 degrees C) in 1 gram aliquots in neutral buffer, for example 100 cc. of 0.005M phosphate buffer solution, pH 7 ("phosphate buffer”) for 15 minutes in a Waring blender, centrifuged at 3000 rpm, and the supernatant concentrated by perevaporation and dialyzed against phosphate buffer in the cold to produce a volume of approximately 15 ml. The volume of this extract solution was noted and an aliquot taken for protein analysis, and the remainder was fractionated to obtain the protein fraction having a pK range between 1 and 4.
  • the preferred method of fractionation is chromatography as follows: The extract solution is fractionated in the cold room (4° C) on a DEAE cellulose (Cellex-D) column 2.5x11.0 cm, which has been equilibrated with 0.005M phosphate buffer. Stepwise eluting solvent changes are made with the following solutions:
  • Solution 1- 4.04 g. NaH2P04 and 0.5gNaH2P04 are dissolved in 15 litres of distilled water (0.005 molar, pH 7);
  • Solution 2 - 8.57 g. NaH2P04 is dissolved in 2,480 ml. of distilled water; Solution 3 - 17.1 g. of NaH2P04 is dissolved in 2480 ml of distilled water
  • Solution 4 - 59.65 g. of NaH2P04 is dissolved in 2470 ml distilled water (0.175 molar); Solution 5 - 101.6 g. of NaH2P04 is dissolved in 2455 ml distilled water
  • Solution 6 - 340.2 g. of NaH2P04 is dissolved in 2465 of distilled water (1.0 molar, pX-i 4.1);
  • Solution 7 - 283.63 g. of 80% phosphoric acid (H3P04) is made up in 2460 ml of distilled water (1.0 molar, pH 1.0).
  • the extract solution in 6 to 10 ml volume, is passed onto the column and overlayed with Solution 1, and a reservoir of 300 ml of Solution 1 is attached and allowed to drip by gravity onto the column.
  • Three ml aliquots of eluant are collected and analyzed for protein content at OD 280 until all of the protein to be removed with Solution 1 has been removed from the column.
  • Solution 2 is then applied to the column, followed in succession by Solutions 3, 4, 5, 6 aid 7 until all of the protein which can, be removed with each Solution is removed from the column.
  • the eluates from Solution 7 are combined, dialyzed against phosphate buffer, the protein content determined of both dialysand and dialyzate, and both analyzed by gel electrophoresis.
  • One or two bands of peptide or protein of molecular weight between 3,000 and 25,000 Daltons are obtained in Solution 7.
  • the algae Caulerpa mexicana, Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpa verticillata, Halimeda tuna, and Penicillos capitatus after extraction and treatment as above, all demonstrated in Solution 7 eluates sharp peptide bands in this molecular weight region with no contaminants.
  • These Solution 7 proteins or their eluted bands are hydrolyzed, and the amino acid composition determined.
  • the peptides so obtained, which have a lysine composition of 6% or greater are Replikin precursors.
  • Replikin peptide precursors are then determined for amino acid sequence and the Replikins are determined by hydrolysis and mass spectrometry as detailed in U.S. Patent 6,242,578 Bl. Those that fulfill the criteria defined by the " 3-point-recognition" method are identified as Replikins. This procedure can also be applied to obtain yeast, bacterial and any plant Replikins. b) Virus
  • Aglyco 1OB or the 16- mer Replikin may be used as antigen to capture and quantify the amount of its corresponding antibody present in serum for diagnostic purposes are as shown in Figures 2, 3, 4 and 7 of U.S. 6,242,578 Bl.
  • Comparison of the Replikin sequences and concentrations to the amino acid sequences obtained from isolates at an earlier time, such as about six months to about three years earlier, provides data that are used to predict the emergence of strains that are most likely to be the cause of influenza in upcoming flu seasons, and that form the basis for seasonal influenza peptide vaccines or nucleic acid based vaccines. Observation of an increase in concentration, particularly a stepwise increase in concentration of Replikins in a given strain of influenza virus for a period of about six months to about three years or more is a predictor of emergence of the strain as a likely cause of influenza epidemic or pandemic in the future.
  • Peptide vaccines or nucleic acid-based vaccines based on the Replikins observed in the emerging strain are generated.
  • An emerging strain is identified as the strain of influenza virus having the highest increase in concentration of Replikin sequences within the hemagglutinin and/or neuraminidase sequence during the time period.
  • the peptide or nucleic acid vaccine is based on or includes any Replikin sequences that are observed to be conserved in the emerging strain.
  • conserveed Replikins are preferably those Replikin sequences that are present in the hemagglutinin or neuraminidase protein sequence for about two years and preferably longer.
  • the vaccines may include any combination of Replikin sequences identified in the emerging strain.
  • the Replikin peptide or peptides identified as useful for an effective vaccine are synthesized by any method, including chemical synthesis and molecular biology techniques, including cloning, expression in a host cell and purification therefrom.
  • the peptides are preferably admixed with a pharmaceutically acceptable carrier in an amount determined to induce a therapeutic antibody reaction thereto. Generally, the dosage is about 0.1 mg to about 10 mg.
  • the influenza vaccine is preferably administered to a patient in need thereof prior to the onset of "flu season.” Influenza flu season generally occurs in late October and lasts through late April. However, the vaccine may be administered at any time during the year.
  • the influenza vaccine is administered once yearly, and is based on Replikin sequences observed to be present, and preferably conserved in the emerging strain of influenza virus.
  • Another preferred Replikin for inclusion in an influenza vaccine is a Replikin demonstrated to have re-emerged in a strain of influenza after an absence of one or more years.
  • Comparison of the Replikin sequences and concentrations to the amino acid sequences obtained from isolates at an earlier time, such as about six months to about three years earlier, provides data that are used to predict the emergence of strains that are most likely to be the cause an outbreak or pandemic, and that form the basis for coronavirus peptide vaccines or nucleic acid based vaccines.
  • Observation of an increase in concentration, particularly a stepwise increase in concentration of Replikins in a given class, or strain, of coronavirus for a period of about six months to about three years or more is a predictor of emergence of the strain as a likely cause of an epidemic or pandemic, such as SARS, in the future.
  • Peptide vaccines or nucleic acid-based vaccines based on the Replikins observed in the emerging strain of coronaviruses are generated.
  • An emerging strain is identified as the strain of coronavirus having the highest increase in concentration of Replikin sequences within the nucleocapsid sequence during the time period.
  • the peptide or nucleic acid vaccine is based on or includes any Replikin sequences that are observed to be conserved in the strain.
  • conserveed Replikins are preferably those Replikin sequences which are present in the nucleocapsid protein sequence for about two years and preferably longer.
  • the vaccines may include any combination of Replikin sequences identified in the emerging strain.
  • the Replikin peptide or peptides identified as useful for an effective vaccine are synthesized by any method, including chemical synthesis and molecular biology techniques, including cloning, expression in a host cell and purification therefrom.
  • the peptides are preferably admixed with a pharmaceutically acceptable carrier in an amount determined to induce a therapeutic antibody reaction thereto. Generally, the dosage is about 0.1 mg to about 10 mg.
  • the coronavirus vaccine may be administered to a patient at any time of the year.
  • the coronavirus vaccine is administered once and is based on Replikin sequences observed to be present, and preferably conserved, in the classes of coronavirus. EXAMPLE 5
  • Peptide vaccines or nucleic acid-based vaccines based on the Replikins observed in the malaria causing organism are generated.
  • the peptide or nucleic acid vaccine is based on or includes any Replikin sequences that are observed to be present on a surface antigen of the organism.
  • the vaccines may include any combination of Replikin sequences identified in the malaria causing strain.
  • the Replikin peptide or peptides identified as useful for an effective vaccine are synthesized by any method, including chemical synthesis and molecular biology techniques, including cloning, expression in a host cell and purification therefrom.
  • the peptides are preferably admixed with a pharmaceutically acceptable carrier in an amount determined to induce a therapeutic antibody reaction thereto. Generally, the dosage is about 0.1 mg to about 10 mg.
  • malaria vaccine is preferably administered to a patient in need thereof at any time during the year, and particularly prior to travel to a tropical environment.
  • Another aspect includes an antisense nucleic acid molecule complementary to the coding strand of the gene or the mRNA encoding organism for the replikins in organisms including, but not limited to, viruses, trypanosomes, bacteria, fungi, algae, amoeba, and plants, wherein said antisense nucleic acid molecules is complementary to a nucleotide sequence of a replikin containing organism.
  • Vaccine V120304U2 KNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK-KLH designated Vaccine V120304U2 was designed by the inventors from the 29 amino acid replikin Scaffold of H5N1 "Bird Flu” Influenza Replikins labeled "2004 H5N1 Vietnam, highly pathogenic” in Table 8 with the addition of two UTOPE units (KKKKHK) on the C-terminal end of the H5N1 scaffold and an additional adjuvant (key limpet hemocyanin (sequence KLH)) covalently linked on the C-terminal end of the two UTOPE units.
  • 100 ug of Vaccine V120304U2 was injected subcutaneously into rabbits and chickens .
  • the antibody response was measured before vaccination and at from one week after injection to eight weeks after injection. An antibody response was noted at one week and reached a peak in the third to fourth week after vaccination. Peak antibody responses ranged from a dilution of 1 -.120,000 to a dilution of greater than 1 :240,000.
  • Antibody titers were determined with an enzyme linked immunosorbent assay (ELISA) with Peptide-GGG (goat gamma globulin) bound in solid phase (0.1 ug/100 ul/well) on high binding 96 well plates. The serum was first diluted 50 fold and then further diluted in 2-fold serial dilutions.
  • ELISA enzyme linked immunosorbent assay
  • the ELISA titer result was determined from the estimated dilution factor that resulted from an optical density at 405 nm of 0.2 and derived from nonlinear regression analysis of the serial dilution curve. Detection was obtained using a horse radish peroxidase conjugated secondary antibody and ABTS substrate (ABTS is a registered trademark of Boehringer Mannheim. GmbH). Results from tests on two chickens and two rabbits are provided in Table 11. Individual well results from the test on rabbit D4500 are provided in Table 12. In combination with the results reported in Example 6, in a total of six tests of Replikin sequences for antibody responses in rabbit or chicken, all six sequences provided a measurable antibody response and have proved antigenic.
  • proc loadParser ⁇ ⁇ ⁇ global env Contents if ⁇ [catch ⁇ load [lindex $env(TCLLIBPATH) end] /ncbiasn.dylib ⁇ rs] ⁇ ⁇ log "load ncbiasn.dylib failed: $rs" uplevel #0 source "$Contents/Resources/Scripts/asn-parser . tcl" log "sourced asn-parser.tcl” ⁇ else ⁇ log "loaded ncbiasn.dylib” ⁇ after idle loadParser set logFile " ⁇ /Library/Preferences/com.
  • A-I 115 # contains, variously, returned HTML pages, parsed sequence data,
  • # number and sequence data page url The page is fetched and saved. #
  • accessionurl http://www.ncbi.nlm.nih.gov: 80$relativeurl set accessionlink " ⁇ a href ' $accessionurl ' >$accessionnumber ⁇ /a>” lappend work $accessionlink $accessionurl $accessionnumber foreach ⁇ accessionlink a ⁇ cessionurl accessionnumber ⁇ ⁇
  • command -label include -command " set criteria ($k. cludelabel) include set criteria ($k. elude) ⁇ 1 ⁇ log ⁇ Change criteria $k cludelabel to 'include' ⁇ triggerSearch
  • proc collectCritera ⁇ ⁇ global criteria set sort ⁇ ⁇ set collected ⁇ ⁇ foreach w [winfo children . ] ⁇ if ⁇ [string match ⁇ .[123456789]* ⁇ $w] ⁇ ⁇ lappend sort [lindex [split $w .] 1] ⁇ ⁇ foreach i [lsort -unique -integer $sort] ⁇ lappend collected $criteria($i. elude) $criteria($i. extent) $criteria($i. words)
  • logmenu . file add command -label Quit -command ⁇ exit 0 ⁇ -accel Command-Q bind .log ⁇ Command-q> ⁇ exit o) menu .
  • logmenu . edit -tearoff 0 .logmenu add cascade -label Edit -menu .logmenu. edit
  • logmenu add cascade -menu . logmenu . help
  • # default smtp host is the sender's host, and the default port
  • # is the smtp port .
  • the message includes any headers and the body. No headers,
  • proc mail ⁇ args ⁇ ⁇ set host ⁇ ⁇ set message ⁇ ⁇ set port 25 set receiver ⁇ ⁇ set sender ⁇ ⁇ while ⁇ [llength $args]>0 ⁇ ⁇ set p [lindex $args 0] ; set args [Irange $args 1 end] switch -glob -- $p ⁇

Abstract

The present invention provides a new class of peptides related to rapid replication and high human mortality, and their use in diagnosing, preventing and treating disease including vaccines and therapeutics for emerging viral diseases and methods of identifying the new class of peptides and related structures.

Description

REPLIKIN PEPTIDES AND USES THEREOF
[0001] This application claims priority to U.S. Provisional Appln. Ser. No.
60/653,083, filed February 16, 2005, and is a continuation-in-part of U.S. Appln. Ser. No. 11/116,203, filed April 28, 2005, which claims priority to U.S. Provisional Appln. Ser. No. 60/565,847, filed April 28, 2004, and is a continuation-in-part of U.S. Appln. Ser. No. 10/860,050, filed June 4, 2004, which claims priority to U.S. Provisional Applns. 60/531,686, filed February 23, 2003, 60/504,958, filed September 23, 2003, and 60/476,186, filed June 6, 2003, and is a continuation-in-part of U.S. Appln. Ser. No. 10/189,437, filed July 8, 2002, which is a continuation-in-part of U.S. Appln. Ser. No. 10/105,232, filed March 26, 2002, which is a continuation-in-part of U.S. Appln. Ser. No. 09/984,057, filed October 26, 2001, which claims priority from U.S. Provisional Applns. 60/303,396, filed July 9, 2001, and 60/278,761, filed March 27, 2001. Each of the foregoing applications is incorporated herein by reference. Technical Field of the Invention
[0002] This invention relates generally to two newly discovered classes of peptides that share structural characteristics and the use of bioinformatics to search databases of amino acids, nucleic acids and other biological information to identify shared structural characteristics. Replikins are a newly discovered class of peptides that share structural characteristics and have been correlated with rapid replication of viruses and organisms. Replikin Scaffolds are a sub-set of the class of Replikin peptides. Exoskeleton Scaffolds are another newly discovered class of peptides that share structural characteristics and have been correlated with a decrease in replication.
Background of the Invention [0003] Rapid replication is characteristic of virulence in certain bacteria, viruses and malignancies, but no chemistry common to rapid replication in different organisms has been described. The inventors have found a family of conserved small protein sequences related to rapid replication, Replikins. Such Replikins offer new targets for developing effective detection methods and therapies. There is a need in the art for methods of identifying patterns of amino acids such as Replikins. Bioinformatic Identification of Amino Acid Sequences
[0004] Identification of amino acid sequences, nucleic acid sequences and other biological structures may be aided with the implementation of bioinformatics. Publicly available databases containing amino acid and nucleic acid sequence information may be searched to identify and define Replikins, Replikin Scaffolds and Exoskeleton Scaffolds within representative proteins or protein fragments or genomes or genome fragments.
[0005] Databases of amino acids and proteins are maintained by a variety of research organizations, including, for example, the National Center for Biotechnology Information (NCBI) at the U.S. National Library of Medicine, and the Influenza Sequence Database at the Los Alamos National Laboratory. These databases are typically accessible via the Internet through web pages that provide a researcher with capabilities to search for and retrieve specific proteins. Amino Acid Search Tools
[0006] As is known in the art, databases of proteins and amino acids may be searched using a variety of database tools and search engines. Using these publicly available tools, patterns of amino acids may be described and located in many different proteins corresponding to many different organisms. Several methods and techniques are available by which patterns of amino acids may be described. One popular format is the PROSITE pattern. A PROSITE pattern description may be assembled according to the following rules:
(1) The standard International Union of Pure and Applied Chemistry (IUPAC) one-letter codes for the amino acids are used (see FIG. 12). (2) The symbol 'x' is used for a position where any amino acid is accepted.
(3) Ambiguities are indicated by listing the acceptable amino acids for a given position, between square parentheses '[ ]'. For example: [ALT] would stand for Alanine or Leucine or Threonine. (4) Ambiguities are also indicated by listing between a pair of curly brackets '{ }' the amino acids that are not accepted at a given position. For example: {AM} stands for any amino acid except Alanine and Methionine.
(5) Each element in a pattern is separated from its neighbor by a '-'.
(6) Repetition of an element of the pattern can be indicated by following that element with a numerical value or a numerical range between parentheses.
Examples: x(3) corresponds to x-x-x, x(2,4) corresponds to x-x or x-x-x or x-x-x-x. (7) When a pattern is restricted to either the N- or C-terminal of a sequence, that pattern either starts with a '<' symbol or respectively ends with a '>' symbol.
(8) A period ends the pattern. [0007] Examples of PROSITE patterns include:
PA [AC]-x-V-x(4)-{ED}. This pattern is translated as: [Alanine or Cysteine] -any- Valine -any-any-any-any-{any but Glutamic Acid or Aspartic Acid}
PA <A-x-[ST](2)-x(0,l)-V. This pattern, which must be in the N- terminal of the sequence ('<'), is translated as: Alanine -any-[Serine or Threonine]-[ Serine or Threonine] -(any or none)- Valine.
[0008] Another popular format for describing amino acid sequence patterns is the regular expression format that is familiar to computer scientists. In computer science, regular expressions are typically used to describe patterns of characters for which finite automata can be automatically constructed to recognize tokens in a language. Possibly the most notable regular expression search tool is the Unix utility grep.
[0009] In the context of describing amino acid sequence patterns, a simplified set of regular expression capabilities is typically employed. Amino acid sequence patterns defined by these simple regular expression rules end up looking quite similar to PROSITE patterns, both in appearance and in result. A regular expression description for an amino acid sequence may be created according to the following rules:
(1) Use capital letters for amino acid residues and put a "-" between two amino acids (not required).
(2) Use "[...]" for a choice of multiple amino acids in a particular position. [LIVM] means that any one of the amino acids L, I, V, or M can be in that position.
(3) Use "{... }" to exclude amino acids, Thus, {CF} means C and F should not be in that particular position. In some systems, the exclusion capability can be specified with a "Λ" character. For example, ΛG would represent all amino acids except Glycine, and [ΛILMV] would represents all amino acids except I, L, M, and V.
(4) Use "x" or "X" for a position that can be any amino acid.
(5) Use "(n)", where n is a number, for multiple positions. For example, x(3) is the same as "xxx". (6) Use "(nl,n2)" for multiple or variable positions. Thus, x(l,4) represents "x" or "xx" or "xxx" or "xxxx".
(7) Use the symbol ">" at the beginning or end of the pattern to require the pattern to match the N or C terminus. For example, ">MDEL" finds only sequences that start with MDEL. "DEL>" finds only sequences that end with
DEL.
[00010] The regular expression, "[LIVM]-[VIC]-X (2)-G-[DENQTA]-x-[GAC]-x (2)-[LIVMFY](4)-x (2)-G" illustrates a 17 amino acid peptide that has: an L, I, V, or M at position 1; a V, I, or C at position 2; any residue at positions 3 and 4; a G at position 5 and so on ....
[00011] Other similar formats are in use as well. For example, the Basic Local Alignment Search Tool (BLAST) is a well-known system available on the Internet, which provides tools for rapid searching of nucleotide and protein databases. BLAST accepts input sequences in three formats: FASTA sequence format, NCBI Accession numbers, or GenBank sequence numbers. However, these formats are even simpler in structure than regular expressions or PROSITE patterns. An example sequence in FASTA format is: >gi|532319|pir|TVFV2E|TVFV2E envelope protein
ELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTT GLLLNGSYSENRT
QIWQKHRTSNDSALILLNKHYNLTVTCKRPGNKTVLPVTIMAGLVFHS
QKYNLRLRQAWC
HFPSNWKGAWKEVKEEIVNLPKERYRGTNDPKRIFFQRQWGDPETANL
WFNCHGEFFYCK MDWFLNYLNNLTVDADHNECKNTSGTKSGNKRAPGPCVQRTYVACHI
RSVIIWLETISKK
TYAPPREGHLECTSTVTGMTVELNYIPKNRTNVTLSPQIESIWAAELDRY
KLVEITPIGF
APTEVRRYTGGHERQKRVPFVXXXXXXXXXXXXXXXXXXXXXXVQS QHLLAGILQQQKNL
LAAVEAQQQMLKLTIWGVK
[00012] Features of the BLAST system include sequence comparison algorithms that are used to search sequence databases for regions of local alignments in order to detect relationships among sequences which share regions of similarity. However, the BLAST tools are limited in terms of the structure of amino acid sequences that can be discovered and located. For example, BLAST is not capable of searching for a sequence that has "at least one lysine residue located six to ten amino acid residues from a second lysine residue," as required by a Replikin pattern, for example. Nor is BLAST capable of searching for amino acid sequences that contain a specified percentage or concentration of a particular amino acid, such as a sequence that has "at least 6% lysine residues." Need for Replikin Search Tools [00013] As can be seen from its definition, a Replikin pattern description cannot be represented as a single linear sequence of amino acids. Thus, PROSITE patterns and regular expressions, both of which are well suited to describing ordered strings obtained by following logical set-constructive operations such as negation, union and concatenation, are inadequate for describing Replikin patterns. [00014] In contrast to linear sequences of amino acids, a Replikin pattern is characterized by attributes of amino acids that transcend simple contiguous ordering. In particular, the requirement that a Replikin pattern contain at least 6% lysine residues, without more, means that the actual placement of lysine residues in a Replikin pattern is relatively unrestricted. Thus, in general, it is not possible to represent a Replikin pattern description using a single PROSITE pattern or a single regular expression. [00015] Accordingly, there is a need in the art for a system and method to scan a given amino acid sequence and identify and count all instances of a Replikin pattern. Similarly, there is a need in the art for a system and method to search protein databases and amino acid databases for amino acid sequences that match a Replikin pattern. Additionally, there is a need in the art for a generalized search tool that permits researchers to locate amino acid sequences of arbitrary specified length that includes any desired combination of the following characteristics: (1) a first amino acid residue located more than N positions and less than M positions away from a second amino acid residue; (2) a third amino acid residue located anywhere in the sequence; and (3) the sequence contains at least R percent of an amino acid residue. Finally, the shortcomings of the prior art are even more evident in research areas relating to disease prediction and treatment. There is a significant need in the art for a system to predict in advance the occurrence of disease (for example, to predict strain-specific influenza epidemics) and similarly to enable synthetic vaccines to be designed based on amino acid sequences or amino acid motifs that are discovered to be conserved over time and which have not been previously detectable by prior art methods of searching proteins and amino acid sequences. SUMMARY OF THE INVENTION
[00016] The present invention provides a method for identifying nucleotide or amino acid sequences that include a Replikin sequence. The method is referred to herein as a 3 -point-recognition method. By use of the "3 -point recognition" method, peptides comprising from 7 to about 50 amino acids including (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues and having replication, transformation, or redox functions may be identified.
[00017] An aspect of the present invention provides a method of identifying a Replikin Scaffold in a virus or organism comprising identifying a series of Replikin Scaffold peptides comprising about 16 to about 30 amino acids comprising (1) a terminal lysine and a lysine immediately adjacent to said terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to said terminal histidine, (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines. [00018] An aspect of the invention may provide a method of identifying a Replikin Scaffold peptide in a virus or organism comprising about 16 to about 30 amino acids comprising (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine, (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines. [00019] An aspect of the invention may also provide a method of making a preventive or therapeutic virus vaccine comprising identifying a Replikin Scaffold comprising about 16 to about 30 amino acids and synthesizing said Replikin Scaffold as a preventive or therapeutic virus vaccine wherein said Replikin Scaffold further comprises: (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine; (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines. The Replikin Scaffold may contain influenza virus peptide Replikins. A Replikin Scaffold may further comprise a group of Replikins comprising : (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine; (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines. [00020] An aspect of the invention may provide a method of identifying an
Exoskeleton Scaffold wherein a Replikin Scaffold is identified in a first strain of virus or organism and the Exoskeleton Scaffold is identified in a later-arising strain of said virus or organism wherein said Exoskeleton Scaffold comprises an amino acid sequence comprising the same number of amino acids as the Replikin Scaffold and further comprising (1) two terminal lysines, (2) two terminal histidines, and (3) no lysine within about 6 to about 10 amino acids from another lysine. [00021] In an aspect of the invention an isolated or synthesized influenza virus peptide is provided with from 7 to about 50 amino acids, at least one lysine residue located six to ten residues from a second lysine residue, at least one histidine residue and at least 6% lysine residues. In a further aspect the peptide comprises a terminal lysine. In yet a further aspect the peptide is present in an emerging strain of influenza virus such as the influenza virus strain H5N1.
[00022] In another aspect of the invention an isolated or synthesized influenza virus peptide is provided comprising the H5N1 peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHH.
[00023] In another aspect of the invention, an isolated or synthesized influenza virus peptide is provided having about 16 to about 30 amino acids; a terminal lysine and a lysine immediately adjacent to the terminal lysine; a terminal histidine and a histidine immediately adjacent to the terminal histidine; a lysine within about 6 to about 10 amino acids from another lysine; and at least 6% lysines.
[00024] In another aspect of the invention, a preventive or therapeutic virus vaccine is provided having at least one isolated or synthesized peptide of influenza virus with at least one lysine residue located six to ten residues from a second lysine residue; at least one histidine residue; and at least 6% lysine residues. In a further aspect of the invention the isolated or synthesized peptide is present in an emerging strain of influenza virus or is present in an H5N1 strain of influenza virus. [00025] In yet a further aspect of the invention, a preventive or therapeutic virus vaccine comprises the peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHH having alternatively a synthetic UTOPE tail, an adjuvant, or a combination thereof. In yet a further aspect, the preventive or therapeutic virus vaccine comprises a pharmaceutically acceptable carrier.
[00026] In a further aspect of the invention the preventive or therapeutic virus vaccine comprises the peptide
KKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHKKLH. [00027] In yet another aspect of the invention a method of stimulating the immune system of a subject to produce antibodies to influenza virus is provided comprising administering an effective amount of at least one isolated or synthesized influenza virus Replikin peptide comprising from 7 to about 50 amino acids comprising (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues. [00028] In a further aspect, in the method of stimulating the immune system the administered Replikin peptide may further comprise a pharmaceutically acceptable carrier and/or adjuvant and prevent or treat an influenza infection. The method of stimulating the immune system may further comprise an isolated or synthesized influenza virus peptide present in an emerging virus or present in an H5N1 strain of influenza virus. The method may further comprise administration of the peptide KKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHKKLH. [00029] An aspect of the invention may also provide a method comprising: applying a plurality of criteria to data representing protein sequences; based on the criteria, identifying an arbitrary sub-sequence within the protein sequences; and outputting the identified sub-sequence to a data file; wherein the criteria include: a set {a}of amino acids to be included in the sub-sequence; a set {b} of amino acids to be excluded from the sub-sequence; and a minimum and a maximum permissible gap between members of sets {a} and {b}. Within the method the protein sequences may be obtained via a network. An aspect of the invention may further comprise a machine-readable medium storing computer-executable instructions to perform such a method. [00030] An aspect of the invention may further provide a method comprising applying a plurality of criteria to data representing protein sequences; based on the criteria, identifying a sub-sequence within the protein sequences, the identified subsequence having a predetermined allowed range of distance between lysine amino acids thereof, and a predetermined allowed range of distance between a histidine amino acid and a farthest Lysine acid thereof; and outputting an identified sub-sequence to a data file. The protein sequences may be obtained via a network. A machine-readable medium storing computer-executable instructions may perform such a method. BRIEF DESCRIPTION OF THE DRAWINGS [00031] Figure 1 is a bar graph depicting the frequency of occurrence of Replikins in various organisms.
[00032] Figure 2 is a graph depicting the percentage of malignin per milligram total membrane protein during anaerobic replication of glioblastoma cells. [00033] Figure 3 is a bar graph showing amount of antimalignin antibody produced in response to exposure to the recognin 16-mer.
[00034] Figure 4A is a photograph of a blood smear taken with ordinary and fluorescent light. Figure 4B is a photograph of a blood smear taken with ordinary and fluorescent light illustrating the presence of two leukemia cells. Figure 4C is a photograph of a dense layer of glioma cells in the presence of antimalignin antibody. Figure 4D and Figure 4E are photographs of the layer of cells in Figure 4C taken at 30 and 45 minutes following addition of antimalignin antibody.
[00035] Figure 4F is a bar graph showing the inhibition of growth of small cell lung carcinoma cells in vitro by antimalignin antibody. [00036] Figure 5 is a plot of the amount of antimalignin antibody present in the serum of patients with benign or malignant breast disease pre-and post surgery.
[00037] Figure 6 is a box diagram depicting an aspect of the invention wherein a computer is used to carry out the 3-point-recognition method of identifying Replikin sequences.
[00038] Figure 7 is a graph showing the concentration of Replikins observed in hemagglutinin of influenza B and influenza A strain, HlNl, on a year by year basis from 1940 through 2001.
[00039] Figure 8 is a graph of the Replikin concentration observed in hemagglutinin of influenza A strains, H2N2 and H3N2, as well as an emerging strain defined by its constituent Replikins, designated H3N2(R), on a year by year basis from 1950 to 2001. [00040] Figure 9 is a graph depicting the Replikin count per year for several virus strains, including the coronavirus nucleocapsid Replikin, from 1917 to 2002. [00041] Figure 10 is a chart depicting the mean Replikin count per year for nucleocapsid coronavirus isolates. [00042] Figure 11 is a chart depicting the Replikin count per year for H5N1 Hemagglutinins.
[00043] Figure 12 is a conversion table that enables amino acids to be encoded as single alphabetic characters according to a standard supplied by the International Union of Pure and Applied Chemistry (IUPAC).
[00044] Figure 13 is a printout of a human cancer protein obtained by searching a protein database maintained by the National Center for Biotechnology Information
(NCBI).
[00045] Figure 14 is a conversion table illustrating a correspondence between nucleic acid base triplets and amino acids.
[00046] Figure 15 is a graph illustrating a rapid increase in the concentration of Replikin patterns in the hemagglutinin protein of the H5N1 strain of influenza prior to the outbreak of three "Bird Flu" epidemics. Figure 15 illustrates that increasing replikin concentration ('Replikin Count') of hemagglutinin protein of H5N1 preceded three 'Bird Flu' Epidemics. In H5N1 influenza, the increasing strain-specific replikin concentration (Replikin Count, Means+/-SD) 1995 to 1997 preceded the Hong Kong H5N1 epidemic of 1997 (El); the increase from 1999 to 2001 preceded the epidemic of 2001 (E2); and the increase from 2002 to 2004 preceded the epidemic in 2004 (E3). The decline in 1999 occurred with the massive culling of poultry in response to the El epidemic in Hong Kong.
[00047] Figure 16 is a table illustrating selected examples of Replikin patterns that have been found in various organisms.
[00048] Figure 17 is a high-level block diagram of a computer system incorporating a system and method for identifying Replikin patterns in amino acid sequences, in accordance with an aspect of the present invention.
[00049] Figure 18 is a simple flow chart illustrating a general method for locating a
Replikin pattern in a sequence of amino acids, according to an aspect of the present invention.
[00050] Figure 19 is a flow chart illustrating a generalized method for locating a plurality of Replikin-like patterns in a sequence of amino acids, according to an aspect of the present invention. [00051] Figure 20 is a source code listing containing a procedure for discovering Replikin patterns in a sequence of amino acids, in accordance with an aspect of the present invention.
[00052] Figure 21 is a table illustrating Replikin Scaffolds occurring in substantially fixed amino acid positions in different proteins.
[00053] Figure 22 is a simplified block diagram of a computer system platform useful with the present invention.
DETAILED DESCRIPTION OF THE INVENTION Definitions [00054] As used herein, the term "peptide" or "protein" refers to a compound of two or more amino acids in which the carboxyl group of one is united with an amino group of another, forming a peptide bond. The term peptide is also used to denote the amino acid sequence encoding such a compound. As used herein, "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. [00055] As used herein, a Replikin peptide or Replikin protein is an amino acid sequence having 7 to about 50 amino acids comprising:
(1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
(2) at least one histidine residue; (3) at least 6% lysine residues.
Similarly, a Replikin sequence is the amino acid sequence encoding such a peptide or protein.
[00056] As used herein, an "earlier-arising" virus or organism 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. A "later-arising" virus or organism is a specimen of a virus or organism collected from a natural source of the virus or organism on a date subsequent to the date on which another specimen of the virus or organism was collected.
I l [00057] As used herein, "emerging strain" as used herein refers to a strain of a virus, bacterium, fungus, or other organism identified as having an increased increasing concentration of Replikin sequences in one or more of its protein sequences relative to the concentration of Replikins in other strains of such organism. The increase or increasing concentration of Replikins occurs over a period of at least about six months, and preferably over a period of at least about one year, most preferably over a period of at least about three years or more, for example, in influenza virus, but may be a much shorter period of time for bacteria and other organisms. [00058] As used herein, "mutation" refers to change in this structure and properties of an organism caused by substitution of amino acids. In contrast, the term
"conservation" as used herein, refers to conservation of particular amino acids due to lack of substitution.
[00059] As used herein, "replikin count" refers to the number of replikins per 100 amino acids in a protein or organism. A higher replikin count in a first strain of virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier- or later-arising strain of the virus or organism having a lower replikin count.
[00060] As used herein "Replikin Scaffold" refers to a series of conserved Replikin peptides wherein each of said Replikin peptide sequences comprises about 16 to about 30 amino acids and further comprises: (1) a terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine; (3) a lysine within 6 to 10 amino acid residues from another lysine; and (4) about 6% lysine. "Replikin Scaffold" peptides may comprise an additional lysine immediately adjacent to the terminal lysine. "Replikin Scaffold" also refers to an individual member or a plurality of members of a series of a "Replikin Scaffold." Identification of Replikins
[00061] The identification of a new family of small peptides related to the phenomenon of rapid replication, referred to herein as Replikins, provides targets for detection of pathogens in a sample and developing therapies, including vaccine development. In general, knowledge of and identification of this family of peptides enables development of effective therapies and vaccines for any organism that harbors Replikins. Identification of this family of peptides also provides for the detection of viruses and virus vaccine development. [00062] For example, identification of this family of peptides provides for the detection of influenza virus and provides new targets for influenza treatment and vaccines including treatment and vaccines for influenza H5N1. Further examples provided by the identification of this family of peptides include the detection of infectious disease Replikins, cancer immune Replikins and structural protein Replikins. [00063] Rapid replication is characteristic of virulence in certain bacteria, viruses and malignancies, but no chemistry common to rapid replication in different organisms has been described. We have found a family of conserved small protein sequences related to rapid replication, which we have named Replikins. Such Replikins offer new targets for developing effective detection methods and therapies. The first Replikin found was the glioma Replikin, which was identified in brain glioblastoma multiforme (glioma) cell protein, called malignin.
[00064] Hydrolysis and mass spectrometry of malignin revealed the novel 16-mer peptide sequence which contains the glioma Replikin. This Replikin was not found in databases for the normal healthy human genome and therefore appeared to be derived from some source outside the body.
[00065] We have devised an algorithm to search for the glioma Replikin or homologue thereof. Homologues were not common in over 4,000 protein sequences, but were found, surprisingly, in all tumor viruses, and in the replicating proteins of algae, plants, fungi, viruses and bacteria.
[00066] We have identified that both 1) Replikin concentration (number of Replikins per 100 amino acids) and 2) Replikin composition correlate with the functional phenomenon of rapid replication. These relationships provide functional basis for the determination that Replikins are related quantitatively as well as qualitatively to the rate of replication.
[00067] The first functional basis for Replikins role to rapid replication was discovered by the Applicants in glioma replication. The fact that glioma malignin was found to be enriched ten-fold compared to the five-fold increase in cell number and membrane protein concentration in rapid replication of glioma cells suggests an integral relationship of the Replikins to replication. When the glioma Replikin was synthesized in vitro and administered as a synthetic vaccine to rabbits, abundant antimalignin antibody was produced. This establishes the antigenic basis of the antimalignin antibody in serum (AMAS) test, and provides the first potential synthetic cancer vaccine and the prototype for Replikin vaccines in other organisms. With the demonstration of this natural immune relationship of the Replikins to replication and this natural immune response to cancer Replikins, which overrides cell type, based upon the shared specificity of cancer Replikins and rapid replication, both passive augmentation of this immunity with antimalignin antibody and active augmentation with synthetic Replikin vaccines now is possible.
[00068] The relationship between the presence of antimalignin antibody and survival in patients was shown in a study of 8,090 serum specimens from cancer patients. The study showed that the concentration of antimalignin antibody increases with age, as the incidence of cancer in the population increases, and increases further two to three-fold in early malignancy, regardless of cell type. In vitro, the antimalignin antibody is cytotoxic to cancer cells at picograms (femtomoles) per cancer cell, and in vivo the concentration of antimalignin antibody relates quantitatively to the survival of cancer patients. As shown in glioma cells, the stage in cancer at which cells have only been transformed to the immortal malignant state but remain quiescent or dormant, now can be distinguished from the more active life-threatening replicating state, which is characterized by the increased concentration of Replikins. In addition, clues to the viral pathogenesis of cancer may be found in the fact that glioma glycoprotein 1OB has a 50% reduction in carbohydrate residues when compared to the normal 1OB. This reduction is associated with virus entry in other instances, and so may be evidence of the attachment of virus for the delivery of virus Replikins to the 1OB of glial cells as a step in the transformation to the malignant state.
[00069] Our study concerning influenza virus hemagglutinin protein sequences and influenza epidemiology over the past 100 years has provided a second functional basis for the relations of Replikins to rapid replication. Only serological hemagglutinin and antibody classification, but no strain-specific conserved peptide sequences have previously been described in influenza. Further, no changes in concentration and composition of any strain-specific peptide sequences have been described previously that correlate with epidemiologically documented epidemics or rapid replication. In this study, a four to ten-fold increase in the concentration of strain-specific influenza Replikins in one of each of the four major strains, influenza B, (A)HINl, (A)H2N2 and, (A)H3N2 is shown to relate to influenza epidemics caused by each strain from 1902 to 2001. [00070] We then showed that these increases in concentration are due to the reappearance of at least one specific Replikin composition from 1 to up to 64 years after its disappearance, plus the emergence of new strain-specific Replikin compositions. Previously, no strain-specific chemical structures were known with which to predict the strains that would predominate in coming influenza seasons, nor to devise annual mixtures of whole-virus strains for vaccines. The recent sharp increase in H3N2 Replikin concentration (1997 to 2000), the largest in H3N2's history, and the reappearance of specific Replikin compositions that were last seen in the high mortality H3N2 pandemic of 1968, and in the two high mortality epidemics of 1975 and 1977, but were absent for 20-25 years, together may be a warning of coming epidemics. This high degree of conservation of Replikin structures observed, whereby the identical structure can persist for 100 years, or reappear after an absence of from one to 64 years, indicate that what was previously thought to be change due to random substitution of amino acids in influenza proteins is more likely to be change due to an organized process of conservation of Replikins.
[00071] The conservation of Replikins is not unique to influenza virus but was also observed in other sources, for example in foot and mouth disease virus, type 0, HIV tat, and wheat. [00072] A third functional basis for Replikins' role in rapid replication is seen in the increase in rapid replication in HIV. Replikin concentration was shown to be related to rapid replication in HIV. We found the Replikin concentration in the slow growing low-titre strain of HIV (NSl, "Bru"), which is prevalent in early stage infection, to be one-sixth of the Replikin concentration in the rapidly-growing high-titre strain of HIV (SI, "Lai")(prevalent in late stage HIV infection). [00073] Further examples demonstrate the relationship of Replikins to rapid replication. In the "replicating protein," of tomato leaf curl gemini virus, which devastates tomato crops, the first 161 amino acids, the sequence that has been shown to bind to DNA, was shown to contain five Replikins. In malaria, legendary for rapid replication when trypanosomes are released from the liver in the tens of thousands from one trypanosome, multiple, novel, almost 'flamboyant' Replikin structures have been found with concentrations of up to 36 overlapping Replikins per 100 amino acids. [00074] The conservation of any structure is critical to whether that structure provides a stable invariant target to attack and destroy or to stimulate. When a structure is tied in some way to a basic survival mechanism of the organism, the structures tend to be conserved. A varying structure provides an inconstant target, which is a good strategy for avoiding attackers, such as antibodies that have been generated specifically against the prior structure and thus are ineffective against the modified form. This strategy is used by influenza virus, for example, so that a previous vaccine may be quite ineffective against the current virulent virus. Replikins as Stable Targets for Treatment
[00075] Both bacteria and HIV have both Replikin and non-Replikin amino acids. In HIV, for example, there has been a recent increase in drug-resistance from 9% to 13% due to mutation, that is, substitution of amino acids not essential to the definition of the Replikin structure. (See detailed analysis of TAT protein of HIV discussed herein). In bacteria, the development of 'resistant strains' is due to a similar mechanism. However, we have found that Replikin structures do not mutate or change to the same degree as non Replikin amino acids (see also discussion of foot and mouth disease virus conservation of Replikins discussed herein; further see discussion of conservation of coronavirus Replikins discussed herein). The Replikin structures, as opposed to the non-Replikin structures are conserved and thus provide new constant targets for treatment. [00076] Certain structures too closely related to survival functions apparently cannot change constantly. Because an essential component of the Replikin structure is histidine (h), which is know for its frequent binding to metal groups in redox enzymes and probable source of energy needed for replication, and since this histidine structure remains constant, this structure remains all the more attractive a target for destruction or stimulation. [00077] From a proteomic point of view, the inventors' construction of a template based on the newly determined glioma peptide sequence led them to the discovery of a wide class of proteins with related conserved structures and a particular function, in this case replication. Examples of the increase in Replikin concentration with virulence of a disease include, influenza, HIV, cancer and tomato leaf curl virus. This newly recognized class of structures is related to the phenomenon of rapid replication in organisms as diverse as influenza, yeast, algae, plants, the gemini curl leaf tomato virus, HIV and cancer. [00078] Replikin concentration and composition provide new quantitative methods to detect and control the process of replication, which is central to the survival and dominance of each biological population. The sharing of immunological specificity by diverse members of the class, as demonstrated with antimalignin antibody for the glioma and related cancer Replikins, suggests that B cells and their product antibodies may recognize Replikins by means of a similar recognition language, [00079] Examples of peptide sequences of cancer Replikins or as containing a Replikin, i.e., a homologue of the glioma peptide, kagvaflhkk, may be found in such cancers of, but not limited to, the lung, brain, liver, soft-tissue, salivary gland, nasopharynx, esophagus, stomach, colon, rectum, gallbladder, breast, prostate, uterus, cervix, bladder, eye, forms of melanoma, lymphoma, leukemia, and kidney. [00080] Replikins provide for: 1) detection of pathogens by qualitative and quantitative determinations of Replikins; 2) treatment and control of a broad range of diseases in which rapid replication is a key factor by targeting native Replikins and by using synthetic Replikins as vaccines; and 3) fostering increased growth rates of algal and plant foods.
[00081] The first Replikin sequence to be identified was the cancer cell Replikin found in a brain cancer protein, malignin, which was demonstrated to be enriched tenfold during rapid anaerobic replication of glioblastoma multiforme (glioma) cells. (Figure 2) Malignin is a 10KDa portion of the 250 KDa glycoprotein 1OB, which was isolated in vivo and in vitro from membranes of glioblastoma multiforme (glioma) cells. Hydrolysis and mass spectroscopy of malignin revealed a 16-mer peptide sequence, ykagvaflhkkndide (SEQ ID NO:4), which is referred to herein as the glioma Replikin and which includes the shorter peptide, kagvaflhkk (SEQ ID NO: 1), both of which apparently are absent in the normal human genome.
Table 1
16-mer peptide sequence YKAGVAFLHKKNDIDE obtained from malignin by hydrolysis and mass spectrometry
Figure imgf000020_0001
[00082] When the 16-mer glioma Replikin was synthesized and injected as a synthetic vaccine into rabbits, abundant antimalignin antibody was produced. (Bogoch et al., Cancer Detection and Prevention, 26 (Suppl. 1): 402 (2002)). The concentration of antimalignin antibody in serum in vivo has been shown to relate quantitatively to the survival of cancer patients. (Bogoch et al., Protides of Biological Fluids, 31 :739-747 (1984). In vitro antimalignin antibodies have been shown to be cytotoxic to cancer cells at a concentration of picograms (femtomolar) per cancer cell. (Bogoch et al., Cancer Detection and Prevention, 26 (Suppl. 1): 402 (2002). [00083] Studies carried out by the inventors showed that the glioma Replikin is not represented in the normal healthy human genome. Consequently, a search for the origin and possible homologues of the Replikin sequence was undertaken by analysis of published sequences of various organisms. [00084] By using the 16-mer glioma Replikin sequence as a template and constructing a recognition proteomic system to visually scan the amino acid sequences of proteins of several different organisms, a new class of peptides, the Replikins, was identified. The present invention provides a method for identifying nucleotide or amino acid sequences that include a Replikin sequence. The method is referred to herein as a 3-point-recognition method. The three point recognition method comprises: a peptide from 7 to about 50 amino acids including (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues. (Replikin). These peptides or proteins constitute a new class of peptides in species including algae, yeast, fungi, amoebae, bacteria, plant, virus and cancer proteins having replication, transformation, or redox functions. Replikin peptides have been found to be concentrated in larger 'replicating' and 'transforming' proteins (so designated by their investigators, See Table 2) and cancer cell proteins. No sequences were found to be identical to the malignin 16-mer peptide. [00085] The present invention further provides a method for identifying nucleotide or amino acid sequences that include a Replikin sequence comprising from 7 to about 50 amino acids including (1) at least one first lysine located at either terminus of the isolated or synthesized peptide, (2) a second lysine located six to ten residues from the first lysine residue; (3) at least one histidine; and (4) at least 6% lysines. In another aspect of the invention the isolated or synthesized peptides are influenza virus peptides. In yet another aspect of the invention, the isolated or synthesized peptides are H5N1 influenza virus peptides. Table 2
Examples of Replikins in various organisms - prototype: Glioma Replikin*
KAGVAFLHKK (SEQ ID NO:!)
Figure imgf000022_0001
Figure imgf000023_0001
[00086] Identification of an amino acid sequence as a Replikin or as containing a Replikin, i.e., a homologue of the glioma peptide, kagvaflhkk, requires that the three following requirements be met. According to the three point recognition system the sequences have three elements: (1) at least one lysine residue located six to ten residues from another lysine residue; (2) at least one histidine residue; and (3) a composition of at least 6% lysine within an amino acid sequence of 7 to about 50 residues. An exemplary non-limiting Replikin comprises a terminal lysine.
[00087] Databases were searched using the National Library of Medicine keyword "PubMed" descriptor for protein sequences containing Replikin sequences. Over 4,000 protein sequences were visually examined for homologues. Sequences of all individual proteins within each group of PubMed-classified proteins were visually scanned for peptides meeting the three above-listed requirements. An infrequent occurrence of homologues was observed in "virus peptides" as a whole (1.5%) (N=953), and in other peptides not designated as associated with malignant transformation or replication such as "brain peptides" and "neuropeptides" (together 8.5%) (N=845). However, surprisingly, homologues were significantly more frequently identified in large "replicating proteins," which were identified as having an established function in replication in bacteria, algae, and viruses. Even more surprising was the finding that Replikin homologues occurred in 100% of "tumor viruses" (N=250), in 97% of "cancer proteins" (N=401), and in 85% of "transforming viruses" (N=248). These results suggest that there are shared properties of cancer pathogenesis regardless of cell type and suggest a role of viruses in carcinogenesis, i.e., conversion of cells from a transformed albeit dormant state to a more virulent actively replicating state. [00088] Homologues of the following amino acid sequence, kagvaflhkk, as defined by the three point recognition method, were found in such viruses, or viral peptides, as, but not limited to, adenovirus, lentivirus, a-virus, retrovirus, andeno-associated virus, human immunodeficiency virus, hepatitis virus, influenza virus, maize streak virus, herpes virus, bovine herpes virus, feline immunodeficiency virus, foot and mouth disease virus, small pox virus, rous sarcoma virus, neuroblastoma RAS viral oncogene, polyamavirus, sindbis, human papilloma virus, myelomonocytic tumor virus, murine acute leukemia, T-cell lymphotropic virus, and tomato leaf curl virus. [00089] Furthermore, homologues of the amino acid sequence kagvafhkk are present in known classes of coronavirus, which are members of a family of enveloped viruses that replicate in the cytoplasm of host cells. Additionally, the homologue of the amino acid sequence kagvatlhkk is present in the recently identified class of coronavirus responsible for severe acute respiratory syndrome, or SARS. The replikin is located in the nucleocapsid whole protein sequence of the SARS coronavirus. In addition, the location of the replikins is present in other members of the coronavirus class and, more specifically, are also present in the nucleocapsid protein sequences from these coronaviruses. [00090] Replikins are present in such bacteria as, but not limited to, Acetobacter, Achromobacter, Actinomyces, Aerobacter, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Chainia, Clostridium, Corynebacterium, Erwinia, Escheria, Lebsiella, Lactobacillus, Haemophilus, Flavobacterium, Methylomonas, Micrococcus, Mycobacterium, Micronomspora, Mycoplasma, Neisseria, Nocardia, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Staphylococcus, Streptocossus, Streptomyces, Streptosporangium, Strepto-virticillium, Vibrio peptide, and Xanthomas. Replikins are present in such fungi as, but not limited to, Penicillium, Diseula, Ophiostoma novo-ulim, Mycophycophta, Phytophthora infestans, Absidia, Aspergillus, Candida, Cephalosporium, Fusarium, Hansenula, Mucor, Paecilomyces, Pichia, Rhizopus, Torulopsis, Trichoderma, and Erysiphe. Replikins are present in such yeast as, but not limited to, Saccharomyces, Cryptococcus, including Cryptococcusneoformas, Schizo-saccharomyces, and Oryza. Replikins are present in algae such as, but not limited to, Caldophera, Isolepisprolifera, Chondrus, Gracilaria, Gelidium, Caulerpa, Laurencia, Cladophexa, Sargassum, Penicillos, Halimeda, Laminaria, Fucus, Ascophyllum, Undari, Rhodymenia, Macrocystis, Eucheuma, Ahnfeltia, and Pteroclasia. Replikins are present in amoeba such as, but not limited to, Entamoeba (including Entamoeba invadens), Amoebidae, Acanthamoeba and Naegleria. Replikins are present in plants such as, but not limited to, Arabidopsis, wheat, rice, and maize. Auxiliary Specifications
[00091] To permit classification of subtypes of Replikins, additional or "auxiliary specifications" to the basic "3-point-recognition" requirements may be added: (a) on a structural basis, such as the common occurrence of adjacent di- and polylysines in cancer cell proteins (e.g., transforming protein P21B(K-RAS 2B), lung, Table 2, SEQ ID NO: 89), and other adjacent di-amino acids in TOLL-like receptors, or b) on a functional basis, such as exhibiting ATPase, tyrosine kinase or redox activity as seen in Table 2. Functional Derivatives [00092] "Functional derivatives" of the Replikins as described herein are fragments, variants, analogs, or chemical derivatives of the Replikins, which retain at least a portion of the immunological cross reactivity with an antibody specific for the Replikin. A fragment of the Replikin peptide refers to any subset of the molecule. 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. Replikins and Replication [00093] As seen in Figure 2, during anaerobic respiration when the rate of cell replication is increased, malignin is enriched. That is, malignin is found to increase not simply in proportion to the increase in cell number and total membrane proteins, but is enriched as much as ten-fold in concentration, starting with 3% at rest and reaching 30% of total membrane protein. This clear demonstration of a marked increase in Replikin concentration with glioma cell replication points to, and is consistent with, the presence of Replikins identified with the 3 -point recognition method in various organisms. For example, Replikins were identified in such proteins as "Saccharomyces cerevisiae replication binding protein" (SEQ ID NO: 2)
(hsikrelgiifdk); the "replication associated protein A of maize streak virus" (SEQ ID NO: 8) (kyivcareahk) and (SEQ ID NO: 9) (kekkpskdeimrdiish); the "replication- associated protein of Staphylococcus aureus" (SEQ ID NO: 10) (kkektthnk); the "DNA replication protein of bovine herpes virus 4" (SEQ ID NO: 11) (hkinitngqk); and the "Mealigrid herpes virus 1 replication binding protein" (SEQ ID NO: 12) (hkdlyrllmk). Previous studies of tomato leaf curl gemini virus show that the regulation of virus accumulation appears to involve binding of amino acids 1-160 of the "replicating protein" of that virus to leaf DNA and to other replication protein molecules during virus replication. Analysis of this sequence showed that amino acids 1-135 of this "replicating protein" contain a replikin count (concentration) as high as 20.7 (see section on tomato leaf curl Gemini virus.)
[00094] Table 2 shows that Replikin-containing proteins also are associated frequently with redox functions, and protein synthesis or elongation, as well as with cell replication. The association with metal-based redox functions, the enrichment of the Replikin-containing glioma malignin concentration during anaerobic replication, and the cytotoxicity of antimalignin at low concentrations (picograms/cell) (Figure 4C- 4F), all suggest that the Replikins are related to central respiratory survival functions, have been found less often subjected to the mutations characteristic of non-Replikin amino acids. Replikins in Influenza Epidemics
[00095] Of particular interest, it was observed that at least one Replikin per 100 amino acids was found to be present in the hemagglutinin proteins of almost all of the individual strains of influenza viruses examined. The Replikin sequences that were observed to occur in the hemagglutinin proteins of isolates of each of the four prevalent strains of influenza virus, influenza B5 HlNl, H2N2, and H3N2, for each year that amino acid sequence data are available (1902-2001), are shown in Tables 3, 4, 5 and 6. [00096] Both the concentration and type, i.e., composition of Replikins observed, were found to relate to the occurrence of influenza pandemics and epidemics. The concentration of Replikins in influenza viruses was examined by visually scanning the hemagglutinin amino acid sequences published in the National Library of Medicine "PubMed" data base for influenza strains isolated world wide from human and animal reservoirs year by year over the past century, i.e., 1900 to 2001. These Replikin concentrations (number of Replikins per 100 amino acids, mean +/- SD) were then plotted for each strain.
[00097] The concentration of Replikins was found to directly relate to the occurrence of influenza pandemics and epidemics. The concentration of Replikins found in influenza B hemagglutinin and influenza A strain, HlNl, is shown in Figure 7, and the concentration of Replikins found in the two other common influenza virus A strains, H2N2 and H3N2 is shown in Figure 8 (H2N2, H3N2). The data in Figure 8 also demonstrate an emerging new strain of influenza virus as defined by its constituent Replikins (H3N2(R)). [00098] Each influenza A strain has been responsible for one pandemic: in 1918, 1957, and 1968, respectively. The data in Figures 7 and 8 show that at least one
Replikin per 100 amino acids is present in each of the influenza hemagglutinin proteins of all isolates of the four common influenza viruses examined, suggesting a function for Replikins in the maintenance of survival levels of replication. In the 1990s, during the decline of the H3N2 strain, there were no Replikins in many isolates of H3N2, but a high concentration of new Replikins appeared in H3N2 isolates, which define the emergence of the H3N2(R) strain. See Tables 3, 4, 5 and 6.
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Table 3
Replikin Sequences present in hemagglutinins of Influenza B viruses in each year for which amino acid sequences were available (1940-2001).
Influenza B Replikins Year Detected in Influenza B strain
Peak in Figure 7:
kshfanlk (SEQ ID NO: 104) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 kshfanlkgtk (SEQ ID NO: 105) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 kshfanlkgtktrgklcpk (SEQ ID NO: 106) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hekygglnk (SEQ ID NO: 107) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hekygglnksk (SEQ ID NO: 108) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hekygglnkskpyytgehak (SEQ ID NO: 109) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hakaigncpiwvk (SEQ ID NO: 110) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hakaigncpiwvktplklangtk (SEQ ID NO: 111) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hakaigncpiwvktplklangtkyrppak (SEQ ID NO: 112) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 hakaigncpϊwvktplklangtkyrppakllk (SEQ ID NO: 113) 1940,43,51,59,75,76,77,89,90,93,97,98,99,00,01 k(a/v)silhevk (SEQ ID NO: 119) 1940, 59, 90,93 kvwcasgrskvikgslpligeadclh (SEQ ID NO: 123) 1940,43, 59,75,76,77,89,90, 98,99,00 kpyytgehak (SEQ ID NO: 124) 1940, 59, 89,90,93,97,98, 01 hgvavaadlkstqeaink (SEQ ID NO: 128) 1940, 59, 00 hgvavaadlkstqeainkdtistqeaink (SEQ ID NO: 129) 1940
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hsdneiqmvklygdsk (SEQ ID NO: 116) hsdneiqdkmvklygdskpqk (SEQ ID NO: 117) kygglπkskpyytgeh (SEQ ID NO: 122) kcmgtipsakasilhevk (SEQ ID NO: 125) 1943, 75,76,77, 93 klygdskpqkftssaπgvtth (SEQ ID NO: 130) 1943, 75,76,77, 93,97, OO hsdnetqmaklygdskpqk (SEQ ID NO: 131) 1943, 75,76,77, 93 hfanlkgtqtrgk (SEQ ID NO: 132) 1959 hfanlkgtktrgk (SEQ ID NO: 114) 1976, 89,90, 99,00,01 hfanlkgtktrgklcpk (SEQ ID NO: 115) 1976, 90 00,01 kprsalkckgfh (SEQ ID NO: 133) 1988 kctgtipsakasϊlhevk (SEQ ID NO: 121) 1993 hnviπaekapggpyk (SEQ ID NO: 126) 1993,97, OO hsdnetqmaklygdsk (SEQ ID NO: 127) 1993,97, OO hsdneiqmvklygdskpqk (SEQ ID NO: 118) 1997,98, 00 kctgtipsakasilh (SEQ IDNO: 120) 2000 kskpyytgehakai(g/a)ncpiwvk (SEQ ID NO: 134) 2000
1. Influenza B has not been responsible for any human pandemic.
2. Abbreviation for years: e.g., "43" = 1943, "01" = 2001.
3. The first year that a given Replikin appears is indicated at the beginning of the series of years in which that Replikin has been found.
4. Overlapping Replikin sequences are listed separately.
5. Return of replikins, absent for several years, in the two years before the epidemic of 1977, underlined, correlates with increased total Replikin concentration (Replikin Count = number of Replikins per 100 amino acid residues). See Figure 7.
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Table 4
HlNl Replikin Sequences present in HINI hemagglutinins of Influenza viruses in each year for which amino acid sequences were available (1918-2000)
HlNl ReplikinYear Detected in Influenza
HlNl Strain
Peak in Figure7 : Pi El EU, 12, 11 ElA )
hp(v/i)tigecpkyv(r/k)(s/t)(t/a)k (SEQ ID NO: 135) 1918,25.28.30.31.35.47.48.51.52.55.56.57.59.63.77.79.80.81.85.87,88,89,91.92,95.96.97.98.99,0O hdsnvknly(e/g)kv(k/r)(n/s)ql(k/r)nnak (SEQ ID NO: 136) 1918, 28,30,31 77,79,80, 88, 91, 95, 98 hdsnvknly(e/g)kv(k/r)(n/s)qlk (SEQ ID NO: 137) 1918. 28,30,31, 77,79,80, 88, 91, 95, 98 hkc(nn/dd)(a/t/e)cmesv(r/k)ngtydypkyseesklnre(e/k)idgvk (SEQ ID NO: 138) 1918. 30, 35, 77, 80, 98 hkc(nn/dd)(a/t/e)cmesv(r/k)ngtydypkyseesk (SEQ ID NO: 139) 1918. 30, 35, 77, 80, 98 hqn(e/g)qgsgyaadqkstqnai(d/n)gitnkvnsviekmntqftavgkefhklek (SEQ ID NO: 140) 1918, 28,30,31,35, 59, 79, 95 hqn(e/g)qgsgyaadqkstqnai(d/n)gitnkvnsviek (SEQ ID NO: 141) 1918, 28,30,31,35, 59, 79, 95 hqn(e/g)qgsgyaadqkstqnai(d/n)gitnk (SEQ ID NO: 142) 1918. 28,30,31,35, 59, 79, 95 kfeifpktsswpnh (SEQ ID NO: 143) 1918. 77 kg(n/s/t)sypkl(n/s)ksy(v/t)nnkgkevlvlwgvh (SEQ ID NO: 144) 1918. 35, 77, 96 ksy(v/t)nnkgkevlvlwgvh (SEQ ID NO: 145) 1918. 35, 77, 96 hkcnnecmesvkngtydypkyseesklnrekidgvk (SEQ ID NO: 146) 1928, 31 95 hkcnnecmesvkngtydypkyseesk (SEQ ID NO: 147) 1928, 31, 9 955 hkcnnecmesvkngtydypk (SEQ ID NO: 148) 1928, 31 9 955 hkcnnecmesvk (SEQ ID NO: 149) 1928, 31 9 955 hngkssfy(k/r)nllwlt(e/g)knglypnlsksyvnnkek (SEQ ID NO: 150) 1928, 9 955,, 0 000 hngkssfy(k/r)nllwlt(e/g)knglypnlsksyvnnk (SEQ ID NO: 151) 1928, H, 9 955,, 0 000
Figure imgf000031_0001
Uϊ"4-Z0.4/-S /O
hngklcrkgϊaplqlgk (SEQ IDNO: 176) 1959, 82 hetnrqvtaacpyagansffrnliwlvkkessypklsk (SEQ ID NO: 177) 1963. 81 hetnrqvtaacpyagansf&nliwlvkkessypk (SEQIDNO: 178) 1963, 81 hpptstdqqslyqnadayifvgsskynrkfk (SEQIDNO: 179) 1963. 81 hpptstdqqslyqnadayifvgsskynrkfkpeia (SEQ ID NO: 180) 1963. 81 hdiyrdeainnrfqiqgvkitqgyk (SEQIDNO: 181) 1977.79. 91 hqneqgsgyaadqkstqnaidgitnkvnsviekmntqftavgk (SEQIDNO: 182) 1977 hqneqgsgyaadqkstqnaidgitnkvnsviek (SEQIDNO: 183) 1977 hqneqgsgyaadqkstqnaingitnkvnsviekmntqftavgkefhklek (SEQIDNO: 184) 1979. 91 hngklcrlkgiaplqlgk (SEQIDNO: 185) 1979 hkcπnecmesvk (SEQ ID NO: 186) 1979 kfeifpkasswpnh (SEQIDNO: 187) 1981 hdsnvknlyekvrsqlrnnak (SEQ ID NO: 188) 1981 kvnsvikkmntqfaavgkefeh (SEQ IDNO: 189) 1981 khngklck (SEQ IDNO: 190) 1981 kkgtsypklsksythπkgkevlvlwgvh (SEQIDNO: 191) 1981 kgtsypklsksythnkgkevlvlwgvh (SEQ ID NO: 192) 1981 klsksythnkgkevlvlwgvh (SEQIDNO: 193) 1981 ksythnkgkevlvlwgvh (SEQ IDNO: 194) 1981 kgvtascshk (SEQIDNO: 195) 1985,87 kgvtascshkgrssfyrnllwlteknglypnlsk (SEQ ID NO: 196) 1985,87 kgnsypklsksyvnnkekevlvlwgih (SEQ ID NO: 197) 1988 kefhhlek (SEQIDNO: 198) 1988 hpptstdqqslyqnadayvfvgsskynkkfkpeiatrpk (SEQIDNO: 199) 1988
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hpptstdqqslyqnadayvfvgsskynkkfk (SEQ ID NO: 200) 1988 hegkssfyrnllwltekegsypklknsyvnk (SEQ ID NO: 201) 1991 hegkssfyrnllwltekegsypk (SEQ ID NO: 202) 1991 hkcdnecmesvrngtydypkyseesk (SEQ ID NO: 203) 1991 kesswpnhtvtk (SEQ ID NO: 204) 1991,92 knllwlteknglypnlsksyvnnkekeilvlwgvh (SEQ ID NO: 205) 1991,92, 96 hngkssfy(k/m)(n/-)llwlt(e/g)(-/k)knglypnlsk (SEQ ID NO: 206) 1991,92, 96, OO hngkssfyknllwltek (SEQ ID NO: 207) 1991,92, 96 htvtkgvtascshngkssfyknllwlteknglypnlsksyvnnkekevlvlwgvh (SEQ ID NO: 208) 1995 htvt(k/g)gv(t/s)ascshngkssfy(k/m)(n/-)llwlt(e/g)k(-n/k)glypnlsk (SEQ ID NO: 209) 1995, OO htvtkgvtascshπgkssfyknllwltek (SEQ ID NO: 210) 1995 kyvrstklrmvtglrnipsiqsrglfgaiagfieggwtgmidgΛvygyh (SEQ ID NO: 211) 1995 hqneqgsgyaadqkstqnaingitnkvnsiiekmntqftavgk (SEQ ID NO: 212) 1995 hqneqgsgyaadqkstqnaingitnkvnsiiek (SEQ ID NO: 213) 1995 hqneqgsgyaadqkstqnaingitnk (SEQ ID NO: 214) 1995 hsgarsfyrnllwivkkgnsypk (SEQ ID NO: 215) 1996 hsgarsfyrnllwivkkgnsypklnk (SEQ ID NO: 216) 1996 hsgarsfymllwϊvkkgnsypklnksytndk (SEQ ID NO: 217) 1996 hsgarsfyrnllvvivkkgnsypklnksytndkgk (SEQ ID NO: 218) 1996 htvskgvttscshngk (SEQ ID NO: 219) 1996 katswpnhettk (SEQ ID NO: 220) 1996 kqvttscshnqk (SEQ ID NO: 221) 1996 kgnsypklnksytπdkgkevlviwgvh (SEQ ID NO: 222) 1996 klnksytndkgkevlviwgvh (SEQ ID NO: 223) 1996
Figure imgf000034_0001
U^tZJ.t /J /O
Table 5
Replikin Sequences present in hemagglutinins of Influenza H2N2 viruses in years 1957-2000 Influenza H2N2 ReplikinsYear Detected in Influenza H2N2 strain
(Peak in Figure 8: P2 E2 )
khfekvkilpk (SEQ ID NO: 230) 1957,58,59,60,61,64,65,68, 78,83,84,91 khllssvkhfekvk (SEQ ID NO: 231) 1957.58.59.60.61. 83,84,91 ha(k/q/m)(d/n)ilekthngk (SEQ ID NO: 232) 1957.58.59.60.61.64.65.68. 78,83,84,91, 95 ha(k/q/m)(d/n)ilekthngklc(k/r) (SEQ ID NO: 233) 1957,58,59,60,61,64,65,68, 78,83,84,91, 95 hnvhpltigecpkyvksek (SEQ ID NO: 234) 1957.58.59. 65,68 hpltigecpkyvksek (SEQ ID NO: 235) 1957.58.59. 65,68,64,65,68,78,83,84,91 khllssvkhfekvkilpk (SEQ ID NO: 236) 1957,58,59,60,61,64,65,68, 78 krqssgimktegtlencetkcqtplgainttlpfhnvh (SEQ ID NO: 237) 1957. 59, 83 kgsnyp(v/i)ak(g/r)synntsgeqmliiwq(v/i)h (SEQ ID NO: 238) 1957,58,59, 61, 83, 91, 95 httlgqsracavsgnpsfϊrnmvwltekgsnypvak (SEQ ID NO: 239) 1957 khfekvk (SEQ ID NO: 240) 1957, 59, 65 kiskrgssgimktegtlencetkcqtplgainttlpfh (SEQ ID NO: 241) 1957. 59, 65, 91 krgssgimktegtlencetkcqtplgainttlpfh (SEQ ID NO: 242) 1957. 59, 65, 91 ktegtlencetkcqtplgainttlpfh (SEQ ID NO: 243) 1957. 59, 65, 91 kiskrgssgimktegtlencetkcqtplgainttlpfh (SEQ ID NO: 244) 1957. 59, 65, 91 ktegtlencetkcqtplgainttlpfhn(v/i)h (SEQ ID NO: 245) 1957. 59, 65, 91 kiskrgssgimktegtlencetkcqtplgainttlpfh (SEQ ID NO: 246) 1957, 59, 65, 91 k(e/g)snypvakgsynntsgeqmliiwgvh (SEQ ID NO: 247) 1957. 60, 65
W4Z5A /ά /b
hpltigecpkyvksek (SEQ DD NO: 248) 1957, 60, 65 kcqtplgaikttlpfh (SEQ ID NO: 249) 1957, 65 hhsndqgsgyaadkestqka(f/i)dgitnkvnsviek-
-mntqfeavgklf(n/s)nleklenlnkk (SEQ ID NO: 250) 1961, 65,68, 83,84 hsndqgsgyaadkestqka(f/i)dgitnkvnsviek-
-mπtqfeavgklf(π/s)nleklenlnkk (SEQ ID NO: 251) 1961, 65,68, 83,84 hsndqgsgyaadkestqka(f/i)dgitnk (SEQ ID NO: 252) 1961, 65,68, 83,84 hdsnvrnlydkvrmqlrdnak (SEQ ID NO: 253) 1964, 68,76, 84,91 hkcddecmnsvkngtydypklnrneϊkgvk (SEQ ID NO: 254) 1964,65,68,76, 83,84,91 hkcddecmnsvkngtydypklnrneik (SEQ ID NO: 255) 1964,65,68,76, 83,84,91 hkcddecmnsvkngtydypk (SEQ ID NO: 256) 1964,65,68,76, 83,84,91 hkcddecmnsvk (SEQ ID NO: 257) 1964,65,68,76, 83,84,91 kgsnypvakgsynntngeqiliϊwgvh (SEQ ID NO: 258) 1976,78 hsndqgsgyaadkestqkavdgitnkvnsviekmntqfeavgk (SEQ ID NO: 259) 1976, 91 krgssgimktegtlencetkcqtplgainttlpfh (SEQ ID NO: 260) 1976,78, 83,84 hpltigecpkyvksek (SEQ ID NO: 261) 1976 hakdilekthngklck (SEQ ID NO: 262) 1976
1. Influenza H2N2 was responsible for the human pandemic (global distribution) of 1957.
2. Abbreviation for years: eg. "58" =1958.
3. The first year that a given Replikin appears is indicated at the beginning of the series of years in which that Replikin has been found.in this work.
4. Overlapping Replikin sequences are listed separately.
5. Increase in number of new Replikin structures occurs in years of epidemics (underlined): eg. 1957 and 1965 and correlates with increased total Replikin concen tration (number of Replikins per 100 amino acid residues). See Figure 8.
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Table 6
H3N2 Replikin Sequences present in H3N2 hemagglutinins of Influenza viruses in each year for which amino acid sequences were available (1968-2000)
Influenza H3N2 RepHkinsYear Detected in Influenza H3N2 strain Influenza Replikins
(Peak in Figure 8: P3 E3 E4)
hdvyrdealnnrfqikgvelksgyk (SEQ ID NO: 263) 1968.72. 75 96,97,98 htidltdsemnklfertrk (SEQ ID NO: 264) kfliqiek (SEQ ID NO: 265) 96,97,98 ktnekfh(g/q)iek (SEQ BD NO: 266) 86 98 klnr(v/l)iektaekfh (SEQ ID NO: 267) 97,98 hqiekefsevegriqdlekyvedtk (SEQ ID NO: 268) 98 kicnnphk (SEQ ID NO: 269) klnrvikktnekfh (SEQ ID NO: 270) hd(i,v)yrdealnnrfqik(g/q)ve(r/k)s(q/g)yk (SEQ ID NO: 271) 86 hqiekefsevegriqdlekyvedtk (SEQ ID NO: 272) kyvedtkidlwsynaellvalenqh (SEQ ID NO: 273)
Figure imgf000037_0001
kyvkqnslklatgmmvpekqtrglfgaiagfiengwegmidgwygfrh (SEQ ID NO: 274) 1975 kefsevegriqdlekyvedtkidlwsynaellvalenqh (SEQ ID NO: 275) 1975 2000 hqn(s/e)(e/q)g(t/s)g(q/y)aad(l/q)k-
-stq(a/n)a(i/l)d(q/g)I(n/t)(g/n)k(yv)n(r/s)vi(e/c)k (SEQ ID NO: 276) 1975 2000 hcd(g/q)f(q,r)nekwdlf(v,/i)er(s/t)k (SEQ ID NO: 277) 1975.76.77.78.80.81.82.83.84.85.86.88.89.90,91,92.93.94.95.96.97.98
WUSA'lό'lb
htidltdsemnkklfertrk (SEQ ID NO: 278) 1977, ksgstypvlkvtmpnndnfdklyiwgvh (SEQ ID NO: 279) 1977 klnwltksgntypvlnvtmpnndnfdklviwgvh (SEQ ID NO: 280) 1982 htidltdsemnklfektrk (SEQ ID NO: 281) 1986 klnrliektnekfhqtek (SEQ ID NO: 282) 1987 htgkssvmrsdapidfcnsecitpnqsipndkpfqnvnkitygacpk (SEQ ID NO: 283) 1994 htgkssvmrsdapidfcnsecitpnqsipndkpfqnvnk (SEQ ID NO: 284) 1994 hpstdsdqtslyvrasgrvtvstkrsqqtvipk (SEQ ID NO: 285) 1994 kyvedtkidlwsynaellvalenqh (SEQ ID NO: 286) 1997,98 klfertrkqlrenaedmgngcfkiyh (SEQ ID NO: 287) 1998 krrsiksffsrlnwlh (SEQ ID NO: 288) 1998 hpvtigecpky(v/r)kstk (SEQ ID NO: 289) 2000 kgnsypklsklsksyiinkkkevlviwgih (SEQ ID NO: 290) 2000 klsklsks(v/y)iinkkkevlviwgih (SEQ ID NO: 291) 2000 klsks(v/y)iinkkkevlviwgih (SEQ ID NO: 292) 2000
1. Influenza H3N2 was responsible for the human pandemic (global distribution) of 1968.
2. Abbreviation for years: eg. "77" =1977.
3. The first year that a given Replikin appears is indicated at the beginning of the series of years in which that Replikin has been found.
4. Overlapping Replikin sequences are listed separately.
5. Increase in number of new Replikin structures occurs in years of epidemics (underlined) : eg. 1975 and correlates with increased total Replikin concentration
(number of Replikins per 100 amino acid residues). See Figure 8.
[00099] Several properties of Replikin concentration are seen in Figure 7 and Figure 8 to be common to all four influenza virus strains. First, the concentration is cyclic over the years, with a single cycle of rise and fall occurring over a period of two to thirty years. This rise and fall is consistent with the known waxing and waning of individual influenza virus strain predominance by hemagglutinin and neuraminidase classification. Second, peak Replikin concentrations of each influenza virus strain previously shown to be responsible for a pandemic were observed to relate specifically and individually to each of the three years of the pandemics. For example, for the pandemic of 1918, where the influenza virus strain, HlNl, was shown to be responsible, a peak concentration of the Replikins in HlNl independently occurred (Pl); for the pandemic of 1957, where H2N2 emerged and was shown to be responsible, a peak concentration of the Replikins in H2N2 occurred (P2); and for the pandemic of 1968, where H3N2 emerged and was shown to be the cause of the pandemic, a peak concentration of the Replikins in H3N2 occurred (P3). Third, in the years immediately following each of the above three pandemics, the specific Replikin concentration decreased markedly, perhaps reflecting the broadly distributed immunity generated in each case. Thus, this post-pandemic decline is specific for HlNl immediately following the pandemic (Pl) for which it was responsible, and is not a general property of all strains at the time. An increase of Replikin concentration in influenza B repeatedly occurred simultaneously with the decrease in Replikin concentration in HlNl, e.g., EBl in 1951 and EB2 in 1976, both associated with influenza B epidemics having the highest mortality. (Stuart-Harris, et al., Edward Arnold Ltd. (1985). Fourth, a secondary peak concentration, which exceeded the primary peak increase in concentration, occurred 15 years after each of the three pandemics, and this secondary peak was accompanied by an epidemic: 15 years after the 1918 pandemic in an HlNl 'epidemic' year (El); eight years after the 1957 pandemic in an H2N2 'epidemic' year (E2); and occurred seven years after the 1968 pandemic in an H3N2 'epidemic' year (E3). These secondary peak concentrations of specific Replikins may reflect recovery of the strain. Fifth, peaks of each strain's specific Replikin concentration frequently appear to be associated with declines in Replikin concentration of one or both other strains, suggesting competition between strains for host sites. Sixth, there is an apparent overall tendency for the Replikin concentration of each strain to decline over a period of 35 years (H2N2) to 60 years (influenza B). This decline cannot be ascribed to the influence of vaccines because it was evident in the case of influenza B from 1940 to 1964, prior to common use of influenza vaccines. In the case of influenza B, Replikin recovery from the decline is seen to occur after 1965, but Replikin concentration declined again between 1997 and 2000 (Figure 7). This correlates with the low occurrence of influenza B in recent case isolates. HlNl Replikin concentration peaked in 1978-1979 (Figure 7) together with the reappearance and prevalence of the HlNl strain, and then peaked in 1996 coincident with an HlNl epidemic. (Figure 7). HlNl Replikin concentration also declined between 1997 and 2000, and the presence of HlNl strains decreased in isolates obtained during these years. For H2N2 Replikins, recovery from a 35 year decline has not occurred (Figure 8), and this correlates with the absence of H2N2 from recent isolates. For H3N2, the Replikin concentration of many isolates fell to zero during the period from 1996 to 2000, but other H3N2 isolates showed a significant, sharp increase in Replikin concentration. This indicates the emergence of a substrain of H3N2, which is designated herein as H3N2(R).
[000100] Figures 7 and 8 demonstrate that frequently, a one to three year stepwise increase is observed before Replikin concentration reaches a peak. This stepwise increase proceeds the occurrence of an epidemic, which occurs concurrently with the Replikin peak. Thus, the stepwise increase in concentration of a particular strain is a signal that particular strain is the most likely candidate to cause an epidemic or pandemic.
[000101] Currently, Replikin concentration in the H3N2(R) strain of influenza virus is increasing (Figure 8, 1997 to 2000). Three similar previous peak increases in H3N2 Replikin concentration are seen to have occurred in the H3N2-based pandemic of 1968 (Figure 8), when the strain first emerged, and in the H3N2-based epidemics of 1972 and 1975 (Figure 8). Each of these pandemic and epidemics was associated with excess mortality. (Ailing, et al., Am J. Epidemiol, 113(l):30-43 (1981). The rapid ascent in concentration of the H3N2(R) subspecies of the H3N2 Replikins in 1997- 2000, therefore, statistically represents an early warning of an approaching severe epidemic or pandemic. An H3N2 epidemic occurred in Russia in 2000 (Figure 8, E4); and the CDC report of December 2001 states that currently, H3N2 is the most frequently isolated strain of influenza virus worldwide. (Morbidity and Mortality Weekly Reports (MMWR), Center for Disease Control; 50(48): 1084-68 (Dec.7, 2001). [000102] In each case of influenza virus pandemic or epidemic new Replikins emerge. There has been no observation of two of the same Replikins in a given hemagglutinin in a given isolate. To what degree the emergence of a new Replikin represents mutations versus transfer from another animal or avian pool is unknown. In some cases, each year one or more of the original Replikin structures is conserved, while at the same time, new Replikins emerge. For example, in influenza virus B hemagglutinin, five Replikins were constantly conserved between 1919 and 2001, whereas 26 Replikins came and went during the same period (some recurred after several years absence). The disappearance and re-emergence years later of a particular Replikin structure suggests that the Replikins return from another virus host pool rather than through de novo mutation.
[000103] In the case of HlNl Replikins, the two Replikins present in the Pl peak associated with the 1918 pandemic were not present in the recovery El peak of 1933, which contains 12 new Replikins. Constantly conserved Replikins, therefore, are the best choice for vaccines, either alone or in combination. However, even recently appearing Replikins accompanying one year's increase in concentration frequently persist and increase further for an additional one or more years, culminating in a concentration peak and an epidemic, thus providing both an early warning and time to vaccinate with synthetic Replikins (see for example, HlNl in the early 1990's, Figure 7; see also, for example, H5N1 1995-2002, Figure 11, "Replikin Count" (number of Replikins per 100 amino acids) refers to Replikin concentration) and Figure 15). [000104] The data in Figures 7, 8 ,11 and 15 demonstrate a direct relationship between the presence and concentration of a particular Replikin in influenza protein sequences and the occurrence of pandemics and epidemics of influenza. Thus, analysis of the influenza virus hemagglutinin protein sequence for the presence and concentration of Replikins provides a predictor of influenza pandemics and/or epidemics, as well as a target for influenza vaccine formulation. It is worth noting again with reference to this data, previously, no strain-specific chemical structures were known with which to predict the strains that would predominate in coming influenza seasons, nor to devise annual mixtures of whole-virus strains for vaccines.
[000105] Similar to the findings of strain-specific Replikin Count increases in the influenza group one to three years prior to the occurrence of a strain-specific epidemics, the increase in Replikin Count of the coronavirus nucleocapsid protein has also been identified. Replikin Counts of the coronavirus nucleocapsid protein has increased as follows: 3.1 (±1.8) in 1999; 3.9(±1.2) in 2000; 3.9 (±1.3) in 2001; and 5.1 (±3.6) in 2002. This pre-pandemic increase supports the finding that a coronavirus is responsible for the current (2003) SARS pandemic. (See Table 7) [000106] Thus, monitoring Replikin structure and Replikin Count provides a means for developing synthetic strain-specific preventive vaccination and antibody therapies against the 1917-1918 Goose Replikin and its modified and accompanying Replikins as observed in both influenza and coronavirus strains. [000107] Figure 10 depicts the automated Replikin analysis of nucleocapsid coronavirus proteins for which the protein sequence is available on isolates collected from 1962 to 2003. Each individual protein is represented by an accession number and is analyzed for the presence of Replikins. The Replikin Count (number of Replikins per 100 amino acid) is automatically calculated as part of the automated Replikin analysis. For each year, the mean (± Standard deviation (S.D.)) Replikin Count per year is automatically calculated for all Replikin Counts that year. This example of early warning of increasing replication, before an epidemic, of a particular protein (the nucleocapsid protein) in a particular virus strain (the coronavirus) is comparable to the increase seen in strains of influenza virus preceding influenza epidemics and pandemics (Figures 7, 8, 11 and 15). It may be seen that the Replikin Count rose from 1999 to 2002, consistent with the SARS coronavirus pandemic, which emerged at the end of 2002 and has persisted into 2003. Figure 9 provides a graph of the Replikin Counts for several virus strains, including the coronavirus nucleocapsid Replikin, from 1917 to 2002.
Uy4ZD/4/-S /0
Table 7
'Multi-K' %
Replikin Sequence Replikins: Untreated ORGANISM Length Mortality
1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2
1 2 3 4 5 6 7 8 9 O 1 2 3 4 5 6 7 8 9 O 1 2 3 4 5 6 7 8 9 Amino Acid position
A. INFLUENZA, SARSAND OTHER CORONAVIRUSES
1917-18 Goose Replikin (SEQ II k k g t S y P k 1 S k S y t n n k g k e V 1 V 1 W g V h h 29 NO: 743)
1918 Human Influenza (SEQ II k k g t S y P k 1 S k S y t n n k g k e V 1 V 1 W g V h h 29 2.5 NO: 744)
1919-2001 HlNl, H1N2 (SEQ II
1 k e d 1 y p k 1 r k S V V h n k k k e V I V ϊ W g I h h 29 NO: 745)
1 k e n S y P k 1 r k S i i i n k k k e V 1 V i W g h h H3N2 Influenza (SEQ ID NO: 746
2001 H1N2 Influenza (SEQ ID NC k k g t S y P k 1 S k S y t n n k k k e V 1 V 1 W g V h h 29 747)
1996-2001 H5N1 Influenza (SE k k n S a y P t I k r S y n n t n q e d 1 1 V 1 W g i h h >37 ID NO: 748)
2000 Human coronavirus 229 k k S a k t g t P k P S r n q S P a S S q t S a k S 1 a h >37 (SEQ ID NO: 794 )'
2003 Cancine coronavirus (SEQ IJ k k 1 g V d t e k q q q r S k S k e r S n S k t r d t t P >37 NO: 795)2 k n g 1 y P n 1 S k S y a n n k e k e V 1 i I W g V h h 28 2002 H1N2 (SEQ ID NO: 749)
Avian bronchitis coronavirus (SE( k k i n S P q P k f e g S g V P d n e n 1 k t S q q h 27 ID NO: 715)
Porcine epidemic diarrhe k t g n a k 1 q r k k e k k n k r e t t 1 q q h 24 coronavirus (SEQ ID NO: 716)
2003 Human SARS nucleocapsi k h 1 d a y k t f P P t e P k k d k k k k 21 (SEQ ID NO: 712)
1 Human coronavirus 229E 2000, SEQ ID NO: 794: kksaktgtpkpsrnqspassqtsakslarsqssetkeqkh
2 Canine coronavirus 2003, SEQ ID NO: 795: kklgvdtekqqqrsrskskersnsktrdttpknenkh
Figure imgf000044_0001
Figure imgf000045_0001
SARS and H3N2-Fujian influenza virus replikins traced back to a 1918 pandemic replikin [000108] The origin of the SARS virus is as yet unknown. We report evidence that certain SARS virus peptides can be traced back through homologous peptides in several strains of influenza virus isolates from 2002 to a sequence in the strain of the 1918 influenza pandemic responsible for the deaths of over 20 million people. [000109] By quantitative analysis of primary protein sequences of influenza virus and other microorganisms recorded through the last century we have found a new class of peptide structures rich in lysines and histidine, related to the phenomenon of rapid replication itself and to epidemics, rather than to the type of organism (eg. Table 1) and named them Replikins. We have found a new class of peptide structures with the following obligatory algorithm: at least two lysines 6 to 10 residues apart, lysine concentration 6% or greater, one histidine, in 7 to 50 amino acids. Because these peptides relate to the phenomenon of rapid replication itself and to epidemics, we named them Replikins. We have found a quantitative correlation of strain-specific replikin concentration (replikin count = number of replikins per 100 amino acids) in the hemagglutinin protein with influenza epidemics and pandemics (Figure 7). No previous correlation of influenza epidemics with strain-specific viral protein chemistry has been reported. Conservation, condensation and concentration of replikin structure also has been found in influenza (eg. in Table 7a), HIV and malaria. The detection of replikins in SARS coronavirus, in addition to tracing its possible evolution, has permitted the synthesis of small SARS antigens for vaccines. [000110] We have found a quantitative correlation of strain-specific replikin concentration (count) in the influenza hemagglutinin proteins with influenza epidemics and with each of the three pandemics of the last century, in 1918, 1957, and 1968. A similar course was observed for each of these three pandemics: after a strain-specific high replikin count, an immediate decline followed, then a 'rebound' increase with an accompanying epidemic occurred. Also, a 1 to 3 year warning increase in count preceded most epidemics.
[000111] We found that the replikin in the hemagglutinin of an influenza virus isolated from a goose in 1917 (which we named the Goose Replikin) appeared in the next year in the HlNl strain of influenza responsible for the 1918 pandemic, with only two substitutions as follows: kkg(t/s)sypklsksy(t/v)nnkgkevlvlwgvhh. Table 7a shows that the influenza 1917 Goose Replikin (GR) then was essentially conserved for 85 years, despite multiple minor substitutions and apparent translocations to other influenza strains. We have found that the 1917 influenza GR demonstrated apparent mobility between several influenza strains, appearing in HlNl (the pandemic of 1918), in H2N2 (pandemic of 1957-58), in H3N2 (pandemic of 1968, epidemic in China and Russia 2000, Fujian strain epidemic 2003) and in H5N1 (epidemic in China 1997). In 1997 its structure was restored in H1N2 exactly to its 1918 structure KKGSSYPKLSKSYVNNKGKEVLVLWGVHH. [000112] The SARS coronavirus first appeared in the 2002-2003 influenza season. The dual origin in 2002 of SARS replikins, from influenza GR and coronavirus replikins (or from some unknown shared precursor) is suggested by the following events, all of which occurred in 2002: 1) a condensation for the first time in 85 years is seen in the GR-H 1N2 Replikin sequence from 29 to 28 amino acids (Table 7a)(A similar condensation was found in H3N2 Fujian from 29 to 27 amino acids in the current epidemic (Table 7a)); 2) the replikin count of GR-H1N2 showed a marked decline consistent with GR moving out of H1N2; 3) the replikin count of coronavirus nucleocapsid proteins showed a marked increase; and 4) SARS coronavirus appeared in 2002-2003 with replikins containing the following motifs: 'kkg' and <k-k', previously seen in GR 1918 and GR-H1N2 2001 ; 'k-kk', 'kk' and 'kl' seen in influenza GR-
H1N2 2001; 'kk' seen in the avian bronchitis coronavirus replikin; and 'kk-kk-k', 'k- k', 'kk', 'kl' and 'kt' seen in the replikin of porcine epidemic diarrhea coronavirus (Table 7a) (SARS is believed to have made its first appearance in humans as the epidemic pneumonia which erupted in a crowded apartment house where there was a severe back-up of fecal sewage, which was then airborne by ventilating fans).
Table 7a.
Goose Replikin (GR) sequences in different influenza strains from 1917 to 2003; SARS and H3N2-Fujian appearance 2002-2003.
Replikins related to the Goose Replikin: Replikin Virus or other organism
Continuous amino acid sequences __^ Length containing replikin
Shared motif and/or position- ^hade^ (Number (Complete replikins
Amino acid substitutions- clear backgroundof amino except for Fujian strain) 'Condensed' indicates condensation of acids) sequence length in H1N2 and H3N2 -Fujian
SEQ.ID
1917 H1N_ Influenza Goose Replikin (GR)
1918 GR in HlNlHuman Influenza
1958 GR H2N2 Influenza
29 1964,1965,1968 GR in H2N2 Influenza
29 1976,'77,'80,'81,'85 GR in HlNl Influenza
29 1996-2001 GR in H5N 1 Influenza
1996 GR in HlNl Influenza
1997,1998 GR in HlNl Influenza
1999 GR in Hl N2 Influenza
2000 GR in H1N2 Influenza
2001 GR in H1N2 Influenza 2001 GR in H1N2 Influenza
28 2002 GR in H1N2 Influenza (condensed) 21 2002-3 Human SARS nucleocapsid protein 1968-2001 GR in H3N2 Influenza (complete) 1996 H3N2 Fujian Influenza (incomplete)
2003 H3N2 Fujian(condensed,incomplete) 24 Porcine epidemic diarrhea coronavirus 27 Avian bronchitis coronavirus 2000 shrimp white spot syndrome virus 27 Avian bronchitis coronavirus 20 2002-3 Human SARS spike protein 14 2002-3 Human SARS nucleocapsid protein 20 2002-3 Human SARS spike protein 14 2002-3 Human SARS nucleocapsid protein 11 2002-3 Human SARS spike protein 9 2002-3 Human SARS envelope protein 9 2002-3 Human SARS spike protein
7 2002-3 Human SARS nucleocapsid protein 11 Nipah virus, v-protein
1 1 Hendra virus, v-protein
10 Sindbis virus
10 EEL leukemia
10 BRCA-I breast cancer
10 Ovarian cancer
10 Glioma Replikin
9 Smallpox virus
9 HIV TAT protein
Figure imgf000048_0001
8 Smallpox virus [jreejhicjk] 8 B.anthracis, HATPase jjatvljkj 8 Ebola virus polymerase
[000113] The recent increasingly high replikin count peaks, including the presence of the 1917 Goose Replikin (Figure 7), now in H1N2 (Table 7a), approaching the 1917 replikin count, could be a warning of a coming pandemic which may already have begun since the SARS virus and the H3N2-Fujian virus are the current carriers of the short replikin derivatives of the Goose Replikin seen in Table 7 and 7a to be associated with high mortality.
[000114] Since the Goose Replikin has at least an 85 year history involving most or all of the A-strains of influenza and SARS, it and its components are conserved vaccine candidates for pan-strain protection. Condensed short SARS replikins, 7 to 21 amino acids long, enriched in % lysine and histidine compared to the Goose Replikin, occurred in association with the higher mortality rate of SARS (10-55%) when compared to that (2.5%) of the Goose Replikin, 29 amino acids long. Short replikins here mixed with long replikins in SARS may be responsible for high mortality. This is also the case for replikins of other organisms such as the ebola and smallpox viruses and anthrax bacteria (Table 7a). These short SARS replikins showed surprising homology with short replikins of other organisms such as smallpox, anthrax, and ebola which are associated with even higher untreated mortality rates (Table 7a). [000115] Short synthetic vaccines, besides being much more rapidly produced (days rather than months), and far less expensive, should avoid the side effects attendant on the contamination and the immunological interference engendered by multiple epitopes of thousands of undesired proteins in current whole virus vaccines in general. In any case for influenza, current whole virus vaccines are ineffective in more than half of the elderly. But would short replikins be sufficiently immunogenic? The short glioma replikin 'kagvaflhkk' proved to be a successful basis for a synthetic anti-glioblastoma multiforme and anti-bronchogenic carcinoma vaccine. It produced anti-malignin antibody, which is cytotoxic to cancer cells at picograms/cell and relates quantitatively to the survival of cancer patients. In order to prepare for a recurrent SARS attack, which appears likely because of the surge we found in the coronavirus nucleocapsid replikin count in 2002, we synthesized four SARS short replikins, found in nucleocapsid, spike, and envelope proteins. We found that these synthetic short SARS replikins when injected into rabbits also produced abundant specific antibody. For example, the 21 amino acid SARS nucleocapsid replikin antibody binds at dilutions greater than 1 in 204,800. Because of previous unsuccessful attempts by others to achieve with various small peptides a strong immune response without the unwanted side effects obtained with a whole protein or the thousands of proteins or nucleic acids as in smallpox vaccine, the ability of small synthetic replikin antigens to achieve strong immune responses is significant for the efficacy of these SARS vaccines. [000116] We examined the relationship of Replikin structure in influenza and SARS viruses to increased mortality, with results as shown in Table 7. The relation of high mortality to short or condensed Replikin sequences is seen in the high mortality organisms shown in Section B of Table 7, in viruses other than influenza and SARS, and in bacteria, malaria and cancer. In support of the unifying concept of Replikin structure and of the relation of Replikins to rapid replication rather than any cell type or infectious organism, in addition to the prevalence of the basic Replikin structure in a broad range of viral, bacterial, malarial and cancer organisms in which replication is crucial to propagation and virulence, the following homologous sequences have been observed: note the "k"s in positions 1 and 2, note the alignment of "k"s as they would present to DNA, RNA or other receptor or ligand for incorporation or to stimulate rapid replication, note the frequency of "double k"s and "multiple k"s , note the frequency of "g" in position 3 and the occurrence of the triplets "kkg", "hek", "hdk" and "hkk" in the most condensed shortened Replikins associated with the highest mortality organisms, cancer cells and genes as diverse as the smallpox virus, the anthrax virus, Rous sarcoma virus and glioblastome multiforme (glioma), c-src in colon and breast cancer, and c-yes in melanoma and colon cancer. Note also the almost identical Replikin structure for two recently emerging high mortality viruses in Australia and Southeast Asia, Nipah and Hendrah viruses. These two viruses are reported to have similar or identical antibodies formed against them but no structural basis has been known for this up till now, with our finding of their two almost identical Replikins, for this similar antibody. [000117] Table 7 also shows the relationship of five SARS Replikins of 2003 which we have found both to the influenza Goose Replikin of 1917 and to two coronaviruses, the avian bronchitis coronavirus and the porcine epidemic diarrhea virus. The first 2003 human SARS Replikin in Table 7 shows certain sequence homologies to the influenza virus goose 1917 and human 1918 Replikins through an intermediary structure of influenza H 1N2 in 2002 (e.g., see Replikin "k" in positions 1, 18 and 19). The 1917 Goose Replikin sequence is seen in Table 7 to have been largely conserved despite many substitutions in amino acids which are not crucial to the definition of Replikins through 1999 (substitutions are show in italics). The original 29 amino acid 1917 Replikin sequence was then found to have been almost exactly restored to its structure of 1917-1918 in the 2001 H1N2 Replikin. However, the 2002 H1N2 influenza Replikin has been shortened from 29 to 28 amino acids and the "shift to the left" of amino acids kevl(i/v)wg (v/i)hh is clearly evident. [000118] In 2003, one Replikin was further shortened (or compacted) to the 21 amino acid Replikin of the first listed 2003 human SARS virus. The % k of the 2003 SARS Replikin is now 38.1 % (8/21) in comparison to 20.7 % of the Goose Replikin and the 1918 Human Pandemic Replikin. Compared to the influenza 29 amino acid Replikin, three SARS Replikins were found to be further shortened (or compacted) to 19, 11 and 9 amino acid long sequences, respectively. In the SARS 9 amino acid sequences shown, the % k is 44.4% (4/9). With the shortening of the SARS Replikin, the SARS mortality rate in humans rose to 10% in the young and 55.5 % in the elderly compared to the 2.5 % mortality in the 1918 influenza pandemic. [000119] The amino acid sequences are shown in Table 7 to emphasize the degree of homology and conservation for 85 years (1917-2002) of the influenza Replikin, for which evidence has first been observed in the 1917 Goose Replikin. No such conservation has ever been observed before. Table 7 also illustrates that the Replikins in the 2003 human SARS virus, in addition to having homologies to the influenza Replikins which first appeared as the 1917 Goose Replikin and the 1918 Human Pandemic influenza Replikin, show certain sequence homologies to both the coronavirus avian bronchitis virus Replikin (e.g. "k" in positions 1 and 2, end in "h") and to the coronavirus acute diarrhea virus Replikin (e.g. "k" in positions 1 and 11, "h" at the end of the Replikin). This evidence of relation to both influenza and coronavirus Replikins is of interest because SARS arose in Hong Kong as did several recent influenza epidemics and earlier pandemics, and the SARS virus has been classified as a new coronavirus partly because of its structure, including nucleocapsid, spike, and envelope proteins. Certain epidemiological evidence also is relevant in that SARS made its first appearance in humans as the epidemic pneumonia, which erupted, in a crowded Hong Kong apartment house where there was a severe back-up of fecal sewage, which was airborne by ventilating fans.
[000120] Composition of Replikins in Strains of Influenza Virus B : Of a total of 26 Replikins identified in this strain (Table 3), the following ten Replikins are present in every influenza B isolate examined from 1940-2001. Overlapping Replikin sequences are listed separately. Lysines and histidines are in bold type to demonstrate homology consistent with the "3-point recognition."
KSHFANLK (SEQ ID NO: 104)
KSHFANLKGTK (SEQ ID NO: 105) KSHFANLKGTKTRGKLCPK (SEQ ID NO: 106)
HEKYGGLNK (SEQ ID NO: 107)
HEKYGGLNKSK (SEQ ID NO: 108)
HEKYGGLNKSKPYYTGEHAK (SEQ ID NO: 10)
HAKAIGNCPIWVK (SEQ ID NO: 110) HAKAIGNCPIWVVKKTPLKLANGTK (SEQ ID NO : 111 )
HAKAIGNCPIWVKTPLKLANGTKYRPPAK (SEQ ID NO: 112)
HAKAIGNCPIWVKTPLKLANGTKYRPPAKLLK (SEQ ID NO: 113) [000121] Tables 3 and 4 indicate that there appears to be much greater stability of the Replikin structures in influenza B hemagglutinins compared with HlNl Replikins. Influenza B has not been responsible for any pandemic, and it appears not to have an animal or avian reservoirs. (Stuart-Harris et al., Edward Arnold Ltd., London (1985)). Replikins in Influenza over Time
[000122] Only one Replikin "hp(v/i)tigecpkyv-(r/k)(s/t)(t/a)k" is present in every HlNl isolate for which sequences are available from 1918, when the strain first appeared and caused the pandemic of that year, through 2000. (Table 4). ("(v/i)" indicates that the amino acid v or i is present in the same position in different years.) Although HlNl contains only one persistent Replikin, HlNl appears to be more prolific than influenza B. There are 95 different Replikin structures in 82 years on HlNl versus only 31 different Replikins in 62 years of influenza B isolates (Table 4). An increase in the number of new Replikin structures occurs in years of epidemics (Tables 3, 4, 5 and 6) and correlates with increased total Replikin concentration (Figures 7, 8, 11 and 15). [000123] Influenza H2N2 Replikins: Influenza H2N2 was responsible for the human pandemic of 1957. Three of the 20 Replikins identified in that strain for 1957 were conserved in each of the H2N2 isolates available for examination on PubMed until 1995 (Table 5). ha(k/q/m)(d/n)ilekthngk (SEQ ID NO : 232) ha(k/q/m)(d/n)ilekthngklc(k/r) (SEQ ID NO: 233) kgsnyp(v/i)ak(g/r)synntsgeqmliiwq(v/i)h (SEQ ID NO: 238) [000124] However, in contrast to HlNl, only 13 additional Replikins have been found in H2N2 beginning in 1961. This paucity of appearance of new Replikins correlates with the decline in the concentration of the H2N2 Replikins and the appearance of H2N2 in isolates over the years. (Figure 8).
[000125] Influenza H3N2 was responsible for the human pandemic of 1968. Five Replikins which appeared in 1968 disappeared after 1977, but reappeared in the 1990s (Table 6). The only Replikin structure which persisted for 22 years was hcd(g/q)f(q/r)nekwdlf(v/i)er(s/t)k, which appeared first in 1977 and persisted through 1998. The emergence of twelve new H3N2 Replikins in the mid 1990s (Table 6) correlates with the increase in Replikin concentration at the same time (Figure 8), and with the prevalence of the H3N2 strain in recent isolates together with the concurrent disappearance of all Replikins from some of these isolates (Figure 8), this suggests the emergence of the new substrain H3N2(R). The current epidemic in November -
December 2003 of a new strain of H3N2 (Fujian) confirms this prediction made first in the Provisional Application US 60/303,396, filed July 9, 2001. [000126] Figures 7, 8, 11 and 15 show that influenza epidemics and pandemics correlate with the increased concentration of Replikins in influenza virus, which is due to the reappearance of at least one Replikin from one to 59 years after its disappearance. Also, in the A strain only, there is an emergence of new strain-specific Replikin compositions (Tables 4-6, see also increase in number of new Replikins, pre- epidemic for H5N1 in Figures 11 and 15). Increase in Replikin concentration by repetition of individual Replikins within a single protein appears not to occur in influenza virus, but is seen in other organisms.
[000127] It has been believed that changes in the activity of different influenza strains are related to sequence changes in influenza hemagglutinins, which in turn are the products of substitutions effected by one of two poorly understood processes: i) antigenic drift, thought to be due to the accumulation of a series of point mutations in the hemagglutinin molecule, or ii) antigenic shift, in which the changes are so great that genetic reassortment is postulated to occur between the viruses of human and non- human hosts. First, the present data suggests that the change in activity of different influenza strains, rather than being related to non-specific sequence changes, are based upon, or relate to the increased concentration of strain-specific Replikins and strain- specific increases in the replication associated with epidemics. In addition, the data were examined for a possible insight into which sequence changes are due to "drift" or "shift", and which are due to conservation, storage in reservoirs, and reappearance. The data show that the epidemic-related increase in Replikin concentration is not due to the duplication of existing Replikins per hemagglutinin, but is due to the reappearance of at least one Replikin composition from 1 to up to 59 years after its disappearance, plus in the A strains only, the emergence of new strain-specific Replikin compositions (Tables 3-6). Thus the increase in Replikin concentration in the influenza B epidemics of 1951 and 1977 are not associated with the emergence of new Replikin compositions in the year of the epidemic but only with the reappearance of Replikin compositions which had appeared in previous years then disappeared (Table 3). In contrast, for the A strains, in addition to the reappearance of previously disappeared virus Replikins, new compositions appear (e.g. in HlNl in the year of the epidemic of 1996, in addition to the reappearance of 6 earlier Replikins, 10 new compositions emerged). Since the A strains only, not influenza B, have access to non-human animal and avian reservoirs, totally new compositions probably derive from non-human host reservoirs rather than from mutations of existing human Replikins which appear to bear no resemblance to the new compositions other than the basic requirements of "3-point recognition" (Tables 2-5). The more prolific nature of HlNl compared with B, and the fact that pandemics have been produced by the three A strains only, but not by the B strain, both may also be a function of the ability of the human A strains to receive new Replikin compositions from non-human viral reservoirs. [000128] Some Replikins have appeared in only one year, disappeared, and not reappeared to date (Tables 3-6). Other Replikins disappear from one to up to 81 years, when the identical Replikin sequence reappears. Key Replikin 'k' and 'h' amino acids, and the spaces between them, are conserved during the constant presence of particular Replikins over many years, as shown in Tables 2 and 3-6 for the following strain- specific Replikins: ten of influenza B, the single Replikin of HlNl, and the single Replikin of H3N2 as well as for the reappearance of identical Replikins after an absence. Despite the marked replacement or substitution activity of other amino acids both inside the Replikin structure and outside it in the rest of the hemagglutinin sequences, influenza Replikin histidine (h) appears never to be, and lysine (k) is rarely replaced. Examples of this conservation are seen in the HlNl Replikin "hp(v/i)tigecpkyv(r/k)(s/t)(t/a)k," (SEQ ID NO: 135) constant between 1918 and 2000, in the H3N2 Replikin "hcd(g/q)f(q,r)nekwdlf(v/i)er(s/t)k" (SEQ ID NO: 277) constant between 1975 and 1998 and in the H3N2 Replikin "hqn(s/e)(e/q)g(t/s)g(q/y)aad(l/q)kstq(a/n)a(i/l)d(q/g)I(n/t)(g/n)k,(l/v)n(r/s) vi(e/c)k" (SEQ ID NO: 276) which first appeared in 1975, disappeared for 25 years, and then reappeared in 2000. While many amino acids were substituted, the basic Replikin structure of 2 Lysines, 6 to 10 residues apart, one histidine, a minimum of 6% lysine in not more than approximately 50 amino acids, was conserved. [000129] Totally random substitution would not permit the persistence of these
HlNl and H3N2 Replikins, nor from 1902 to 2001 in influenza B the persistence of 10 Replikin structures, nor the reappearance in 1993 of a 1919 18-mer Replikin after an absence of 74 years. Rather than a random type of substitution, the constancy suggests an orderly controlled process, or in the least, protection of the key Replikin residues so that they are fixed or bound in some way: lysines, perhaps bound to nucleic acids, and histidines, perhaps bound to respiratory redox enzymes. The mechanisms, which control this conservation, are at present unknown. H5N1 Influenza Conservation of Replikin Scaffold [000130] There is concern that the current outbreak of high mortality H5N1 "bird flu" in several countries may represent the first phase of an overdue influenza pandemic. A recent report suggests that in the first probable person-to-person transmission of H5N1, "sequencing of the viral genes identified no change in the receptor-binding site of hemagglutinin or other key features of the virus. The sequences of all eight viral gene segments clustered closely with other H5N1 sequences from recent avian isolates in Thailand." Phylogenetic analysis suggested that from the absence of evidence of "reassortment with human influenza viruses" that H5N1 is not a new variant. However, we now report three recent changes in a specific H5N1 protein sequence at sites which had not been changed in the last two H5N1 epidemics and in fact had been conserved since 1959.
[000131] Previously, there has been no protein chemistry which correlated with virus epidemics and dormancy. We found that each of the three influenza pandemics of the last century, HlNl , H2N2 and H3N2, retrospectively was predicted by and correlated with an increase in the concentration of a specific class of peptides in the virus, rich in lysine and histidine, associated with rapid replication, called replikins. We have now again found the replikins to be predictive in each of the three H5N1 epidemics, in 1997, 2001, and 2003-2004 (Figure 15). Each year that they appear in isolates, the replikins can now be counted per 100 amino acids as in Figure 15, and their sequences analyzed and compared as in Table 9. Analysis of replikins may be accomplished manually or in a preferred aspect of the present invention automatically by software designed by the inventors for the purpose of counting replikin concentration in available sequence information. [000132] A graph illustrating a rapid increase in the concentration of Replikin patterns in the hemagglutinin protein of the H5N1 strain of influenza prior to the outbreak of three "Bird Flu" epidemics may be seen in Figure 15. A review of Figure 15 illustrates that an increasing replikin concentration ('Replikin Count') in the hemagglutinin protein of H5N1 preceded three 'Bird Flu' Epidemics. For example, an increase in the Replikin Count (Means+/-SD) in 1995 to 1997 preceded the Hong Kong H5N1 epidemic of 1997 (El). An increase in the Replikin Count from 1999 to 2001 preceded the epidemic of 2001 (E2). And an increase in Replikin Count from 2002 to 2004 preceded the epidemic in 2004 (E3). The decline in 1999 occurred with the massive culling of poultry in response to the El epidemic in Hong Kong. [000133] In addition to the total number of replikins in the virus protein, the structure of each replikin through time is informative. Table 8 shows a replikin first observed in a goose infected with influenza in 1917 (Goose Replikin). Constant length, constant lysines at the amino terminal and histidine residues at the carboxy terminal were conserved in different strains in a fixed scaffold for decades. Homologies of the Goose Replikin appeared from 1917 to 2006 in strains including each responsible for the three pandemics of 1918, 1957, and 19681, HlNl, H2N2 and H3N2, and with further substitutions between H1N2, H7N7, H5N2 and H5N1. Even certain substitutions which have occurred in the Goose Replikin tend to be selective and retained for years, rather than random. Thus despite the common assumption that amino acid substitutions should occur at random, it would appear that not all substitutions in influenza are, in fact, random. This replikin conservation over decades allows the production of synthetic influenza vaccines which rapidly and inexpensively can be prepared in advance and can be effective for more than one year.
[000134] Therefore a target for synthetic influenza vaccines is the conserved Replikin Scaffold in influenza virus. A Replikin Scaffold comprises a series of conserved peptides comprising a sequence of about 16 to about 30 amino acids and further comprising ( 1 ) a terminal lysine ;
(2) a terminal histidine and another histidine in the residue portion immediately adjacent to the terminal histidine;
(3) at least one lysine within about 6 to about 10 amino acid residues from at least one other lysine; and (4) at least about 6% lysines within the 16 to about 30 amino acid peptide.
A Replikin Scaffold may further comprise a an additional lysine immediately adjacent to the terminal lysine. "Replikin Scaffold" peptides may comprise an additional lysine immediately adjacent to the terminal lysine. "Replikin Scaffold" peptide also refers to an individual member or a plurality of members of a series of a "Replikin Scaffold." [000135] A non-limiting and preferred target for synthetic influenza vaccines may be a Replikin Scaffold in influenza virus further comprising a sequence of about 29 amino acids and a lysine immediately adjacent to the terminal lysine. [000136] A non-preferred target for synthetic influenza may be an Exoskeleton Scaffold in a first strain of influenza virus comprising a first peptide of about 29 amino acids and
(1) a terminal lysine and a lysine immediately adjacent to the terminal lysine;
(2) a terminal histidine and a histidine immediately adjacent to the terminal histidine;
(3) no lysine within 6 to 10 amino acid residues from any other lysine wherein an earlier-arising specimen of the first strain or another strain of virus comprises a Replikin Scaffold of about 29 amino acids. [000137] In the 1997 H5N1 Hong Kong epidemic, the human mortality rate was approximately 27%. In 2004, of the fifty-two people reported to have been infected by H5N1 in Asia approximately 70% died. Most recently, nine of the eleven cases in Vietnam from December 28, 2004 to January 27, 2005 died. Although the virulence of the virus appears to have increased, any changes thought to be required for further spread human to human, had been thought not yet to have occuredl . However, we now have observed recent substitutions in three H5N1 replikin amino acid residues at position numbers 18, 24 and 28 of the Goose Replikin scaffold from isolates in Vietnam, Thailand and China in 2004 (see Table 1). Substitution at site number 24 has not occurred since the appearance of H5N1 in 1959 but was present in the last two influenza pandemics caused by other strains, H2N2 in 1957 and H3N2 in 1968, together responsible for over two million human deaths, and in a recent virulent epidemic caused by H7N7 (see Table 8). While these are only hints of possible danger, these data on substitution, combined with the rising Replikin Count shown in Figure 15, and the past correlation of such replikin data with pandemics, does not give the same reassurance as that obtained from phylogenetic analysis that the virus is unlikely to spread human to human.
[000138] With respect to the H5N1 influenza, FIG. 15 illustrates a rapid increase in the concentration of Replikins per 100 amino acids just prior to epidemics in 1997 (indicated as El), 2001 (indicated as E2) and 2004 (indicated as E3).
TABLE 8; Replikin Scaffold showing ordered substitution in the 89 year conservation of influenza virus replikin peptides related to rapid replication, from a 1917 goose influenza replikin and the 1918 human pandemic replikin to 2006
H5N1 "Bird Flu" homologues.
[< 29 Amino Acids ->] Year Strain Influenza Goose Replikin Human Influenza Pandemic
Figure imgf000058_0001
kkgSsypklpls^ tgkevlvlwgvhh 1933 HONl kkgtsypklsksytnnkgkevlvlwgvhh 1976 HlNl kkgtsypklsksytnnkgkevlvlwgvhh 1977 HlNl kkg sypklsksytnnkgkevlvjiwgvhh 1979 HlNl kkg sypklsksytnnkgkevlvαwgvhh 1980 HlNl kkgtsypklsksytnnkgkevlvlwgvhh 1980 HlNl kkgjjsypklsksytnnkgkevlvαwgvhh 1981 HlNl kkgt sypkl sksy tnnkgkevlvl wgvhh 1981 HlNl kkgtsypklsksytnnkgkevlvlwgvhh 1985 HlNl kkg Isypklsksytnnkgkevlvdwgvhh 1991 HlNl kkg isypklsksytnnkgkevlviwgvhh 1992 HlNl kkg ϊsypklsksytnnkgkevlvi'wgvhh 1996 HlNl
Pandemic
goose)
Figure imgf000059_0001
* Residues identical to Goose Replikin amino acids unshaded; amino acid substitutions shaded lightly and darkly to show scaffold pattern across years and strains.
[000139] Table 8, above, provides further support for the role of replikins in epidemics and pandemics in humans and birds. In Table 8, the history of the Goose Replikin and its homologues are tracked from 1917 to the present outbreak of avian H5N1 virus. Table 8 demonstrates conservation of the "scaffold" homology of the Goose Replikin in virulent strains of influenza.
[000140] Table 8 illustrates the history, by year or smaller time period, of the existence in the protein structure of the Goose Replikin and its homologues in other influenza Replikins. Table 8 further illustrates the history of amino acid substitutions in those homologues and the conservation of certain amino acids of the Replikin structure which are essential to the definition of a Replikin and the function of rapid replication supplied by Replikins. [000141] A review of Table 8 illustrates that if random substitution of amino acids were to occur in virulent strains of influenza from 1917 through the present, certain framework amino acids of the Goose Replikin would not be conserved from year to year in strains in which epidemics occurred. However, contrary to what would result from random substitution, virulent strains of influenza from year to year consistently contain conserved amino acids at those positions that define a Replikin. That is, if a substitution were to occur in one of the amino acids that define a Replikin, e.g. lysine or a histidine, the definition of the Replikin would be lost. Nevertheless, the Replikin sequence is conserved over more than 85 years. Thus, since there is conservation of certain amino acids over decades, substitution cannot be said to be completely at random. The fact that substitutions do occur in amino acids that are not essential to the definition of a Replikin (i.e., amino acids other than lysines or histidines) demonstrates the importance of the Replikin in the pathogenicity of the strain. [000142] It may be further noted from Table 8 that when substitutions do occur, they are seen to occur at certain apparently preferred positions of the Replikin Scaffold. Table 8 illustrates recurring substitutions at positions 1, 3-24 and 26-27. Further, while substitutions occur throughout these positions, a lysine continues to exist at a position 6 to 10 amino acids from the second lysine (which has not been substituted in these virulent strains). [000143] Even when there is a substitution of a lysine position within the 29 amino acid stretch, as is seen in 1957, when K at position 11 shifts to position 10, that new position has been maintained until 2005, as have YP, AY, N (position 15), and LVLWG to conserve the homologous structure of the Replikin Scaffold with few exceptions. [000144] Table 8 demonstrates the integrity of the Replikin Scaffold in virulent strains of influenza. As discussed above, degeneration of the Replikin Scaffold into an Exoskeleton Scaffold is seen to decrease pathogenicity. The integrity and conservation of the Replikin Scaffold, therefore, is seen by the fact that there is generally a fixed 29 amino acid sequence that begins with two lysines and ends with two histidines. [000145] It is important to note that an extra K has appeared in the Replikin Scaffold of a 2006 strain of H5N1 in China (Anhui). This presence of an extra K signals an increase in the Replikin count within the Replikin Scaffold. The 2006 China (Anhui) strain has a Replikin count of 6.6 (as discussed below). A Replikin count of 6.6 is the highest ever observed for an H5N1 strain and is comparable in the entire A strain of influenza only to the Replikin count of the influenza strain that caused the 1918 Pandemic. If this initial 2006 report is repeated and maintained, it may indicate that the Counts of 4.5 and 4.0 in 2004 and 2005 respectively will be substantially increased, and foretell a continuing or increased epidemic of H5N1 'Bird Flu'. [000146] An aspect of the present invention is a combination of replikin structure and function to track the pathogenicity or rate of replication of a virus, epidemic or pandemic or to predict the occurrence of epidemics or pandemics. An example of this combination is the ability of the Replikin algorithm of the invention to be used to count increases in Replikin counts in influenza strains such as the strain of 1918 and the current H5N 1 strain of H5N 1. The Replikin Count of the 1918 influenza pandemic and the current outbreak of "Bird Flu" demonstrate the predictive capacity of this exemplary aspect in accordance with and made possible by the invention.
Relation of Some Shrimp White Spot Virus Replikins to Influenza Fixed Scaffold Replikin Structures [000147] The inventors have also established a relationship between virulent influenza virus and white spot virus in the Replikin Scaffold portions of the viruses. No relationship between these two viruses has been suggested previously. Although there is extensive substitution, the applicants' finding of several short Replikins of the Shrimp White Spot Syndrome Virus demonstrate significant homologies to the influenza virus Replikin sequences, especially with regard to length and key lysine (k) and histidine (h) residues (Fixed Scaffold or Replikin Scaffold), suggesting that similar mechanisms of Replikin production are used in both virus groups. Table 10 - Shrimp White Spot Scaffolding
1917 HlN_Influenza goose peptide
Figure imgf000062_0001
Figure imgf000062_0002
[000148] In addition, since many species, including but not limited to swine and birds, are known to provide animal "reservoirs" for human influenza infection, marine forms such as the shrimp virus can now be examined, with early warning diagnostic benefits possible for outbreaks such as swine flu and bird flu. While similarities of some influenza viruses were noted between species, and the transfer of these viruses interspecies was known, there was no previous quantitative method to gauge virus activity. It has not been possible previously to examine potential reservoirs for increased activity which might move into a different species; thus providing an advanced warning. The activity of the Replikins in each species can now be monitored constantly for evidence of increased viral replication rate and thus emergence of epidemics in that species which may then transfer to other species. [000149] This data further supports the Replikins as a new class of peptides, with a history of its own, and a shared function of rapid replication and disease of its hosts. With the high mortality for its shrimp host, white spot syndrome virus can now have its Replikins examined as earlier forms of the virus Replikins, or as parallel morphological branches, which in either case may well act as reservoirs for bird and animal Replikins such as those in influenza viruses. The diagnostic and preventive uses of these Replkin findings in shrimp follow as they do in influenza and for other organisms containing Replikins. Conservation of Replikin Structures
[000150] Whether Replikin structures are conserved or are subject to extensive natural mutation also was examined by scanning the protein sequences of various isolates of foot and mouth disease virus (FMDV), where mutations in proteins of these viruses have been well documented worldwide for decades. Protein sequences of
FMDV isolates were visually examined for the presence of both the entire Replikin and each of the component Replikin amino acid residues observed in a particular Replikin. [000151] Rather than being subject to extensive substitution over time as occurs in neighboring amino acids, the amino acids which comprise the Replikin structure are substituted little or not at all, that is the Replikin structure is conserved.
[000152] For example, in the protein VPl of FMDV type O, the Replikin (SEQ ID NO: 3) "hkqkivapvk" was found to be conserved in 78% of the 236 isolates reported in PubMed, and each amino acid was found to be conserved in individual isolates as follows: his, 95.6%; lys, 91.8%; gin 92.3%; lys, 84.1%; ile, 90.7%; val, 91.8%; ala, 97.3%; pro, 96.2%; ala, 75.4%; and lys, 88.4%. The high rate of conservation suggests structural and functional stability of the Replikin structure and provides constant targets for treatment.
[000153] Similarly, sequence conservation was found in different isolates of HIV for its Replikins, such as (SEQ ID NO: 5) "kcfncgkegh" or (SEQ ID NO: 6) "kvylawvpahk" in HIV Type 1 and (SEQ ID NO: 7) "kcwncgkegh" in HIV Type 2 (Table 2). Further examples of sequence conservation were found in the HIV tat proteins, such as (SEQ ID NO: 613) "hclvckqkkglgisygrkk," wherein the key lysine and histidine amino acids are conserved. (See Table 9). [000154] Similarly, sequence conservation was observed in plants, for example in wheat, such as in wheat ubiquitin activating enzyme E (SEQ ID NOs. 614-616). The Replikins in wheat even provided a reliable target for stimulation of plant growth as described within. Other examples of conservation are seen in the constant presence of malignin in successive generations, over ten years of tissue culture of glioma cells, and by the constancy of affinity of the glioma Replikin for antimalignin antibody isolated by immunoadsorption from 8,090 human sera from the U.S., U.K., Europe and Asia (e.g., Figure 5 and U.S. Patent 6,242,578 Bl).
[000155] Similarly, conservation was observed in trans-activator (Tat) proteins in isolates of HIV. Tat (trans-activator) proteins are early RNA binding proteins regulating lentiviral transcription. These proteins are necessary components in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (HIV). Tat is a transcriptional regulator protein that acts by binding to the trans-activating response sequence (TAR) RNA element and activates transcription Initiation and/or elongation from the LTR promoter. HIV cannot replicate without tat, but the chemical basis of this has been unknown. In the HIV tat protein sequence from 89 to 102 residues, we have found a Replikin that is associated with rapid replication in other organisms. The amino acid sequence of this Replikin is ΗCLVCKQKKGLGISYGRKK." In fact, we found that this Replikin is present in every HIV tat protein. Some tat amino acids are substituted frequently by alternate amino acids (in small size fonts lined up below the most frequent amino acid (Table 9), the percentage of conservation for the predominant Replikin ΗCLVCFQKKGLGIS YGRKK"). These substitutions have appeared for most of the individual amino acids. However, the key lysine and histidine amino acids within the Replikin sequence, which define the Replikin structure, are conserved 100% in the sequence; while substitutions are common elsewhere in other amino acids, both within and outside the Replikin, none occurs on these key histidine amino acids. [000156] As shown in Table 9 it is not the case that lysines are not substituted in the tat protein amino acid sequence. From the left side of the table, the very first lysine in the immediate neighboring sequence, but outside the Replikin sequence, and the second lysine (k ) in the sequence inside the Replikin, but "extra" in that it is not essential for the Replikin formation, are both substituted frequently. However, the 3rd, 4th and 5th lysines, and the one histidine, in parentheses, which together set up the Replikin structure, are never substituted. Thus, these key amino acid sequences are 100% conserved. As observed in the case of the influenza virus Replikins, random substitution would not permit this selective substitution and selective non-substitution to occur due to chance.
Table 9
% Replikin CONSERVATION of each constituent amino acid in the first 117 different isolates of HIV tat protein as reported in PubMed:
38 (100) 57 86 (10O) (IOO) 66 76 (100) 99 57 49 (100) 94 (100) 97 98 85 97 99 (100)(l 00)(l 00)%
Neighboring
Amino acids tat Replikin k (c) s y [(H) (C) l v (c) f q k (k) g (1) g i s y g (r) (k) (k)] below are the amino acid substitutions observed for each amino acid above: h c f q ϊ 1 h t a a 1 y h q r w p 1 1 i h q V y S S 1 ni r S i S m S
S r n
V a f
P q
[000157] The conservation of the Replikin structure suggests that the Replikin structure has a specific survival function for the HIV virus which must be preserved and conserved, and cannot be sacrificed to the virus 'defense' maneuver of amino acid substitution created to avoid antibody and other 'attack.' These 'defense' functions, although also essential, cannot 'compete' with the virus survival function of HIV replication.
[000158] Further conservation was observed in different isolates of HIV for its Replikins such as "kcfncgkegh" (SEQ ID NO: 5) or "kvylawvpahk" (SEQ ID NO: 6) in HIV Type 1 and "kcwncgkegh" (SEQ ID NO: 7) in HIV Type 2. The high rate of conservation observed in FMVD and HIV Replikins suggests that conservation also observed in the Replikins of influenza Replikins is a general property of viral Replikins. This conservation makes them a constant and reliable targeted for either destruction, for example by using specific Replikins such as for influenza, FMVD or HIV vaccines as illustrated for the glioma Replikin, or stimulation. [000159] Similarly, as provided in examples found in viruses including influenza viruses, FMDV, and HIV, where high rates of conservation in Replikins suggest that conservation is a general property of viral Replikins and thus making Replikins a constant and reliable target for destruction or stimulation, conservation of Replikin structures occurs in plants. For example, in wheat plants, Replikins are conserved and provide a reliable target for stimulation. Examples of conserved Replikins in wheat plants ubiquitin activating enzyme E include: E3 HKDRLTKKVVDIAREVAKVDVPEYRRH (SEQ ID NO : 614) E2 HKERLDRKVVDVAREVAKVEVPSYRRH (SEQ ID NO: 615)
E 1 HKERLDRKVVDVAREVAKMEVPSYRRH (SEQ ID NO : 616) [000160] Similarly to conservation found in the HIV tat protein, the Replikin in the wheat ubiquitin activating enzyme E is conserved. As with the HIV tat protein, substitutions of amino acids (designated with an '*') adjacent to the Replikin variant forms in wheat ubiquitin activating enzyme E are common. The key k and h amino acids that form the Replikin structure, however, do not vary whereas the 'unessential' k that is only 5 amino acids (from the first k on the left) is substituted. Anti-Replikin Antibodies [000161] An anti-Replikin antibody is an antibody against a Replikin. Data on anti-Replikin antibodies also support Replikin class unity. An anti-Replikin antibody response has been quantified by immunoadsorption of serum antimalignin antibody to immobilized malignin (see Methods in U.S. Patent No. 5,866,690). The abundant production of antimalignin antibody by administration to rabbits of the synthetic version of the 16-mer peptide whose sequence was derived from malignin, absent carbohydrate or other groups, has established rigorously that this peptide alone is an epitope, that is, provides a sufficient basis for this immune response (Figure 3). The 16-mer peptide produced both IgM and IgG forms of the antibody. Antimalignin antibody was found to be increased in concentration in serum in 37% of 79 cases in the U.S. and Asia of hepatitis B and C, early, in the first five years of infection, long before the usual observance of liver cancer, which develops about fifteen to twenty-five years after infection. Relevant to both infectious hepatitis and HIV infections, transformed cells may be one form of safe haven for the virus: prolonging cell life and avoiding virus eviction, so that the virus remains inaccessible to anti-viral treatment. [000162] Because administration of Replikins stimulates the immune system to produce antibodies having a cytotoxic effect, peptide vaccines based on the particular influenza virus Replikin or group of Replikins observed to be most concentrated over a given time period provide protection against the particular strain of influenza most likely to cause an outbreak in a given influenza season, e.g., an emerging strain or re-emerging strain For example, analysis of the influenza virus hemagglutinin amino acid sequence on a yearly or bi-yearly basis, provides data which are useful in formulating a specifically targeted influenza vaccine for that year. It is understood that such analysis may be conducted on a region-by-region basis or at any desired time period, so that strains emerging in different areas throughout the world can be detected and specifically targeted vaccines for each region can be formulated.
Influenza Vaccines, Treatments and Therapeutics [000163] Currently, vaccine formulations for influenza are changed twice yearly at international WHO and CDC meetings. Vaccine formulations are based on serological evidence of the most current preponderance of influenza virus strain in a given region of the world. However, prior to the present invention there has been no correlation of influenza virus strain specific amino acid sequence changes with occurrence of influenza epidemics or pandemics.
[000164] The observations of specific Replikins and their concentration in influenza virus proteins provides the first specific quantitative early chemical correlates of influenza pandemics and epidemics and provides for production and timely administration of influenza vaccines tailored specifically to treat the prevalent emerging or re-emerging strain of influenza virus in a particular region of the world. By analyzing the protein sequences of isolates of strains of influenza virus, such as the hemagglutinin protein sequence, for the presence, concentration and/or conservation of Replikins, influenza virus pandemics and epidemics can be predicted. Furthermore, the severity of such outbreaks of influenza can be significantly lessened by administering an influenza peptide vaccine based on the Replikin sequences found to be most abundant or shown to be on the rise in virus isolates over a given time period, such as about one to about three years. [000165] An influenza peptide vaccine of the invention may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in influenza virus strains. Preferably, the peptide vaccine is based on Replikin sequence(s) shown to be increasing in concentration over a given time period and conserved for at least that period of time. However, a vaccine may include a conserved Replikin peptide(s) in combination with a new Replikin(s) peptide or may be based on new Replikin peptide sequences. The Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences, although vaccines based on peptides containing only Replikin sequences are preferred. Preferably, vaccine compositions of the invention also contain a pharmaceutically acceptable carrier and/or adjuvant.
[000166] The influenza vaccines of the present invention can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
[000167] The influenza vaccine of the present invention may be administered to any animal capable of producing antibodies in an immune response. For example, the influenza vaccine of the present invention may be administered to a rabbit, a chicken, a pig or a human. Because of the universal nature of replikin sequences, an influenza vaccine of the invention may be directed at a range of strains of influenza or a specific strain of influenza. [000168] In a non-limiting aspect in accordance with the present invention, an influenza vaccine may be directed to an immune response against animal or human strain of influenza including influenza B, (A)HlNl, (A)H2N2 and (A)H3N2, or any human variant of the virus that may arise hereafter, as well as strains of influenza predominantly in animals such as the current avian H5N1. An influenza vaccine may further be directed to a particular replikin amino acid sequence in any portion of an influenza protein.
[000169] In a non-limiting aspect in accordance with the present invention, an influenza vaccine may comprise a Replikin Scaffold of the H5N1 virus such as KKNSTYPTIKRSYNNTNQEDLLVLWGIHH. In a further non-limiting aspect, an influenza vaccine may comprise a UTOPE such as KKKKH or KKKKHKKKKKH. In a further alternative, a vaccine may comprise the addition of an adjuvant such as the well known key limpet hemocyanin having amino acid sequence KLH. In yet a further preferred non-limiting aspect, an influenza vaccine may comprise a Replikin Scaffold of influenza H5N1 further comprising two UTOPES and an adjuvent sequence such as KKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK-KLH (Vaccine V120304U2). An aspect of the present invention may comprise the Replikin Scaffold previously constructed and shown in Table 8 as one of the Bird Flu Replikins labelled"2004 H5N1 Vietnam, highly pathogenic." With administration of 100 ug of the peptide of Vaccine V120304U2 injected subcutaneously into rabbits and chickens an antibody response was observed from unvaccinated dilutions of less than 1 :50 to reach a peak in the third to fourth week after vaccination of from a dilution of 1 : 120,000 to greater than 1 :240,000. (See Example 7.) Repition and Overlapping Replikin Structures
[000170] Analysis of the primary structure of a Plasmodium farciparum malaria antigen located at the merozoite surface and/or within the parasitophorous vacuole revealed that this organism, like influenza virus, also contains numerous Replikins. However, there are several differences between the observation of Replikins in Plasmodium falciparum and influenza virus isolates. For example, Plasmodium falciparum contains several partial Replikins. Another difference seen in Plasmodium falciparum is a frequent repetition of individual Replikin structures within a single protein, which was not observed with influenza virus. Repetition may occur by (a) sharing of lysine residues between Replikins, and (b) by repetition of a portion of a Replikin sequence within another Replikin sequence.
[000171] Another significant difference between Replikin structures observed in influenza virus isolates and Plasmodium falciparum is a marked overlapping of Replikin structures throughout malarial proteins, e.g., there are nine overlapping Replikins in the 39 amino acid sequence of SEQ ID NO: 393 (Replikin concentration = 23.1/100 amino acids); and 15 overlapping Replikins in the 41 amino acids of SEQ ID NO: 467 (Replikin concentration = 36.6/100 amino acids). Both of these overlapping Replikin structures occur in blood stage trophozoites and schizonts. In contrast, influenza virus Replikins are more scattered throughout the protein and the maximum Replikin concentration is about 7.5/100 amino acids (Figure 7); and tomato leaf curl gemini virus, which was also observed to have overlapping Replikins.
High Concentrations of Replikin Correlates with Rapid Replication [000172] Tomato leaf curl Gemini virus has devastated tomato crops in China and in many other parts of the world. Its replikins reach high counts because of overlapping replikins as illustrated below in a virus isolated in Japan where the replikin count was 20.7
[000173] The relationship of higher Replikin concentration to rapid replication is also confirmed by analysis of HIV isolates. It was found that the slow-growing low titer strain of HIV (NSI, "Bru," which is prevalent in early stage HIV infection) has a Replikin concentration of 1.1 (+/- 1.6) Replikins per 100 amino acids, whereas the rapidly-growing high titer strain of HIV (Sl, "Lai", which is prevalent in late stage HIV infection) has a Replikin concentration of 6.8 (+/- 2.7) Replikins per 100 amino acid residues.
Passive Immunity
[000174] In another aspect of the invention, isolated Replikin peptides may be used to generate antibodies, which may be used, for example to provide passive immunity in an individual. Passive immunity to the strain of influenza identified by the method of the invention to be the most likely cause of future influenza infections may be obtained by administering antibodies to Replikin sequences of the identified strain of influenza virus to patients in need. Similarly, passive immunity to malaria may be obtained by administering antibodies to Plasmodium falciparum Replikin(s). [000175] 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 an 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. Furthermore, combinations of antibodies to different Replikins may be administered as an antibody cocktail.
[000176] For the production of antibodies, various host animals or plants may be immunized by injection with a Replikin peptide or a combination of Replikin peptides, including but not limited to rabbits, mice, rats, and larger mammals. [000177] 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 (US 4,946,778) can be adapted to produce Replikin-specific single chain antibodies.
[000178] Particularly useful antibodies of the invention are those that specifically bind to Replikin sequences contained in peptides and/or polypeptides of influenza virus. For example, antibodies to any of peptides observed to be present in an emerging or re-emerging strain of influenza virus and combinations of such antibodies are useful in the treatment and/or prevention of influenza. Similarly, antibodies to any Replikins present on malaria antigens and combinations of such antibodies are useful in the prevention and treatment of malaria. [000179] Antibody fragments which contain binding sites for a Replikin may be generated by known techniques. For example, such fragments include but are not limited to F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be generated (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. [000180] The fact that antimalignin antibody is increased in concentration in human malignancy (see Figure 5), regardless of cancer cell type, and that this antibody binds to malignant cells regardless of cell type now may be explained by the presence of the Replikin structures herein found to be present in most malignancies (Figure 1 and Table 2). Population studies have shown that antimalignin antibody increases in concentration in healthy adults with age, and more so in high-risk families, as the frequency of cancer increases. An additional two-fold or greater antibody increase, which occurs in early malignancy, has been independently confirmed with a sensitivity of 97% in breast cancers 1-10 mm in size. Shown to localize preferentially in malignant cells in vivo, histochemically the antibody does not bind to normal cells but selectively binds to (Figure 4A5B) and is highly cytotoxic to transformed cells in vitro (Figure 4C-F). Since in these examples the same antibody is bound by several cell types, that is, brain glioma, hematopoietic cells (leukemia), and small cell carcinoma of lung, malignant Replikin class unity is again demonstrated.
[000181] Antimalignin does not increase with benign proliferation, but specifically increases only with malignant transformation and replication in breast in vivo and returns from elevated to normal values upon elimination of malignant cells (Figure 5). Antimalignin antibody concentration has been shown to relate quantitatively to the survival of cancer patients, that is, the more antibody, the longer the survival. Taken together, these results suggest that anti-Replikin antibodies may be a part of a mechanism of control of cell transformation and replication.
Augmentation of this immune response may be useful in the control of replication, either actively with synthetic Replikins as vaccines, or passively by the administration of anti-Replikin antibodies, or by the introduction of non-immune based organic agents, such as for example, carbohydrates, lipids and the like, which are similarly designed to target the Replikin specifically.
[000182] In another aspect of the invention, 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.
Synthetic Replikin Vaccines (Active Immunity)
[000183] Synthetic Replikin vaccines, based on Replikins such as the glioma Replikin (SEQ ID NO: 1) "kagvaflhkk" or the hepatitis C Replikin (SEQ ID NO: 18) "hyppkpgcivpak", or HIV Replikins such as (SEQ ID NO: 5) "kcfncgkegh" or (SEQ ID NO: 6) "kvylawvpahk" or preferably, an influenza vaccine based on conserved and/or emerging or re-emerging Replikin(s) over a given time period may be used to augment antibody concentration in order to lyse the respective virus infected cells and release virus extracellularly where chemical treatment can then be effective. Similarly, a malaria vaccine, based on Replikins observed in Plasmodium falciparum malaria antigens on the merozoite surface or within the parasitophorous vacuole, for example, can be used to generate cytotoxic antibodies to malaria. Table 7 shows the relation of shortening or compacting of Replikin sequences to mortality rate caused by the organisms which contain these Replikins, to as short as seven amino acids. This correlation has been found by us to be a general phenomenon regardless of the type of organism. We have also found that there may be a progression over time to the shortened Replikin structure, as in influenza and SARS viruses. [000184] There is abundant evidence that there are constant evolutionary and competitive pressures for the emergence of constantly increasing "efficacy" of each infectious organism. Based upon these observations, and by projection, it would appear that if evolutionary pressures are towards shorter and shorter Replikins, with higher and higher concentrations of lysine (k), to as high as 70% as in EEL leukemia (Table 7), then the projected theoretical ideal would be the shortest possible
Replikin permitted by the algorithm which defines a Replikin, that is six amino acids (two ks six to ten amino acids apart), with the highest possible % k ( see deduced Replikin "kkkklik", which contains 83.3% k, 5/6, and one obligatory "h"). We have therefore, so-to-speak, taken what appears to be, or might be, the next evolutionary step, not apparently as yet taken by the organisms themselves, and devised the resultant deduced Replikins to use as general vaccines. [000185] These Replikins which we have deduced have maximum %'k's, therefore maximum potential binding capacity, plus the constituent 'h' by definition required for the Replikin, giving the potential for 'h' connection to redox energy systems. These devised Replikins are least likely to be cleaved by organisms because of their short length (proteins are cleaved to 6 to 10 amino acids long in processing for presentation to and recognition by immune cells), therefore most likely to present intact to immune-forming apparatuses in the organism to which they are administered, and, because of their high k content, they are most likely to generate a maximum immune response which mimics and may increase the maximum such response which can be generated against short homologous high mortality Replikins.
[000186] Further, we have found that high % k Replikins generate the highest antibody responses when administered to rabbits. These synthetic peptides, designed by us, are designated as Universal synthetic epitopes, or "UTOPE's" , and the vaccines based upon these UTOPEs, are designated "UVAX"s. UVAXs, deduced synthetic vaccines, may be used as sole vaccines or as adjuvants when administered with more specific Replikin vaccines oi • other vaccines. The following are examples of deduced UTOPEs and UVAXs:
DEVISED SYNTHETIC REPLIKIN SEQ ID NO:
(UTOPE OR UVAX)
KKKKHK 732
KKKHKK 733
KKHKKK 734
KHKKKK 735
KKKKKKH 736
KKKKKHK 737
KKKKHKK 738
KKKHKKK 739
KKHKKKK 740
KHKKKKK 741
HKKKKKK 742
[000187] Recognin and/or Replikin peptides may be administered to a subject to induce the immune system of the subject to produce anti-Replikin antibodies. Generally, a 0.5 to about 2 mg dosage, preferably a 1 mg dosage of each peptide is administered to the subject to induce an immune response. Subsequent dosages may be administered if desired.
[000188] The Replikin sequence structure is associated with the function of replication. Thus, whether the Replikins of this invention are used for targeting sequences that contain Replikins for the purpose of diagnostic identification, promoting replication, or inhibiting or attacking replication, for example, the structure-function relationship of the Replikin is fundamental. [000189] It is preferable to utilize only the specific Replikin structure when seeking to induce antibodies that will recognize and attach to the Replikin fragment and thereby cause destruction of the cell. Even though the larger protein sequence may be known in the art as having a "replication associated function," vaccines using the larger protein often have failed or proven ineffective. [000190] Although the present inventors do not wish to be held to a single theory, the studies herein suggest that the prior art vaccines are ineffective because they are based on the use of the larger protein sequence. The larger protein sequence invariably has one or more epitopes (independent antigenic sequences that can induce specific antibody formation); Replikin structures usually comprise one of these potential epitopes. The presence of other epitopes within the larger protein may interfere with adequate formation of antibodies to the Replikin, by "flooding" the immune system with irrelevant antigenic stimuli that may preempt the Replikin antigens, See, e.g., Webster, R.G., J. Immunol, 97(2):177-183 (1966); and Webster et al, J. Infect. Dis, 134:48-58, 1976; Klenerman et al, Nature 394:421-422 (1998) for a discussion of this well-known phenomenon of antigenic primacy whereby the first peptide epitope presented and recognized by the immune system subsequently prevails and antibodies are made to it even though other peptide epitopes are presented at the same time. This is another reason that, in a vaccine formulation, it is important to present the constant Replikin peptide to the immune system first, before presenting other epitopes from the organism so that the Replikin is not pre- empted but lodged in immunological memory.
[000191] The formation of an antibody to a non-Replikin epitope may allow binding to the cell, but not necessarily lead to cell destruction. The presence of structural "decoys" on the C-termini of malaria proteins is another aspect of this ability of other epitopes to interfere with binding of effective anti-Replikin antibodies, since the decoy epitopes have many lysine residues, but no histidine residues. Thus, decoy epitopes may bind anti-Replikin antibodies, but may keep the antibodies away from histidine-bound respiratory enzymes. Treatment may therefore be most efficacious in two stages: 1) proteases to hydro lyze decoys, then; 2) anti-Replikin antibodies or other anti-Replikin agents. [000192] It is well known in the art that in the course of antibody production against a "foreign" protein, the protein is first hydrolyzed into smaller fragments. Usually fragments containing from about six to ten amino acids are selected for antibody formation. Thus, if hydrolysis of a protein does not result in Replikin- containing fragments, anti-Replikin antibodies will not be produced. In this regard, it is interesting that Replikins contain lysine residues located six to ten amino acids apart, since lysine residues are known to bind to membranes. [000193] Furthermore, Replikin sequences contain at least one histidine residue. Histidine is frequently involved in binding to redox centers. Thus, an antibody that specifically recognizes a Replikin sequence has a better chance of inactivating or destroying the cell in which the Replikin is located, as seen with anti-malignin antibody, which is perhaps the most cytotoxic anti-cancer antibody yet described, being active at picograms per cell. [000194] 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 (such as foot and mouth vaccine which has been developed against the VPl protein or large segments of the VPl protein) is that the best antibodies have not been produced, that is - it is likely that the antibodies to the Replikins have not been produced. Replikins have not been produced. That is, either epitopes other than Replikins present in the larger protein fragments may interfere according to the phenomenon of antigenic primacy referred to above, and/or because the hydrolysis of larger protein sequences into smaller sequences for processing to produce antibodies results in loss of integrity of any Replikin structure that is present, e.g., the Replikin is cut in two and/or the histidine residue is lost in the hydrolytic processing. The present studies suggest that for an effective vaccine to be produced, the Replikin sequences, and no other epitope, should be used as the vaccine. For example, a vaccine of the invention can be generated using any one of the Replikin peptides identified by the three-point recognition system. [000195] Particularly preferred peptides - for example - an influenza vaccine include peptides that have been demonstrated to be conserved over a period of one or more years, preferably about three years or more, and/or which are present in a strain of influenza virus shown to have the highest increase in concentration of Replikins relative to Replikin concentration in other influenza virus strains, e.g., an emerging strain. The increase in Replikin concentration preferably occurs over a period of at least about six months to one year, preferably at least about two years or more, and most preferably about three years or more. Among the preferred Replikin peptides for use in an influenza virus vaccine are those Replikins observed to "re- emerge" after an absence from the hemagglutinin amino acid sequence for one or more years.
[000196] The Replikin peptides of the invention, alone or in various combinations are administered to a subject, preferably by i.v. or intramuscular injection, in order to stimulate the immune system of the subject to produce antibodies to the peptide. Generally the dosage of peptides is in the range of from about 0.1 μg to about 10 mg, preferably about 10 μg to about 1 mg, and most preferably about 50 μg to about 500 ug. The skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.
Quantitative Measurement Early Response(s) to Replikin Vaccines [000197] The ability to measure quantitatively the early specific antibody response in days or a few weeks to a Replikin vaccine is a major practical advantage over other vaccines for which only a clinical response months or years later can be measured. Adjuvants
[000198] Various adjuvants may be used to enhance the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, key limpet hemocyanin, dintrophenol, and potentially useful human adjuvants such as BCG and Corynebacterium parvum. In addition to the use of synthetic UTOPEs as vaccines in themselves, UTOPEs can be used as adjuvants to other Replikin vaccines and to non-Replikin vaccines. Replikin Nucleotide Sequences
[000199] Replikin DNA or RNA may have a number of uses for the diagnosis of diseases resulting from infection with a virus, bacterium or other Replikin encoding agent. For example, Replikin 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 a particular organism in a tissue sample or an environmental sample, for example. The present invention also 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.
[000200] Also within the scope of the invention are oligoribonucleotide sequences, that include antisense RNA and DNA molecules and ribozymes that function to inhibit the translation of Replikin- or recognin-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 i.v. or i.m. delivery is routine. The dosage amount is also readily ascertainable.
[000201] Particularly preferred antisense nucleic acid molecules are those that are complementary to a Replikin sequence contained in a mRNA encoding, for example, an influenza virus polypeptide, wherein the Replikin sequence comprises from 7 to about 50 amino acids including (1) at least one 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. More preferred are antisense nucleic acid molecules that are complementary to a Replikin present in the coding strand of the gene or to the mRNA encoding the influenza virus hemagglutinin protein, wherein the antisense nucleic acid molecule is complementary to a nucleotide sequence encoding a Replikin that has been demonstrated to be conserved over a period of six months to one or more years and/or which are present in a strain of influenza virus shown to have an increase in concentration of Replikins relative to Replikin concentration in other influenza virus strains. The increase in Replikin concentration preferably occurs over a period of at least six months, preferably about one year, most preferably about two or three years or more.
[000202] Similarly, antisense nucleic acid molecules that are complementary to mRNA those that are complementary to a mRNA encoding bacterial Replikins comprising a Replikin sequence of from 7 to about 50 amino acids including (1) at least one 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. More preferred are antisense nucleic acid molecules that are complementary to the coding strand of the gene or to the mRNA encoding a protein of the bacteria. Further Aspects of Replikins [000203] In an aspect of the present invention a method of preventing or treating ■ a virus infection comprising administering to a patient in need thereof a preventive or therapeutic virus vaccine is provided comprising at least one isolated Replikin present in a protein of an emerging strain of the virus and a pharmaceutically acceptable carrier and/or adjuvant. In a further aspect of the invention the isolated or synthesized peptides are influenza virus peptides. In yet a further aspect of the invention, the isolated or synthesized peptides are H5N1 influenza virus peptides [000204] The present invention also provides a method of making a preventive or therapeutic virus vaccine comprising: (1) identifying a strain of a virus as an emerging strain,
(2) selecting at least one Replikin sequence present in the emerging strain as a peptide template for the virus vaccine manufacture,
(3) synthesizing peptides having the amino acid sequence of the at least one Replikin sequence selected in step (2), and (4) combining a therapeutically effective amount of the peptides of step
(3) with a pharmaceutically acceptable carrier and/or adjuvant. In a further aspect of the method of making a preventive or therapeutic virus vaccine, the isolated Replikin is from influenza virus. In still a further aspect, the isolated Replikin is from an influenza H5N1 virus. [000205] In another aspect, the invention is directed to a method of identifying an emerging strain of a virus for diagnostic, preventive or therapeutic purposes comprising:
(1) obtaining at least one isolate of each strain of a plurality of strains of the virus; (2) analyzing the amino acid sequence of the at least one isolate of each strain of the plurality of strains of the virus for the presence and concentration of Replikin sequences;
(3) comparing the concentration of Replikin sequences in the amino acid sequence of the at least one isolate of each strain of the plurality of strains of the virus to the concentration of Replikin sequences observed in the amino acid sequence of each of the strains from at least one earlier time period to provide the concentration of Replikins for at least two time periods, said at least one earlier time period being within about six months to about three years prior to step (1); and
(4) identifying the strain of the virus having the highest increase in concentration of Replikin sequences during the at least two time periods. [000206] In another aspect of the invention there is provided a process for stimulating the immune system of a subject to produce antibodies that bind specifically to a Replikin sequence, said process comprising administering to the subject an effective amount of a dosage of a composition comprising at least one Replikin peptide. A further aspect of the present invention comprises at least one peptide that is present in an emerging strain of the organism if such new strain emerges. Another aspect of the present invention comprises at least one peptide that is present in influenza H5N1. [000207] The present invention also provides antibodies that bind specifically to a Replikin, as defined herein, as well as antibody cocktails containing a plurality of antibodies that specifically bind to Replikins. Another aspect of the present invention provides compositions comprising an antibody or antibodies that specifically bind to a Replikin and a pharmaceutically acceptable carrier. [000208] In one aspect of the invention there are provided isolated, or separated from other proteins, recombinant, or synthesized peptides or other methods containing a viral Replikin sequence.
[000209] The present application also provides isolated, or separated from nucleocapsid proteins, amongst others, recombinant, or synthesized peptides or other methods containing a viral Replikin sequence. [000210] In another aspect of the invention there is provided a process for stimulating the immune system of a subject to produce antibodies that bind specifically to a viral Replikin sequence, said process comprising administering to the subject an effective amount of a dosage of a composition comprising at least one Replikin peptide. Another aspect of the present invention comprises at least one peptide that is present in an emerging strain of the virus if such new strain emerges. [000211] The present invention also provides antibodies that bind specifically to a viral Replikin, as defined herein, as well as antibody cocktails containing a plurality of antibodies that specifically bind to viral Replikins. Another aspect of the present invention provides compositions comprising an antibody or antibodies that specifically bind to a viral Replikin and a pharmaceutically acceptable carrier. [000212] The present invention also provides therapeutic compositions comprising one or more of isolated Replikin virus peptides and a pharmaceutically acceptable carrier. [000213] In another aspect of the invention there is provided an antisense nucleic acid molecule complementary to a virus Replikin mRNA sequence, said Replikin mRNA sequence denoting from 7 to about 50 amino acids comprising:
(1) at least one 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.
[000214] In yet another aspect of the invention there is provided a method of simulating the immune system of a subject to produce antibodies to viruses, said method comprising: administering an effective amount of at least one virus Replikin peptide.
[000215] In another aspect, there is provided a method of selecting a virus peptide for inclusion in a preventive or therapeutic virus vaccine comprising:
(1) obtaining at least on isolate of each strain of a plurality of strains of said virus;
(2) analyzing the amino acid sequence of the at least one isolate of each strain of the plurality of strains of the virus for the presence and concentration of Replikin sequences;
(3) comparing the concentration of Replikin sequences in the amino acid sequence of the at least one isolate of each strain of the plurality of strains of the virus to the concentration of Replikin sequences observed in the amino acid sequence of each of the strains at least one earlier time period to provide the concentration of Replikins for at least two time periods, said at least one earlier time period being within about six months to about three years prior to step (1);
(4) identifying the strain of the virus having the highest increase in concentration of Replikin sequences during the at least two time periods; and
(5) selecting at least one Replikin sequence present in the strain of the virus peptide identified in step (4) as a peptide for inclusion in the virus vaccine.
[000216] In one aspect of the invention there are provided isolated or synthesized influenza virus peptides comprising a Replikin sequence. [000217] In another aspect of the invention, there is provided a process for stimulating the immune system of a subject to produce antibodies that bind specifically to an influenza virus Replikin sequence, said process comprising administering to the subject an effective amount of dosage of a composition comprising at least one influenza virus Replikin peptide. A further aspect of the present invention comprises at least one Replikin peptide that is present in an emerging strain of influenza virus. Yet another aspect of the present invention comprises a composition comprising at least one influenza H5N1 Replikin peptide. [000218] The present invention also provides antibodies that bind specifically to an influenza virus Replikin, as defined herein, as well as antibody cocktails containing a plurality of antibodies that specifically bind to influenza virus Replikins. In another aspect of the present invention, there are provided compositions comprising an antibody or antibodies that specifically bind to an influenza Replikin and a pharmaceutically acceptable carrier. [000219] The present invention also provides therapeutic compositions comprising one or more of isolated influenza virus peptides having from 7 to about 50 amino acids comprising:
(1) at least one lysine residue located six to ten residues form a second lysine residue; (2) at least one histidine residue; and
(3) at least 6% lysine residues, and a pharmaceutical acceptable carrier. [000220] In another aspect of the invention there is provided an antisense nucleic acid molecule complementary to an influenza virus hemagglutinin Replikin mRNA sequence, said Replikin mRNA sequence denoting from 7 to about 50 amino acids comprising:
(1) at least one 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. [000221] In yet another aspect of the invention there is provided a method of simulating the immune system of a subject to produce antibodies to influenza virus comprising administering an effective amount of at least one influenza virus Replikin peptide having from 7 to about 50 amino acids comprising: (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
(2) at least one histidine residue; and
(3) at least 6% lysine residues. [000222] In another aspect, there is provided a method of selecting an influenza virus peptide for inclusion in a preventive or therapeutic influenza virus vaccine comprising:
(1 ) obtaining at least one isolate of each strain of a plurality of strains of influenza virus; (2) analyzing the hemagglutinin amino acid sequence of the at least one isolate of each strain of the plurality of strains of influenza virus for the presence and concentration of Replikin sequences;
(3) comparing the concentration of Replikin sequences in the hemagglutinin amino acid sequence of the at least one isolate of each strain of the plurality of strains of influenza virus to the concentration of Replikin sequences observed in the hemagglutinin amino acid sequence of each of the strains at least one earlier time period to provide the concentration of Replikins for at least two time periods, said at least one earlier time period being within about six months to about three years prior to step (1);
(4) identifying the strain of influenza virus having the highest increase in concentration of Replikin sequences during the at least two time periods;
(5) selecting at least one Replikin sequence present in the strain of influenza virus peptide identified in step (4) as a peptide for inclusion in an influenza virus vaccine.
[000223] The present invention also provides a method of making a preventive or therapeutic influenza virus vaccine comprising:
(1) identifying a strain of influenza virus as an emerging strain; (2) selecting at least one Replikin sequence present in the emerging strain as a peptide template for influenza virus vaccine manufacture, (3) synthesizing peptides having the amino acid sequence of the at least one Replikin sequence selected in step (2), and (4) combining a therapeutically effective amount of the peptides of step
(3) with a pharmaceutically acceptable carrier and/or adjuvant. [000224] In another aspect, the invention is directed to a method of identifying an emerging strain of influenza virus for diagnostic, preventive or therapeutic purposes comprising:
(1) obtaining at least one isolate of each strain of a plurality of strains of influenza virus;
(2) analyzing the hemagglutinin amino acid sequence of the at least one isolate of each strain of the plurality of strains of influenza virus for the presence and concentration of Replikin sequences;
(3) comparing the concentration of Replikin sequences in the hemagglutinin amino acid sequence of the at least one isolate of each strain of the plurality of strains of influenza virus to the concentration of Replikin sequences observed in the hemagglutinin amino acid sequence of each of the strains at least one earlier time period to provide the concentration of Replikins for at least two time periods, said at least one earlier time period being within about six months to about three years prior to step (1); and
(4) identifying the strain of influenza virus having the highest increase in concentration of Replikin sequences during the at least two time periods.
[000225] In yet another aspect of the invention, there is provided a preventive or therapeutic influenza virus vaccine comprising at least one isolated Replikin present in the hemagglutinin protein of an emerging strain of influenza virus and a pharmaceutically acceptable carrier and/or adjuvant.
[000226] Also provided by the present invention is a method of preventing or treating influenza virus infection comprising administering to a patient in need thereof a preventive or therapeutic vaccine comprising at least one isolated Replikin present in the hemagglutinin protein of an emerging strain of influenza virus and a pharmaceutically acceptable carrier and/or adjuvant.
COMPUTER SOFTWARE FOR IDENTIFYING REPLIKINS AND RELATED STRUCTURES [000227] Identification of Replikin structures, Replikin Scaffold structures and degenerate Exoskeleton Scaffold structures may be accomplished with the aid of bioinformatics.
[000228] Embodiments of the present invention are directed to a system and method for identifying and/or locating complex patterns in an amino acid sequence such as Replikin patterns, Replikin Scaffold structures, Exoskeleton Scaffold structures and other complex patterns in amino acid and nucleic acid sequences. According to an aspect of the present invention, techniques are provided to facilitate queries of protein databases. For protein descriptions received in response to the queries, aspects of the present invention may include a scan of the received protein descriptions to identify and locate Replikin patterns. According to an aspect of the present invention, a Replikin pattern is a sequence of from 7 to about 50 amino acids that include the following three (3) characteristics, each of which may be recognized as an aspect of the present invention: (1) the sequence has at least one lysine residue located six to ten amino acid residues from a second lysine residue; (2) the sequence has at least one histidine residue; and (3) at least 6% of the amino acids in the sequence are lysine residues. Another aspect of the present invention may identify and/or locate a complex amino acid sequence having specified length constraints, which further includes any combination of the following characteristics: (1) a first amino acid residue located more than N positions and less than M positions away from a second amino acid residue; (2) a third amino acid residue located anywhere in the sequence; and (3) at least R percent of a fourth amino acid residue. According to yet another aspect, the present invention may count occurrences of the identified amino acid sequences and may report the counted occurrences, either as raw absolute values or as ratios of the number of identified amino acid sequences per N amino acids in the protein. Still another aspect of the present invention may analyze the evolution of identified amino acid sequence patterns in variants of a given protein over time, and may also analyze the similarities and differences between instances of identified amino acid sequence patterns across a plurality of different proteins over time. As a result of the analysis, yet another aspect of the present invention may identify potential amino acid scaffolding structures that appear to be preserved over time and across different proteins, as component elements of the identified amino acid sequence patterns mutate and/or evolve. [000229] Embodiments of the present invention will be described with reference to the accompanying drawings, wherein like parts are designated by like reference numerals throughout, and wherein the leftmost digit of each reference number refers to the drawing number of the figure in which the referenced part first appears. [000230] FIG. 17 is a high-level block diagram of a computer system incorporating a system and method for identifying Replikin patterns in amino acid sequences, in accordance with an aspect of the present invention. As shown in FIG. 17, computer workstation 610 may be a computer having a processor and a memory configured to permit a researcher to search protein databases and to scan protein descriptions for selected amino acid patterns. To accomplish these functions, computer workstation 610 may include protein and amino acid research system 630, which may receive instructions from a user/researcher to conduct protein searching and amino acid scanning operations. According to an aspect, protein and amino acid research system 630 may further include amino acid sequence scanner 640 that scans and searches retrieved protein and amino acid sequences for specific patterns of amino acids, including Replikin patterns. Protein and amino acid research system 630 may communicate with network interface 620 to obtain protein sequences and amino acid sequences from resources on network 660, which may include the Internet. Alternatively, protein and amino acid research system 630 may obtain protein sequences and amino acid sequences from a local protein database 650. In addition, protein and amino acid research system 630 may obtain protein sequences and amino acid sequences directly from other input means, such as keyboard input. Protein and amino acid research system 630 may also communicate with network interface 620 to transmit results to other computers on network 660. Automated Scanning for Replikin Patterns
[000231] Embodiments of the present invention may include a generalized method and system for identifying complex patterns of amino acids within proteins. For any protein definition identified or selected by protein and amino acid research system 630, the user may direct aspects of the invention to search for a variety of complex patterns of amino acids. As an example of one pattern of amino acids, the present invention provides a method for identifying nucleotide or amino acid sequences that include a Replikin pattern. FIG. 18 is a simple flow chart illustrating a general method for locating a Replikin pattern in a sequence of amino acids, according to an aspect of the present invention. The method 700 may begin after a sequence of amino acids has been obtained. Typically, the sequence of amino acids may be represented by alphabetic characters according to the code supplied in FIG. 12. However, other encodings are envisioned by the present invention as well. [000232] Referring to FIG. 18, once a sequence of amino acids has been obtained, the sequence is searched for a Replikin pattern (710), which comprises a subsequence (or string) of amino acids that includes the following characteristics:
(1) the string contains from 7 to about 50 amino acids;
(2) the string contains at least one lysine residue located 6 to 10 positions from a second lysine residue;
(3) the string contains at least one histidine residue; and
(4) the string contains at least 6% lysine residues.
[000233] Once a string of amino acids is found to match the Replikin pattern, the string may be identified or marked (720) accordingly. [000234] A given sequence of amino acids may contain many subsequences or strings that match the Replikin pattern. Additionally, Replikin patterns may overlap each other. Thus, to locate and identify all possible Replikin patterns in a sequence of amino acids, method 700 may be invoked iteratively for each subsequence of amino acids contained within the original sequence of amino acids. [000235] When method 700 is invoked iteratively to identify and locate all possible Replikin patterns in an amino acid sequence, an aspect of the present invention may count the number of resulting Replikin patterns. A Replikin count may be reported as an absolute number. Additionally, aspects of the invention may also determine a ratio of the number of Replikins per N amino acids in the sequence. For example, an aspect of the present invention may determine that a given protein contains a ratio of 6 Replikins for every 100 amino acids. Replikin ratios have been shown by laboratory experiment and by epidemiological evidence to correlate directly to the rate that a given protein replicates. Rapid replication of proteins may be an indication of disease. For example, the presence of relatively high ratios of Replikin patterns has been correlated to epidemics of influenza. Similarly, an increase in the count of Replikin patterns observed in a protein over time may also be an indication of future disease caused by the organism from which the protein was obtained (see, e.g., FIG. 15). Thus, the ability to detect and count Replikin patterns within sequences of amino acids is a significant advantage of the present invention.
[000236] Still referring to FIG. 18, aspects of the present invention may utilize method 700 to identify and locate other complex patterns of amino acids, which exhibit characteristics similar to Replikin patterns. That is, although some aspects of the present invention may specify exact values for: (1) distances between amino acids, (2) acceptable lengths of recognized amino acid sequences, and (3) the percentage or concentration of specific amino acids, these exact values may also be expressed as variables. Thus a researcher may employ an aspect of the present invention to identify sequences of amino acids in a protein that have the following characteristics:
(1) the sequence contains from rmin to rmax amino acids;
(2) the sequence contains at least one lysine residue located kmin to kmax amino acid residues from a second lysine residue; (3) the sequence contains at least one histidine residue; and
(4) the sequence contains at least kpercent lysine residues. [000237] FIG. 19 is a flow chart illustrating a generalized method 800 for locating a plurality of Replikin-like patterns in a given sequence of amino acids, according to an aspect of the present invention. The method 800 begins by locating a first lysine residue in the given sequence (810). Then, the method 800 may determine whether a second lysine residue resides within kmin to kmax positions of the first lysine residue (820). As indicated in FIG. 19, kmin and kmax define the limits on the distance between the first and second lysine residues. For a typical Replikin pattern, kmin will equal 6 and kmax will equal 10. However, these values may be varied by a researcher interested in discovering other similar patterns. [000238] Once method 800 has identified two lysine residues that are close enough to each other (820), the method 800 may examine every histidine residue that resides within rmax positions of both the first and second lysine residues (830). When method 800 is employed to identify and locate typical Replikin patterns, rmax will usually be set to equal 50. For every histidine residue that resides within rmax positions of the two lysine residues identified in steps (810) and (820), method 800 will construct the shortest string of amino acid residues that includes the first lysine residue, the second lysine residue, and the identified histidine residue (840). Then, method 800 will determine whether the length of that shortest string is within the desired range - that is, whether it contains at least rmin amino acid residues and no more than rmax amino acid residues (850). Finally, if the identified string of amino acids also contains at least kpercent of lysine residues (860), the string will be identified as matching the desired Replikin-like pattern (870).
[000239] Still referring to FIG. 19, it is apparent that method 800 may identify several Replikin-like patterns from a single given amino acid sequence. This may happen because method 800 may examine more than one histidine residue that resides within rmax positions of the two identified lysine residues. Each identified histidine residue may, in combination with the two lysine residues, match the desired Replikin-like pattern.
[000240] One aspect of the method illustrated by FIG. 19 is shown in FIG. 20, which is a source code listing containing a procedure for discovering all Replikin patterns present in a given sequence of amino acids, in accordance with an aspect of the present invention. The "match" procedure shown in FIG. 20 is programmed in an interpreted shell language called "TcI" and recognizes Replikins in a straightforward fashion. As known in the art, the "Tool Command Language" or TcI (pronounced "tickle") is a simple interpreted scripting language that has its roots in the Unix command shells, but which has additional capabilities that are well-suited to network communication, Internet functionality and the rapid development of graphical user interfaces.
[000241] Alternative methods of recognizing Replikin patterns are also covered by the teachings of the present invention. For example, the match procedure shown in FIG. 20 could be implemented in other programming languages such as Java or C or C++. Additionally, alternative aspects of the Replikin recognizing algorithm may identify the characteristics of a Replikin pattern in any order, and may also traverse component amino acid sequences and subsequences using recursive techniques, iterative techniques, parallel processing techniques, divide-and-conquer techniques or any combination thereof. Protein Search Engine
[000242] Returning to FIG. 17, the present invention may include a search engine to access and interact with amino acid and protein databases, either locally or over a network such as the Internet, to retrieve protein definitions. For example, protein and amino acid research system 630 may accept protein search criteria from a user, and may then access a plurality of on-line amino acid and protein database search engines to retrieve protein definitions that match the supplied search criteria. Protein database search criteria may comprise any text string that may form a valid search term in any of the on-line protein or amino acid search engines. Typically, these search criteria relate to text that may be found in the printout that describes each specific protein. For example, if the user supplied the search criteria "influenza type A," aspects of the present invention may forward this text string to a plurality of Internet protein and amino acid search engines, each of which may then return any protein descriptions found in their databases that contained the terms "influenza type A." Employing amino acid sequence scanner 640, each of the returned protein descriptions may be scanned for the presence of Replikin patterns. [000243] Additional aspects of the present invention may permit a user to select or de-select a plurality of Internet protein search engines and to customize the search criteria and protein retrieval capabilities of the present invention for each of the selected on-line protein search engines. Moreover, aspects of the invention may also permit a user to access a local protein database 650 or to supply a specific protein definition directly, for example, by supplying a local file name containing the protein definition, or by other methods known in the art for supplying parameters to computer software.
[000244] Another aspect of the present invention may include a search engine to access and interact with amino acid and protein databases on the Internet to retrieve protein definitions or amino acid sequence definitions. After accepting protein or amino acid sequence search criteria from a user, the present invention may access a plurality of amino acid and protein database search engines, through on-line access, to retrieve protein definitions or amino acid sequence definitions that match the supplied search criteria.
[000245] Initial existing protein search criteria based on existing definitions may comprise any text string that may form a valid search term in any of the on-line protein or amino acid search engines. Typically, these search criteria relate to text that may be found in the printout that describes each specific protein. For example, if the user supplied the search criteria "influenza type A," the present invention would forward this text string to the plurality of Internet protein and amino acid search engines, each of which would then return any protein definitions in their databases that contained the terms "influenza type A."
[000246] A non-limiting aspect of the present invention comprising a protein search engine entitled "Genome Explorer" is included in Appendix A. The TcI procedure named "GenomalEnquirer" may control the macro level operation of the protein search engine (see "proc GenomalEnquirer {database term additionalCriteria})." Within the procedure GenomalEnquirer, a series of specific on-line protein search engines may be accessed and queried using the user-supplied protein search terms and additional criteria. Additional aspects of the invention may permit a user to select or de-select a plurality of Internet protein search engines and to customize the search criteria and protein retrieval capabilities of the present invention for each of the selected on-line protein search engines. Moreover, aspects of the invention may also permit a user to access local protein databases or to supply a specific protein definition directly, for example, by supplying a local file name containing the protein definition, or by other methods known in the art for supplying parameters to computer software.
[000247] Instructions for running the Genome Explorer are included in Appendix B. Screen snapshots of the Genome Explorer application are included in Appendix C. Replikin Analysis
[000248] Embodiments of the present invention may be employed not only to identify and locate Replikin patterns in amino acid sequences. Embodiments may also be used to discover and analyze similarities in the structure of Replikin patterns occurring in different proteins, or to analyze different Replikin patterns occurring in the same protein over time. FIG. 21 for example, is a table illustrating a Replikin Scaffold or "fixed scaffold" structure that was preserved in a "Bird Flu" influenza virus over an 87 year period from 1917 to 2004. Embodiments of the present invention may assemble a number of discovered Replikin patterns in proteins, including Replikin patterns discovered in variants of the same protein. Along with each Replikin pattern, aspects of the present invention may also associate a date when each protein was first identified. When directed by a researcher, an aspect of the present invention may include sorting and displaying a plurality of selected Replikin patterns according to content, date or other criteria, in order to reveal substantially fixed amino acid structures that have been preserved in Replikin patterns over time and which may be present in different proteins as well as variants of the same protein. Further, when directed by a researcher, an aspect of the invention may employ known methods of pattern analysis to compare a plurality of selected Replikin patterns in order to identify such fixed amino acid structures automatically. As an example, in FIG. 21, the illustrated Replikin patterns appear to demonstrate - in this case - a relatively fixed scaffold structure of (usually) 29 amino acids that begins with a pair of lysine residues (kk) at the amino terminal, ends with a pair of histidine residues (hh) at the carboxyl terminal, and contains a lysine residue in either position 8, 10 or 11. This conservation of scaffold structure over decades permits synthetic vaccines to be prepared rapidly and inexpensively. To synthesize such vaccines after a Replikin scaffolding structure has been identified, a researcher may select elements of that scaffolding structure that are conserved over time and which are also present in a current variant of a protein. A vaccine may then be prepared based on the selected elements from the scaffolding structure. Because such vaccines are based on conserved scaffolding structures, they may be effective for multiple years and may also be developed well in advance of an anticipated outbreak. [000249] The discovery of Replikins themselves, as well as aspects of the present invention for identifying and locating Replikin patterns, provides targets for the identification of pathogens, as well as facilitates the development of anti-pathogen therapies, including vaccines. In general, knowledge of and identification of the Replikin family of peptides enables development of effective therapies and vaccines for any organism that harbors Replikins. Specifically, identification of Replikins provides for the detection of viruses and virus vaccine development, including the influenza virus. Further, identification of Replikins also provides for the detection of other pathogens, such as malaria, anthrax and small pox virus, in addition to enabling the development of therapies and vaccines that target Replikin structures. Additional examples provided by the identification of Replikins include the detection of infectious disease Replikins, cancer immune Replikins and structural protein Replikins.
[000250] Embodiments of the present invention enable important Replikin patterns of amino acids to be recognized, located and analyzed in manners that are not found in the prior art. Using prior art capabilities, researchers have been limited in by existing techniques for describing sequences of amino acids. Indeed, limitations of the prior art have in some ways dampened research in this field, since heretofore it has not been possible to specify sequences of amino acids that comprise non-linear attributes. Until the development of the methods and aspects of the present invention, descriptions of amino acid sequences were limited to linear sequences containing, at most, repetitive substrings and logical constraints on substring content. Embodiments of the present invention enable a new class of amino acid sequences to be discovered, located and analyzed using tools not found in the prior art. This new class of amino acids is characterized by attributes such as specific amino acid concentration and distance relationships between specific amino acids. These attributes transcend simple contiguous ordering and thus are not easily described, discovered or located by existing methods known in the art. [000251] For example, rather than examining strict amino acid sequence matches (homologies) as is done by other widely used programs such as BLAST, the present inventors have discovered a unique quantitative "language" related to rapid replication which defines a new class of amino acid grouping. Novel computer programs described herein detect instances of this new language. [000252] These programs include functionality to search electronic data for amino acid sub-sequences meeting predetermined criteria. The data, which may be obtained online, may include data defining a specified group of protein sequences. The criteria may include: i) the occurrence within a protein sequence of two amino acids, in this case Lysine(K) and histidine(H) in specific concentrations in the sequence ii) the spacing of one of these (K) to a second K in the sequence, and iii) the concentration of one or more amino acids (e.g. K) in a percentage greater than a defined percentage.
[000253] Amino acid sequences meeting the above criteria relate to a particular biological function such as rapid replication. [000254] The programs include the capability to identify Replikin sub-sequences in genome sequences. One source of the genome sequences may be published genome sequences obtained from online, electronic databases, using search criteria provided by a user. In aspects of the invention, the databases may be NCBI (National Center for Biotechnology Information) or LANL (Los Alamos National Laboratory) databases. The programs further include the capability to search for arbitrary sub-sequences (i.e., not only Replikin sub-sequences), based on user- supplied criteria. [000255] In one aspect, a program herein entitled "Genome Explorer" may generate a user interface to prompt a user for search terms. Genome Explorer may apply the search terms to online databases, such as NCBI or LANL databases, to obtain raw sequence data. Additional data may be further obtained, such as article names, protein source, strain, serotype and year of discovery for all the raw sequences which match the search terms. Once the raw data has been acquired, Genome Explorer may further apply additional search criteria to identify Replikin sub-sequences within the raw sequences. The search criteria can be specified by the user in such a way as to implement relatively strict, or relatively relaxed definitions of what can be included in the set of matching sub-sequences to be reported by Genome Explorer. As it identifies Replikin sub-sequences, Genome Explorer may compile ongoing statistics and display a progress bar in a user interface. When Genome Explorer completes its processing, it may save resulting statistics in a data file. For example, the data file may be an HTML file that can be opened in any word processor for inspection of results. [000256] In another aspect, a program herein entitled "Dr. Peptide," search criteria may be applied to identify sub-sequences other than Replikin sub-sequences. With Dr. Peptide it is possible to search for, e.g., all instances of the sequence hlk hlk, separated by not more than 15 amino acids, in publicly available genome databases. Such searches allow the creation of new statistical profiles and new groupings of proteins based on meeting these criteria. Dr. Peptide may include much the same functionality as Genome Explorer. For example, like Genome Explorer, Dr. Peptide may, via a user interface, prompt a user for search terms and apply the search terms to online databases, such as NCBI or LANL databases, to obtain raw sequence data. Additional data may be further obtained, such as article names, protein source, strain, serotype and year of discovery for all the raw sequences which match the search terms. Once the raw data has been acquired, Dr. Peptide may further process the data to identify arbitrary sub-sequences and present its output in a data file, for example in the form of HTML pages that can be opened in any word processor.
[000257] Below is a description of one example of a logic sequence that could be included in the Genome Explorer program. In the description, an "initial server inquiry" refers to search criteria to be applied to one or more network elements, such as server computers, storing electronic data representing protein sequences. The network elements may be included in private networks or, for example, the Internet. The data may be in the form of a "protein page," i.e., a quantum of data representing protein sequences. The character "k" represents a lysine amino acid, and the character "h" represents a histidine amino acid. Genome Explorer Logic Sequence
[000258] Initialize user interface procedures and input fields for search parameters.
Construct user interface. wait for user to specify search parameters. Search parameters include:
(1) words or phrases to be matched in the initial server inquiry to obtain summaries and protein pages,
(2) The allowed distance between k's, expressed as range kmin...kmax for a sub- sequence to qualify for a set.
(3) The allowed range of distances between an h and the farthest k, expressed as kmin+l..hmax, for a sub-sequence to qualify for the set.
(4) The allowed fraction of k's in the sub-sequence, expressed as x percent or larger, for the sub-sequence to qualify for the set. [000259] Once search parameters are specified,
Initialize output files in HTML format - these will be used to display reports.
Compare specified search parameters with previous search.
If the search parameters are identical, reuse cached protein pages as data input. If the search parameters are not identical (cached protein pages are not relevant),
Send the inquiry to the server (NCBI or LANL).
If it did not return all summaries,
Re-send the inquiry requesting all summaries.
For each summary, Fetch and save the protein page retrieved.
For each protein page retrieved,
If from NCBI,
Parse ASN page.
Extract found sequence data (seq-data.ncbieaa). Extract article names (descrΛarticle.titleΛname).
Extract protein source (source. org.taxname).
Extract strain (subtype).
Derive year discovered.
Derive serotype. If from LANL5
Parse HTML page for strain, definition, source, year, serotype, and raw nucleotide sequence. Convert nucleotides to amino acids by mapping every three nucleotides in sequence to the corresponding amino acid. Save parsed value for this protein.
For each parsed page, update user interface as to progress via progress bar, and: For each sequence data found on the page, Scan the amino acid sequence data for each sub-sequence matching
(a) The distance between k's is in the range kmin...kmax as defined in parameter (2) from the user interface above.
(b) The distance between an h and the farthest k is in the range kmin+1..hmax as defined in parameter (3) from the user interface above. (c) The fraction of k units in the sub-sequence, expressed as x percent or larger as defined in parameter (4) from the user interface above. and save the range of each matching sub-sequence, including overlaps.
Ignore sequences with no matches.
Accept the sequence with the most sub-sequence matches. If a sequence was accepted,
Catalog each sequence by the year it was discovered.
For each additional set of criteria,
Check the additional criteria against other parsed fields.
If does not match, do not accept the page. If the page was accepted,
Add it as a passed page.
Create an HTML page showing the full sequence and all matched sub-sequences.
If the page was not accepted,
Add it as a failed page. For each unique matched replikin sequence,
Create an amino acid history HTML page,
Show every protein it occurs in ordered by year.
Create a statistics HTML page displaying the following:
For each year, Show number of matched proteins and replikin sub-sequences.
Update user interface to reflect that the operation is complete;
Re-initialize input fields to allow next set of search parameters to be specified by user.
[000260] In view of the foregoing description, it may be understood that Genome Explorer implements a method including applying a plurality of criteria to data representing protein sequences, and based on the criteria, identifying a sub-sequence within the protein sequences, the identified sub-sequence having a predetermined allowed range of distance between Lysine amino acids thereof, and a predetermined allowed range of distance between a histidine amino acid and a farthest Lysine acid thereof. An identified sub-sequence may be output to a data file. [000261] The functionality of the herein aspects may be provided on various computer platforms executing program instructions. One such platform 1100 is illustrated in the simplified block diagram of FIG. 22. There, the platform 1100 is shown as being populated by a processor 1160, which communicates with a number of peripheral devices via a bus subsystem 1150. These peripheral devices typically include a memory subsystem 1110, a network interface subsystem 1170, and an input/output (I/O) unit 1180. The processor 1160 may be any of a plurality of conventional processing systems, including microprocessors, digital signal processors and field programmable logic arrays. In some applications, it may be advantageous to provide multiple processors (not shown) in the platform 1100. The processor(s) 1160 execute program instructions stored in the memory subsystem 1110. The memory subsystem 1110 may include any combination of conventional memory circuits, including electrical, magnetic or optical memory systems. As shown in FIG. 22, the memory system may include read only memories 1120, random access memories 1130 and bulk storage 1140. Memory subsystem 1110 not only stores program instructions representing the various methods described herein but also may store the data items on which these methods operate. Network interface subsystem 1170 may provide an interface to outside networks, including an interface to communications network 1190 comprising, for example, the Internet. I/O unit 1180 would permit communication with external devices, which are not shown.
[000262] Several aspects of the present invention are specifically illustrated and described herein. However, it will be appreciated that modifications and variations of the present invention are covered by the teachings of the present invention without departing from the spirit and intended scope of the invention. Additionally, the teachings of the present invention may be adaptable to other sequence- recognizing problems that have heretofore been addressed using sequential linear analyses limited to the identification of specific sequences of component elements. [000263] Using the exemplary software contained in Appendix A, the inventors have discovered in a non-limiting aspect in accordance with the present invention that the nucleocapsid protein of the shrimp white spot virus has an exceptionally high Replikin Count as compared to all other viruses and organisms surveyed for replikins up to the present time (with the exception of malaria). While Replikins have been shown to be essential accompaniments of rapid replication in fungi, yeast, viruses, bacteria, algae, and cancer cells, the inventors have provided the first demonstration of the presence of replikins in marine organisms other than algae. And, as with algae, the presence of replikins is again related to rapid infestations. In shrimp, the white spot virus has destroyed millions of dollars of harvest of shrimp, first in eastern countries, and now in western hemisphere countries. At present, there is no effective prevention or treatment. Other examples of Replikin high mortality marine viral disease have been demonstrated by us in fish such as carp and hemorrhagic disease in salmon, and are probably widespread in marine ecology and disease.
[000264] The presence of repeat sequences of the Replikins of the nucleocapsid protein of shrimp white spot syndrome virus (WSSV) accounts for the unusually high Replikin Count of 103.8. This virus Replikin Count is much higher than the Replikin Counts of for example influenza viruses which usually range from less than 1 up to 5 or 7, and is comparable only to the record Replikin Count (so far) observed in Plasmodium Falciparum (malaria) of 111. Interestingly, while the shrimp white spot syndrome organism is a virus, and the Pl. Falciparum is a trypanosome, both spend an essential part of their reproductive cycles in red blood cells, an unusual host cell whether in shrimp (white spot virus) or man (malaria), both are fulminating rapidly replicating diseases with high mortality rates of their hosts, and both appear to use the same methods of increasing their high Replikin Counts to such record highs, namely, Replikin Repeats and Replikin Overlap.
[000265] As illustrated in Table 10, examples of Replikin Repeats and Replikin Overlap were found by the applicants in the above nucleocapsid protein of the shrimp white spot syndrome virus as seen below. 497 Replikins were discovered in the white spot virus using the exemplary software provided in Appendix A. Of those 497, the replikins illustrated below in Table 10 were selected for their short sequences and high concentration of lysine which, as demonstrated throughout this application, appears to be associated with high mortality. The chosen sequences are easier and less expensive to synthesize than the longer sequences that are not included in Table 10.
[000266] Table 10 illustrates intramolecular Replikin Repeats and Replikin Overlap in shrimp white spot syndrome virus (WSSV) nucleocapsid protein (VP35) gene with a Replikin Count (number of replikins per 100 amino acids) of 103.8 (497 total replikins per 479 amino acids).
TABLE 10 - Intramolecular Replikin Repeats and Replikin Overlap in shrimp white spot syndrome virus (WSSV) nucleocapsid protein (VP35) gene with
Replikin Count = Number of Replikins per 100 amino acids = 497/479 = 103.8 and with thymidine kinase and thymidylate kinase activity. Individual Replikins at Different Positions in the same Molecule, in order of appearance in the sequence
Replikin ID Number
_^_ ___, _ _ (to be assigned)
[k24 q25 "126 127 h28 Ϊ29 k30Δ
Figure imgf000099_0001
*Note in the shrimp virus the repeated use of identical whole Replikin sequences (underlined) and partial Replikin sequences (shaded) in different positions in the one molecule (each amino acid is numbered according to its order in the sequence).
[000267] Now that we have been able to identify these Replikins using the software described in this application, we can synthesize each of them and use them as targets for antibody and other inhibitory products and for specific synthetic vaccines against the shrimp white spot syndrome virus, specifically directed against each repeating Replikin.
[000268] The phenomenon of repeats is well known in protein structure. What is unique and specific in this case is that these are Replikin repeats. Thus while repeat of a specific Replikin sequence increases the Replikin Count within a specific molecule and is associated with more rapid replication as in the case of ATPase in Pl.Falciparum in malaria, thus has apparent survival value for the molecule and the organism which contains it, at the same time it provides an increasing vulnerability, an 'Achilles Heel' so to speak. Thus the Replikin Repeat provides a higher concentration per molecule, additional target sites for attack by specific antibodies as generated by specific synthetic vaccines produced against these Replikins and other specific anti-Replikin agents. These new targets were previously unavailable because they could not be identified. Complex Amino Acid Analysis
[000269] A further aspect of the present invention comprises a protein search engine directed to recognizing generalized amino acid and nucleic acid patterns online databases. Appendix D is an exemplary protein search engine directed to recognizing complex amino acid patterns such as Scaffold Exoskeletons. Appendix D is entitled "Dr. Peptide." Appendix D is an exemplary non-limiting aspect of the present invention and is designed to recognize generalized amino acid patterns in addition to the Replikin pattern. [000270] Below is a description of one example of a logic sequence that could be included in the Dr. Peptide program. In the description, an "initial server inquiry" refers to search criteria to be applied to one or more network elements, such as server computers, storing electronic data representing protein sequences. The network elements may be included in private networks or, for example, the Internet. The data may be in the form of a "protein page," i.e., a quantum of data representing protein sequences .
Dr. Peptide Logic Sequence
[000271] Initialize user interface procedures and input fields for search parameters.
Construct user interface. wait for user to specify search parameters, including:
(1) words or phrases to be matched in the initial server inquiry to obtain summaries and protein pages,
(2) a set of specific amino acids which must be included in any sub-sequences qualifying for a set. (3) a set of specific amino acids which must be excluded from any sub-sequences qualifying for the set.
(4) minimum m and maximum n sizes for the permissible size spacing gap which is to be applied to the set inclusion and exclusion criteria (2) and (3). [000272] Once search parameters are specified, Query:
If the saved protein pages are not relevant,
Send the inquiry to the server (NCBI or LANL).
If it did not return all summaries,
Re-send the inquiry requesting all summaries. For each summary, Fetch and save the protein page.
For each protein page,
If from NCBI,
Parse ASN page. Extract found sequence data (seq-data.ncbieaa).
Extract article names (descr.*.article.title.*.name).
Extract protein source (source.org.taxname).
Extract strain (subtype).
Derive year discovered. Derive serotype.
If from LANL,
Parse HTML page for strain, definition, source, year, serotype, and raw nucleotide sequence.
Convert nucleotides to amino acids by mapping every three nucleotides in sequence to the corresponding amino acid.
Save parsed value for this protein.
For each parsed page,
For each sequence data found on the page, Scan the amino acid sequence data for each sub-sequence matching.
The match patterns are a sequence of alternative steps:
(a) An amino acid in the amino acid sequence data is in a set of specific amino acids as defined in user parameter (2) above.
(b) An amino acid in the amino acid sequence data is not in the set of specific amino acids defined in user parameter (3) above.
(c) An amino acid in the amino acid sequence data has a spacing gap of m to n amino acids from another amino acid in the amino acid sequence data as defined in user parameter (4) above.
The initial sub-sequence set is all possible terminal sequences, or "tails" of the sequence data at the first pattern step,
While the set of sub-sequences is not empty,
Remove one sub-sequence and record how far in the pattern string its evaluation has reached.
If the amino acid at the current pattern step - Is in a set of specific amino acids,
If the next amino acid of the sub-sequence is also in the set of amino acids,
Add the elongated sub-sequence and next pattern step to the sub-sequence set.
- Is not in a set of specific amino acids. If the next amino acid of the sub-sequence is not one of the set of amino acids, Add the elongated sub-sequences and next pattern step to the sub-sequence set.
- Has a gap of m to n any amino acids.
First, elongate each sub-sequence for each possible length m through n Then add each elongated version of the sub-sequence to the sub-sequence set
If the above pattern is exhausted, The sub-sequence is a matched sub-sequence. Ignore sequences with no matches. Accept the sequence with the most matches. If a sequence has been accepted, Catalog each sub-sequence by the year it was discovered. For each additional criteria, Check the additional criteria against other parsed fields. If it does not match, do not accept the page. If the page was accepted, Add it as a passed page.
Create an HTML page showing the full sequence and all matched subsequences. If the page was not accepted, Add it as a failed page.
[000273] In view of the foregoing description, it may be understood that Dr. Peptide implements a method including applying a plurality of criteria to data representing protein sequences, and based on the criteria, identifying arbitrary subsequences within the protein sequences. An identified sub-sequence may be output to a data file. The criteria may include: a set {a}of amino acids to be included in the sub-sequence; a set of amino acids to be excluded from the sub-sequence; and a minimum and a maximum permissible gap between members of sets {a} and {b}.
[000274] A non-limiting and exemplary aspect of the invention employs the complex amino acid analysis aspect of the invention to analyze Replikin Scaffold sequences in earlier strains of influenza that have degenerated into non-rep likin sequences but maintained the scaffold structure of the Replikin Scaffold. As an example of the use of the exemplary and non-limiting software program in Appendix D to recognize generalized amino acid patterns, the inventors first discovered by visual scanning of protein sequences (now by Dr. Peptide software) that what was in earlier-arising specimens of a particular influenza species a Replikin Scaffold, was in later specimens changed as follows:
1) The length of 29 amino acids was preserved;
2) The first two amino acid positions (1 and 2) were preserved, i.e. KK;
3) The last two amino acid positions (28 and 29) were preserved, i.e. HH;
4) But there was no longer a K which was 6 to 10 amino acids from KK (needed for the definition of a Replikin). [000275] Thus this Scaffold is no longer a Replikin Scaffold, but now is a Scaffold Exoskeleton so to speak. While Replikin Scaffolds are associated with high Replikin Counts and the occurrence of epidemics, Scaffold Exoskeletons are associated with virus dormancy and the reduction or end of the epidemic. Thus Scaffold Exoskeletons appear to be degenerative structures left as residues when Replikin Scaffolds and specific viral outbreaks are declining, thus a useful diagnostic structure for this purpose. This confirms the revelation and use of Replikin Scaffolds as 1) targets for anti-rapid replication agents such as antibodies or small inhibitory RNAs and 2) the basis of anti- viral vaccines. Software according to aspects of the present invention may comprise logic to obtain and analyze protein sequences to identify sequences having characteristics 1, 2, 3 and 4 above. For example, Scaffold Exoskeletons can now be detected and counted in any protein sequence by the exemplary software in Appendix D. [000276] Another non-limiting aspect in accordance with the present invention is a method of identifying a Replikin Scaffold comprising indentifying a series of peptides comprising about 17 to about 30 amino acids and further comprising
(1) identifying a terminal lysine;
(2) identifying a terminal histidine and another histidine in the residue potion immediately adjacent to the terminal histidine; (3) identifying at least one lysine within about 6 to about 10 amino acid residues from at least one other lysine; and (4) identifying at least about 6% lysines.
[000277] In a non-limiting aspect in accordance with the present invention the method of identifying a Replikin Scaffold may comprise identifying a single or plurality of individual members of the series of a Replikin Scaffold.
[000278] In a preferred non-limiting aspect in accordance with the present invention the method of identifying a Replikin Scaffold further comprises the identification of a second lysine immediately adjacent to the terminal lysine. Software according to aspects of the present invention may comprise logic to obtain and analyze protein sequences to identify sequences using steps 1, 2, 3 and 4 above. The TcI Programming Language
[000279] TcI (the "Tool Command Language," pronounced "tickle") is a simple interpreted scripting language that has its roots in the Unix command shells, but which has additional capabilities that are well-suited to network communication, Internet functionality and the rapid development of graphical user interfaces. TcI was created by John K. Ousterhout at the University of California at Berkeley in 1988. Originally conceived as a reusable, embeddable language core for various software tools, it is now widely used in applications including web scripting, test automation, network and system management, and in a variety of other fields. [000280] In aspects, Genome Explorer and Dr. Peptide may be coded in Tcl/Tk, a scripting programming language that includes powerful facilities for internet access, user interface design, and string manipulation. Because Tcl/Tk has been ported to nearly all available computer architectures and is familiar to those skilled in the art, programs written in Tcl/Tk can be run on nearly any operating system. Source code for specific implementations of Genome Explorer and Dr. Peptide are provided in Appendices A and D. The specific implementations are provided by way of illustration and example only, and the present invention is not in any way limited to the specific implementations illustrated.
OTHER USES OF THE THREE POINT RECOGNITION METHOD [000281] Since "3-point-recognition" is a proteomic method that specifies a particular class of proteins, using three or more different recognition points for other peptides similarly should provide useful information concerning other protein classes. Further, the "3-point- recognition" method is applicable to other recognins, for example to the TOLL 'innate' recognition of lipopolyssacharides of organisms. The three point recognition method may also be modified to identify other useful compounds of covalently linked organic molecules, including other covalently linked amino acids, nucleotides, carbohydrates, lipids or combinations thereof. In this aspect of the invention a sequence is screened for subsequences containing three or more desired structural characteristics. In the case of screening compounds composed of covalently linked amino acids, lipids or carbohydrates the subsequence of 7 to about 50 covalently linked units should contain (1) at least one first amino acid, carbohydrate or lipid residue located seven to ten residues from a second of the first amino acid, carbohydrate or lipid residue; (2) encoding at least one second amino acid, lipid or carbohydrate residue; and (3) at least 6% of the first amino acid, carbohydrate or lipid residue. In the case of screening nucleotide sequences, the subsequence of about 21 to about 150 nucleotides should contain (1) at least one codon encoding a first amino acid located within eighteen to thirty nucleotides from a second codon encoding the first amino acid residue; (2) at least one second amino acid residue; and (3) encodes at least 6% of said first amino acid residue. [000282] Several aspects of the present invention are specifically illustrated and described herein. However, it will be appreciated that modifications and variations of the present invention are encompassed by the above teachings and within the purview of the appended claims without departing from the spirit and intended scope of the invention. EXAMPLE 1 PROCESS FOR EXTRACTION, ISOLATION AND
IDENTIFICATION OF REPLIKINS AND THE USE OF REPLIKINS TO TARGET, LABEL OR DESTROY REPLIKIN-CONΥ AINING ORGANISMS a) Algae
[000283] The following algae were collected from Bermuda water sites and either extracted on the same day or frozen at -20 degrees C and extracted the next day. The algae were homogenized in a cold room (at 0 to 5 degrees C) in 1 gram aliquots in neutral buffer, for example 100 cc. of 0.005M phosphate buffer solution, pH 7 ("phosphate buffer") for 15 minutes in a Waring blender, centrifuged at 3000 rpm, and the supernatant concentrated by perevaporation and dialyzed against phosphate buffer in the cold to produce a volume of approximately 15 ml. The volume of this extract solution was noted and an aliquot taken for protein analysis, and the remainder was fractionated to obtain the protein fraction having a pK range between 1 and 4.
[000284] The preferred method of fractionation is chromatography as follows: The extract solution is fractionated in the cold room (4° C) on a DEAE cellulose (Cellex-D) column 2.5x11.0 cm, which has been equilibrated with 0.005M phosphate buffer. Stepwise eluting solvent changes are made with the following solutions:
Solution 1- 4.04 g. NaH2P04 and 0.5gNaH2P04 are dissolved in 15 litres of distilled water (0.005 molar, pH 7);
Solution 2 - 8.57 g. NaH2P04 is dissolved in 2,480 ml. of distilled water; Solution 3 - 17.1 g. of NaH2P04 is dissolved in 2480 ml of distilled water
(0.05 molar, pH 4.7);
Solution 4 - 59.65 g. of NaH2P04 is dissolved in 2470 ml distilled water (0.175 molar); Solution 5 - 101.6 g. of NaH2P04 is dissolved in 2455 ml distilled water
(pH 4:3);
Solution 6 - 340.2 g. of NaH2P04 is dissolved in 2465 of distilled water (1.0 molar, pX-i 4.1);
Solution 7 - 283.63 g. of 80% phosphoric acid (H3P04) is made up in 2460 ml of distilled water (1.0 molar, pH 1.0).
[000285] The extract solution, in 6 to 10 ml volume, is passed onto the column and overlayed with Solution 1, and a reservoir of 300 ml of Solution 1 is attached and allowed to drip by gravity onto the column. Three ml aliquots of eluant are collected and analyzed for protein content at OD 280 until all of the protein to be removed with Solution 1 has been removed from the column. Solution 2 is then applied to the column, followed in succession by Solutions 3, 4, 5, 6 aid 7 until all of the protein which can, be removed with each Solution is removed from the column. The eluates from Solution 7 are combined, dialyzed against phosphate buffer, the protein content determined of both dialysand and dialyzate, and both analyzed by gel electrophoresis. One or two bands of peptide or protein of molecular weight between 3,000 and 25,000 Daltons are obtained in Solution 7. For example the algae Caulerpa mexicana, Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpa verticillata, Halimeda tuna, and Penicillos capitatus, after extraction and treatment as above, all demonstrated in Solution 7 eluates sharp peptide bands in this molecular weight region with no contaminants. These Solution 7 proteins or their eluted bands are hydrolyzed, and the amino acid composition determined. The peptides so obtained, which have a lysine composition of 6% or greater are Replikin precursors. These Replikin peptide precursors are then determined for amino acid sequence and the Replikins are determined by hydrolysis and mass spectrometry as detailed in U.S. Patent 6,242,578 Bl. Those that fulfill the criteria defined by the " 3-point-recognition" method are identified as Replikins. This procedure can also be applied to obtain yeast, bacterial and any plant Replikins. b) Virus
[000286] Using the same extraction and column chromatography separation methods as above in a) for algae, Replikins in virus-infected cells are isolated and identified. c) Tumor cells in vivo and in vitro tissue culture
[000287] Using the same extraction and column chromatography separation methods as above in a) for algae, Replikins in tumor cells are isolated and identified. For example, Replikin precursors of Astrocytin isolated from malignant brain tumors, Malignin (Aglyco lOB) isolated from glioblastoma tumor cells in tissue culture, MCF7 mammary carcinoma cells in tissue culture, and P3 J Lymphoma cells in tissue culture each treated as above in a) yielded Replikin precursors with lysine content of 9,1%, 6.7%, 6.7%, and 6.5% respectively. Hydrolysis and mass spectrometry of Aglyco 1OB as described in Example 10 U.S. 6,242,578 Bl produced the amino acid sequence, ykagvaflhkkndiide the 16-mer Replikin. EXAMPLE 2:
[000288] As an example of diagnostic use of Replikins: Aglyco 1OB or the 16- mer Replikin may be used as antigen to capture and quantify the amount of its corresponding antibody present in serum for diagnostic purposes are as shown in Figures 2, 3, 4 and 7 of U.S. 6,242,578 Bl.
[000289] As an example of the production of agents to attach to Replikins for labeling, nutritional or destructive purposes: Injection of the 16-mer Replikin into rabbits to produce the specific antibody to the 16-mer Replikin is shown in Example 6 and Figures 9A and 9B of U.S. 6,242,578 Bl. [000290] As an example of the use of agents to label Replikins: The use of antibodies to the 16-mer Replikin to label specific cells which contain this Replikin is shown in Figure 5 and Example 6 of U.S. 6,242,578 Bl . [000291] As an example of the use of agents to destroy Replikins: The use of antibodies to the 16-mer Replikin to inhibit or destroy specific cells which contain this Replikin is shown in Figure 6 of U.S. 6,242,578 Bl. EXAMPLE 3
[000292] Analysis of sequence data of isolates of influenza virus hemagglutinin protein or neuraminidase protein for the presence and concentration of Replikins is carried out by visual scanning of sequences or through use of a computer program based on the 3-point recognition system described herein. Isolates of influenza virus are obtained and the amino acid sequence of the influenza hemagglutinin and/or neuraminidase protein is obtained by any art known method, such as by sequencing the hemagglutinin or neuraminidase gene and deriving the protein sequence therefrom. Sequences are scanned for the presence of new Replikins, conservation of Replikins over time and concentration of Replikins in each isolate. Comparison of the Replikin sequences and concentrations to the amino acid sequences obtained from isolates at an earlier time, such as about six months to about three years earlier, provides data that are used to predict the emergence of strains that are most likely to be the cause of influenza in upcoming flu seasons, and that form the basis for seasonal influenza peptide vaccines or nucleic acid based vaccines. Observation of an increase in concentration, particularly a stepwise increase in concentration of Replikins in a given strain of influenza virus for a period of about six months to about three years or more is a predictor of emergence of the strain as a likely cause of influenza epidemic or pandemic in the future.
[000293] Peptide vaccines or nucleic acid-based vaccines based on the Replikins observed in the emerging strain are generated. An emerging strain is identified as the strain of influenza virus having the highest increase in concentration of Replikin sequences within the hemagglutinin and/or neuraminidase sequence during the time period. Preferably, the peptide or nucleic acid vaccine is based on or includes any Replikin sequences that are observed to be conserved in the emerging strain. Conserved Replikins are preferably those Replikin sequences that are present in the hemagglutinin or neuraminidase protein sequence for about two years and preferably longer. The vaccines may include any combination of Replikin sequences identified in the emerging strain.
[000294] For vaccine production, the Replikin peptide or peptides identified as useful for an effective vaccine are synthesized by any method, including chemical synthesis and molecular biology techniques, including cloning, expression in a host cell and purification therefrom. The peptides are preferably admixed with a pharmaceutically acceptable carrier in an amount determined to induce a therapeutic antibody reaction thereto. Generally, the dosage is about 0.1 mg to about 10 mg. [000295] The influenza vaccine is preferably administered to a patient in need thereof prior to the onset of "flu season." Influenza flu season generally occurs in late October and lasts through late April. However, the vaccine may be administered at any time during the year. Preferably, the influenza vaccine is administered once yearly, and is based on Replikin sequences observed to be present, and preferably conserved in the emerging strain of influenza virus. Another preferred Replikin for inclusion in an influenza vaccine is a Replikin demonstrated to have re-emerged in a strain of influenza after an absence of one or more years. EXAMPLE 4
[000296] Analysis of sequence data of isolates of coronavirus nucleocapsid, or spike, or envelope, or other protein for the presence and concentration of Replikins is carried out by visual scanning of sequences or through use of a computer program based on the 3-point recognition method described herein. Isolates of coronavirus are obtained and the amino acid sequence of the coronavirus protein is obtained by any method known in the art, such as by sequencing the protein's gene and deriving the protein sequence therefrom. Sequences are scanned for the presence of new Replikins, conservation of Replikins over time and concentration of Replikins in each isolate. Comparison of the Replikin sequences and concentrations to the amino acid sequences obtained from isolates at an earlier time, such as about six months to about three years earlier, provides data that are used to predict the emergence of strains that are most likely to be the cause an outbreak or pandemic, and that form the basis for coronavirus peptide vaccines or nucleic acid based vaccines. Observation of an increase in concentration, particularly a stepwise increase in concentration of Replikins in a given class, or strain, of coronavirus for a period of about six months to about three years or more is a predictor of emergence of the strain as a likely cause of an epidemic or pandemic, such as SARS, in the future. [000297] Peptide vaccines or nucleic acid-based vaccines based on the Replikins observed in the emerging strain of coronaviruses are generated. An emerging strain is identified as the strain of coronavirus having the highest increase in concentration of Replikin sequences within the nucleocapsid sequence during the time period. Preferably, the peptide or nucleic acid vaccine is based on or includes any Replikin sequences that are observed to be conserved in the strain. Conserved Replikins are preferably those Replikin sequences which are present in the nucleocapsid protein sequence for about two years and preferably longer. The vaccines may include any combination of Replikin sequences identified in the emerging strain. [000298] For vaccine production, the Replikin peptide or peptides identified as useful for an effective vaccine are synthesized by any method, including chemical synthesis and molecular biology techniques, including cloning, expression in a host cell and purification therefrom. The peptides are preferably admixed with a pharmaceutically acceptable carrier in an amount determined to induce a therapeutic antibody reaction thereto. Generally, the dosage is about 0.1 mg to about 10 mg. [000299] The coronavirus vaccine may be administered to a patient at any time of the year. Preferably, the coronavirus vaccine is administered once and is based on Replikin sequences observed to be present, and preferably conserved, in the classes of coronavirus. EXAMPLE 5
[000300] Analysis of sequence data of isolates of Plasmodium falciparum antigens for the presence and concentration of Replikins is carried out by visual scanning of sequences or through use of a computer program based on the 3-point recognition method described herein. Isolates of Plasmodium falciparum are obtained and the amino acid sequence of the protein is obtained by any art known method, such as by sequencing the gene and deriving the protein sequence therefrom. Sequences are scanned for the presence of Replikins, conservation of Replikins over time and concentration of Replikins in each isolate. This information provides data that are used to form the basis for anti-malarial peptide vaccines or nucleic acid based vaccines.
[000301] Peptide vaccines or nucleic acid-based vaccines based on the Replikins observed in the malaria causing organism are generated. Preferably, the peptide or nucleic acid vaccine is based on or includes any Replikin sequences that are observed to be present on a surface antigen of the organism. The vaccines may include any combination of Replikin sequences identified in the malaria causing strain. [000302] For vaccine production, the Replikin peptide or peptides identified as useful for an effective vaccine are synthesized by any method, including chemical synthesis and molecular biology techniques, including cloning, expression in a host cell and purification therefrom. The peptides are preferably admixed with a pharmaceutically acceptable carrier in an amount determined to induce a therapeutic antibody reaction thereto. Generally, the dosage is about 0.1 mg to about 10 mg. [000303] Then malaria vaccine is preferably administered to a patient in need thereof at any time during the year, and particularly prior to travel to a tropical environment. [000304] Another aspect includes an antisense nucleic acid molecule complementary to the coding strand of the gene or the mRNA encoding organism for the replikins in organisms including, but not limited to, viruses, trypanosomes, bacteria, fungi, algae, amoeba, and plants, wherein said antisense nucleic acid molecules is complementary to a nucleotide sequence of a replikin containing organism. EXAMPLE 6
[000305] Amino acid sequences of five short SARS Replikins found in nucleocapsid, spike, and envelope proteins of the SARS coronavirus were synthesized and tested on rabbits to test immune response to Replikin sequences in the SARS coronavirus. The following Replikin sequences were tested: (1) 2003 Human SARS nucleocapsid (SEQ ID NO: 712); (2) 2003 Human SARS spike protein (SEQ ID NO: 717); (3) 2003 Human SARS spike protein (SEQ ID NO: 718); 2003 Human SARS spike protein; (SEQ ID NO: 719); (4) 2003 SARS envelope protein (SEQ ID NO: 720); and (5) 2003 Human SARS nucleocapsid protein (SEQ ID NO: 743). Each synthesized peptide was injected subcutaneously into a rabbit. The tested rabbits produced measurable specific antibody to each of the five sequences that bound at dilutions of greater than 1 in 10,0000. The 21 amino acid SARS nucleocapsid replikin antibody (SEQ. ID NO: 712) was demonstrated to bind at dilutions greater than 1 in 204,800. Because of previous unsuccessful attempts by others to achieve with various small peptides a strong immune response without the unwanted side effects obtained with a whole protein or the thousands of proteins or nucleic acids as in smallpox vaccine, the ability of small synthetic replikin antigens to achieve strong immune responses was shown to be significant for the efficacy of SARS vaccines. EXAMPLE 7
[000306] A 43 amino acid replikin sequence
KNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK-KLH designated Vaccine V120304U2 was designed by the inventors from the 29 amino acid replikin Scaffold of H5N1 "Bird Flu" Influenza Replikins labeled "2004 H5N1 Vietnam, highly pathogenic" in Table 8 with the addition of two UTOPE units (KKKKHK) on the C-terminal end of the H5N1 scaffold and an additional adjuvant (key limpet hemocyanin (sequence KLH)) covalently linked on the C-terminal end of the two UTOPE units. 100 ug of Vaccine V120304U2 was injected subcutaneously into rabbits and chickens . The antibody response was measured before vaccination and at from one week after injection to eight weeks after injection. An antibody response was noted at one week and reached a peak in the third to fourth week after vaccination. Peak antibody responses ranged from a dilution of 1 -.120,000 to a dilution of greater than 1 :240,000. Antibody titers were determined with an enzyme linked immunosorbent assay (ELISA) with Peptide-GGG (goat gamma globulin) bound in solid phase (0.1 ug/100 ul/well) on high binding 96 well plates. The serum was first diluted 50 fold and then further diluted in 2-fold serial dilutions. The ELISA titer result was determined from the estimated dilution factor that resulted from an optical density at 405 nm of 0.2 and derived from nonlinear regression analysis of the serial dilution curve. Detection was obtained using a horse radish peroxidase conjugated secondary antibody and ABTS substrate (ABTS is a registered trademark of Boehringer Mannheim. GmbH). Results from tests on two chickens and two rabbits are provided in Table 11. Individual well results from the test on rabbit D4500 are provided in Table 12. In combination with the results reported in Example 6, in a total of six tests of Replikin sequences for antibody responses in rabbit or chicken, all six sequences provided a measurable antibody response and have proved antigenic.
Table 11
Chickens injected with 100 μg V120304U2 on day 1. ELISA titer of antibody production on da 18
Figure imgf000113_0001
Rabbits injected with 100 μg V120304U2 on day 1. ELISA titer of antibody production on day 20
Figure imgf000113_0002
Table 12
Rabbits injected with 100 μg V120304U2 on day 1. OD450 results for titers on days
7, 20 and 28 in individual wells
Figure imgf000114_0001
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# #
# GENOME-EXPLORER #
# # ##################################################################### #
# Copyright (C) 2003 by Samuel Bogoch and Elenore Bogoch
# All rights reserved.
# The basic design style is aggunlative. Originally a batch tool to do the search,
# with a Tk gui tacked on, and then various parts, such as being able to stop a
# search, additional criteria, etc. set Contents [file dir [file dir [lindex $tcl_libPath 0] ] ] set env(PATH) $env(PATH) :$Contents/Resources/bin lappend env(TCLLIBPATH) $Contents/Resources/lib set versionDate 2004-01-24 proc : : tk : :mac : : OpenDocument {args } { global Contents set files {} set paths { } foreach document $args { set path [exec osascript -e "get POSIX path of file \"$document\""] if {[string equal [file extension $path] .tcl]} { lappend files [file tail $path] lappend paths $path } if {[string equal [tk_messageBox -message "Install upgrade: [join $files {, }]?" -type yesno -title "Install Upgrade"] yes] } { foreach path $path { file copy -force $path $Contents/Resources/Scripts/ [file tail $path]
} exit
} proc loadParser { } { global env Contents if {[catch {load [lindex $env(TCLLIBPATH) end] /ncbiasn.dylib} rs] } { log "load ncbiasn.dylib failed: $rs" uplevel #0 source "$Contents/Resources/Scripts/asn-parser . tcl" log "sourced asn-parser.tcl" } else { log "loaded ncbiasn.dylib" } after idle loadParser set logFile "~/Library/Preferences/com. omyx.genome.log" proc log message { global log logFile puts $message if { ! [info exists log] } {set log [open $logFile w] } set message "[clock format [clock seconds] -format {%Y/%m/%d %H:%M:%S}]. $message" puts $log $message flush $log catch {
.log.t.t insert end $message\n . log . t . t see end
}
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# #
# PREFERENCES #
# # ##################################################################### array set preference {debuggingMode 0 outputDirectory -/Desktop openHTML 0} catch {source $prefsFile} if {[catch {cd $ρreference (outputDirectory) }]} { cd -/Desktop set preference (outputDirectory) -/Desktop set ch [open $prefsFile w] puts $ch "array set preference {" foreach {p v} [array get preference] { puts $ch " [list $p] [list $v] " puts $ch " } " close $ch
} foreach p [lsort [array names preference] ] { log "preference $p = $preference ($p) " } proc : : tk : : mac : : ShowPreferences { } { global preference preferencel toplevel .preferences wm title .preferences Preferences wm geometry .preferences 400x150+100+100 array set : :preferencel [array get : :preference] place [ label .preferences.lwd -text "Working directory:" -anchor e ] -x 0 -y 0 -relwidth 0.45 -height 25 place [ button .preferences .bwd -textvar preferencel (outputDirectory) -anchor w -command { set T [tk_chooseDirectory -initialdir $preferencel (outputDirectory) \
-title "Output Directory" -mustexist 0] if { [string length $T] } { set preferencel (outputDirectory) $T }
] -relx .50 -y 0 -relwidth 0.45 -height 25 place [ label .preferences .ldebug -text "Save fetched pages:" -anchor e ] -x 0 -y 30 -relwidth 0.45 -height 25 place [ checkbutton .preferences .cdebug -text " " -variable preferencel (debuggingMode) ] -relx .50 -y 30 -relwidth 0.45 -height 25 place [ label .preferences, lcache -text "Purge query cache:" -anchor e ] -x 0 -y 55 -relwidth 0.45 -height 25 set cachesize [llength [glob -nocomplain ".cache/*"]] place [ button .preferences.bcache -text "Purge $cachesize pages" -command { catch { foreach fn [glob -nocomplain ".cache/*" " .cache/ .query" { . cache/ . [A- S] * } ] {file delete -force $fn}
}
.preferences. bcache configure -state disabled
}
] -relx .50 -y 55 -relwidth 0.45 -height 25 if { ! $cachesize} {
.preferences.bcache configure -state disabled
} place [ label .preferences. lopen -text "Open results in browser:" -anchor e ] -x 0 -y 80 -relwidth 0.45 -height 25 place [ checkbutton .preferences, copen -text " " -variable preferencel (openHTML) ] -relx .50 -y 80 -relwidth 0.45 -height 25 place [ button .preferences. cancel -text Cancel -command { destroy .preferences unset -nocomplain preferencel
}
] -x 0 -rely 1.0 -y -35 -width 100 place [ button .preferences. reset -text Reset -command { array set preferencel [array get preference]
} ] -relx 1.0 -x -210 -rely 1.0 -y -35 -width 100 button . pref erences . okay - text Okay - co-imand { array set pref erence [array get pref erencel] f ile mkdir $preference ( outputDirectory) cd $pref erence ( outputDirectory) set ch [open $pref sFile w] puts $ch ' array set preference { " f oreach (p v} [array get pref erence] { puts $ch " [list Sp] [list $v] ;
} puts $ch ' } close $ch destroy .preferences unset -nocomplam preferencel foreach p [lsort [array naπes preference] ] { log 'preference $p = $preference ($p) '
] -relx 1.0 -x -100 -rely 1.0 -y -35 -width 100 place [ button .preferences crap -text " -command
} "
] -relx 1.0 -x -100 -rely 1.0 -y -35 -width 100 lower .preferences . crap
≠ #
# INITIALISE GLOBALS #
# #
set seen 0; # Nunber of entries examined. set pass 0; # Nurber of those passing the criteria. set fail 0; # Number of those failing the criteria, set stop 0; # If the search was terminated, array set replikinRef { } ; # All references to each specific replikin, array set coding {
GCT a GCC a GCA a GCG
TGT b TGC c
GAT d GAC d
GAA e GAG e
TTT f TTC f
GGT g GGC g GGA g GGG g
CAT h CAC h
ATT i ATC i ATA i
AAA k AAG k
TTG l TTA l CTT l CTC k CTA l CTG l
ATG m
AAT n AAC n
CCT p CCC p CCA p CCG P
CAA q CAG q
CGT r CGC r CGA r CGG r AGA r AGG r
TCT s TCC s TCA s TCG s AGT s AGC s
ACT t ACC ACA t ACG t
GTT v GTC v GTA v GTG v
TGG w
TAT y TAC y
TAA * TAG * TGA *
set db "NCBI ; # Data base searched, NCBI or LANL. set query "", # T.-.e query string. set passpercent " " , # 100 * pass/seen. set failpercent ' ' ; # 100 * fail/seen
<t ≠ if DATABASE QUERY # f % if
A-I 115 # contains, variously, returned HTML pages, parsed sequence data,
# and the query term that fetched all these pages. #
# Drive the query. If the query term has changed from that cached
# term or there is no cache, the cache is cleared and new pages are
# fetched from NCBI or LANL as requested. The term is sent to the
# query page and the initial response parsed. If there are more 20
# returns, the query is repeated with the all the returns requested. #
# Each return is a short paragraph which is parsed for the accession
# number and sequence data page url. The page is fetched and saved. #
# The second pass then parses the fetched pages . NCBI and LANL
# pages have different parsers. #
# If the third pass if the cache had to be filled, or the first
# pass if the cache was okay and no pages were fetched, the parsed
# sequence data is checked against the criteria. #
# Matching goes through three steps. (1) The query term is used
# to fetch sequence data from NCBI or LANL. Only pages fetched
# contribute to the 'seen' count. (2) The peptide sequence is
# compared to repilikin criteria. (3) Those matching can then be
# passed through the additional criteria, if any. proc GenomalEnquirer {database term additionalCriteria} { global seen pass fail stop failing coding replikinRef global kmin kmax hmax percent log "GenomalEnquirer database=$database term='$term' additionalCriteria=' $additionalCriteria' " set seen 0 set pass 0 set fail 0 set stop 0 status " " 0 0 array unset replikinRef * set failing [open fail.htm w] puts $failing {<htmlxheadxtitle>Failing Sequences</titlex/headxbody>} puts $failing "<pxb>query: </b> $term" regsub -all {\s+} $term {+} term set queryerror 0 if {[catch {open . cache/ . query r} fd] } { set fetching 1 } else { foreach {olddatabase oldterm} [read $fd] break close $fd set fetching [expr { ! [string equal $olddatabase $database] | | ! [string equal $oldterm $term] }]
} if {$fetching} { set previousTerm $term package require http file mkdir .cache foreach fn [glob -nocomplain ".cache/*" ". cache/ . query" ] {file delete -force $fn} set re [catch { while 1 { status "Fetching from $database" 0 0 update; if {$stop} {status "Stopped." 0 0; return} switch $database { NCBI { set url http : //www . ncbi . nlm . nih. gov : 80/entrez/query. fcgi?cmd=Search&db=Protein&term=$termS:doptcmdl=Doc Sum
}
LANL { set url http: //www. flu. lanl.gov/search2/resultN, html?search=l&field=ALL&num=20&hspecies=Any&seg=any&nu corpro=nuc&orderby=dateasc&text=$term
} set token [ : : http : : geturl $url] set queryPage [;: http :: data $token]
:: http: : cleanup $token log "got [string length $queryPage] bytes" set fn [open query.htm w] puts $fn $queryPage close $fn if { [regexp {Your request could not be processed due to a problem} $queryPage] } status "Server busy, retrying in 5 seconds..." 0 0 after 5000 continue
} switch $database { NCBI { regexp {<td align="center" width="50%"><div class="? (?:pager|medium2) "?>Items l-[0-9]+ of ( [0-9] +) </divx/td>} $queryPage all numentries
LANL { regexp {Got <B> ( [0-9] +) </B> hits total} $queryPage all numentries } } if { ! [info exists numentries] || [string equal $numentries ""] || $numentries==θ} { status "Nothing matched the query." 0 0 return
} if {$numentries>20} { log "Fetching $numentries entries from $database" update; if {$stop} {status "Stopped." 0 0; return} status "Fetching $numentries queries from $database." 0 0 switch $database { NCBI { set token [ : : http : : geturl
"http://www.ncbi.nlm.nih.gov: 80/entrez/query. fcgi?SUBMIT=y" \
-query [ : :http: : formatQuery \ db Protein \ term [string map {+ { }} $term] \ dispmax $numentries \ ]]
}
LANL { set token [ : : http : : geturl
"http: //www. flu. lanl.gov/search2/resultN. html?search=l&field=ALL&num=$numentries&hspecies=Any& seg=any&nucorpro=nuc&orderby=dateasc&text=$term"]
} set queryPage [ : : http : : data $token]
: : http : : cleanup $token log "got [string length $queryPage] bytes" if { $ : preference (debuggingMode) } { set fn [open query.htm w] puts $fn $queryPage close $fn
} if { [regexp {Your request could not be processed due to a problem} $queryPage] } { status "Server busy: retrying in 5 seconds..." 0 0 after 5000 continue } } break
} set work { } set queryPage [string map {»\»» '} $queryPage] file mkdir . cache/ . $database switch $database { NCBI { foreach {uselesslink relativeurl accessionnumber} [regexp -all -inline {<a href=' (/entrez/viewer . fcgi [?] db=protein&val= [Λ<>' ] *) ' > ( [Λ<] *) </a>} $queryPage] \
{ set accessionurl http://www.ncbi.nlm.nih.gov: 80$relativeurl set accessionlink "<a href=' $accessionurl ' >$accessionnumber</a>" lappend work $accessionlink $accessionurl $accessionnumber foreach {accessionlink aσcessionurl accessionnumber} \
[regexp -all -inline { <A HREF= 1 (view_record . html [?] [Λ ' <>] * ) ' [A<>] *> ( [Λ<] * ) </A> } $queryPage] \
{ set accessionurl "http: //www. flu. lanl .gov/search2/$accessionurl" set accessionlink [string map ("HREF=1" "HREF= ' http : //www . flu. lanl . gov/search2/ " } $accessionlink] lappend work $accessionlink $accessionurl $accessionnumber
} , ' while { [llength $work]>θ} { set working $work set work { } set i 0; set n [expr { [llength $working] /3}] foreach {accessionlink accessionurl accessionnumber} $working { if {$stop} {status "Stopped." 0 0; return} if { ! [info exists repeat ($accessionnumber) ]} { set repeat ($accessionnumber) 0
} incr i; status "Fetching $accessionnumber" $i $n set aσcessionurl [string map { Scamp; &} $accessionurl] set token [ : :http: :geturl [string map {query. fcgi viewer. fcgi} $accessionurl] &view=asn] set page [:: http :: data $token]
::http: : cleanup $token if {$: :preference(debuggingMode) } { set fn [open page.htm w] puts $fn $page close $fn
} if { [regexp {try again later} $page] } { lappend work $accessionlink $accessionurl $accessionnumber log "server busy. $accessionnumber" continue
} set ch [open . cache/ . $database/$accessionnumber w] puts $ch [list $accessionlink $accessionurl $page] close $ch
} if {[llength $work]>0} { status "Retrying [expr {[llength $work]/3}] queries in 5 seconds..." 0 0 after 5000 } set fd [open . cache/ . query w] puts $fd [list $database $term] close $fd } rs] set queryerror $rc if {$rc ScSc ! [string equal {couldn't open socket: host is unreachable} $rs] } { error $rs } } set paths [glob -nocomplain -join .cache {. [A-Z] *} *] set i 0; set n [llength $paths] foreach path $paths { if {$stop} {status "Stopped." 0 0; return} set databasel [file tail [file dirname $path] ] set accessionnumber [file tail $path] incr i; status "Parsing $accessionnumber" $i $n set ch [open $path r] set re [catch {foreach {accessionlink accessionurl page} [read $ch] break}] close $ch if {$rc} { file delete $path continue
} unset -nocomplain PAGE set PAGE (%accession) $accessionnumber set PAGE(%link) $accessionlink set PAGE(%uri) $accessionurl switch $databasel { .NCBI { if {$starts>=0} { incr starts 5 set stops [string first "</pre>" $page $starts] if {$stops>0} {incr stops -l} else {set stops end} set page [string range $page $starts $stops] array set PAGE [ncbiasn $page] set PAGE(fcseqdata) {} foreach {p seqdata} [array get PAGE * .seq-data.ncbieaa.*J { set q [string map {inst . seq-data.ncbieaa id.0.ddbj .accession} $p] if {[info exists PAGE($q)] && ! [string equal $PAGE($q) $accessionnumber] \ continue regsub -all {\s+} $seqdata {} seqdata lappend PAGE (%seqdata) [string tolower $seqdata] if { [llength $PAGE (%seqdata) ] ==0} { log "sequence-data not found: $accessionnumber" continue set PAGE(%definition) {} foreach {p v} [array get PAGE *.descr.* .article. title.* .name. >] {lappend PAGE(%definition) $v} set PAGE(%definition) [join $PAGE (%definition) " "] set PAGE{%source) unknown foreach {p v} [array get PAGE * .source. org.taxname. >] { set PAGE{%source) $v
} set PAGE (%strain) unknown foreach {p v} [array get PAGE *. subtype. >] { if { [string equal $v strain] } { set PAGE(%strain) $PAGE ( [string map {. subtype. > .subname.>} $p] )
} if { [string equal $PAGE (%strain) unknown]
&& [regexp { \ ( ( [Λ { ) ] * (\ ( [Λ ( ) ] *\) ) ? ) \) \s*$ } $PAGE (%source) -> strain]
} { set PAGE (%strain) $strain
} set slash [string last / $PAGE (%strain) ] if {$slash>=0 && [regexp -start $slash {/([0-9]+)} $PAGE (%strain) -> y] } { if {$y<30} {incr y 2000} elseif {$y<100} {incr y 1900} set PAGE(%year) $y } else { set PAGE(%year) unknown
} foreach {p x} [array get PAGE *. subtype. >] { if { [string equal $x isolate] } { set p [string map {. subtype. > .subname.>} $p] if {[info exists PAGE($p)]} { set dates [regexp -inline -all {/(\d+)} $PAGE($p)] if {[llength $dates]>θ} { set y [lindex $dates end] regexp {*[*/]+} $PAGE($p) PAGE(%country) if {$y<30} {incr y 2000} elseif {$y<100} {incr y 1900} if ($PAGE(%year) eq "unknown" || $PAGE (fcyear) >$y} {set PAGE(%year) $y}
}
} foreach {p y} [array get PAGE *.year.>] { if {$y<30} {incr y 2000} elseif {$y<100} {incr y 1900} if j$PAGE(%year) eq "unknown" || $PAGE (%year) >$y} {set PAGE(fcyear) $y) if {[regexp {\ ( ( T 0 ] +) \) \s*$} $PAGE(%strain) -> s] } { set PAGE (%serotype) $s } elseif {[regexp {\ ( ( [Λ () ] +) \) \) \s*$} $PAGE (%source) -> s] } { set PAGE(%serotype) $s } else { set PAGE(%serotype) unknown foreach {p x} [array get PAGE *. subtype. >] { if { [string equal $x serotype] } { set p [string map {. subtype. > ,subname.>} $p] if {[info exists PAGE($p)]} { break } } }
. LANL { foreacϊi {all attribute value} \
[regexp -all -inline { <tr><tdxspan class="bold" > ( [Λ<>] +) </spanx/td>\s*<td> ( { ? : [A<] | < [Λ/] | </ [At] | </t [Ad] | </td p>] ) * ) </td>\s*</tr> } $page] \
{ regsub -all {<[Λ<>]*>} $value {} value set value [string trim [string map {fcribsp; " " fcamp,- & &lt; < &gt,- > &apos; ' fcquot " \ " " } $value] ] regsub -all {\s+} $value { } value set PAGE ( [string map {" " -} $attribute] ) $value switch $attribute {
Strain {set PAGE (%strain) $value} Definition {set PAGE (%definition) $value} Source {set PAGE (%source) $value} "Collection Year" {set PAGE(%year) $value} Serotype {set PAGE (%serotype) $value} "Raw Sequence" { regsub -all {\s+} $value {} value set PAGE(%seqdata) "" for {set c 0} {$c< [string length $value] } {incr c 3} { set codon [string range $value $c [expr {$c+2}]] if {[info exists coding ($codon) ]} { append PAGE (%seqdata) $coding($codon) } else { append PAGE (fcseqdata) ? }
} } set ch [open . cache/$accessionnumber w] foreach {p v} [array get PAGE] { puts $ch " [list $p] [list $v] "
} close $ch file delete $path } set paths [glob -nocomplain ".cache/*"] set i 0; set n [llength $paths] if {$n==0 ScSc $queryerror} { error "could not connect to $database"
} foreach path $paths { set accessionnumber [file tail $path] if {$stop} {status "Stopped." 0 0; return} incr i status "Searching $accessionnumber" $i $n unset -nocomplain PAGE ' set ch [open .cache/$accessionnumber r] array set PAGE [read $ch] close $ch if { ! [info exists PAGE(%kmin)]
$PAGE(%kmin) !=$kmin
$PAGE(%kmax) !=$kmax
$PAGE(%hmax) !=$hmax
$PAGE (%percent) !=$percent
} { set results { } set sequence { } foreach sequence $PAGE (%seqdata) { set result [match $sequence] if { [llength $result] } { lappend results [list [llength $result] $result $sequence] } set results [lsort -integer -index 0 $results] set PAGE(%result) [lindex $results end 1] set PAGE(%sequence) [lindex $results end 2] } else { set PAGE(%result) {} set PAGE(%sequence) {}
} array set PAGE [list \
%kmin $ktnin \
%kmax $kmax \
%hmax $hmax \
%percent $percent \ ]
} incr seen set PAGE(%accept) [expr { [llength $PAGE(%result) ] >o}] if {$PAGE(%accept) } { foreach {kind descr string} $additionalCriteria { if { ! [string equal $kind =] } continue catch {unset exists} foreach {p extent} [array get PAGE $descr] { regsub -all { [Λ- [:alnum:] @%\ (\)_+=/3 +} $extent { } extent foreach word [split [string tolower [string trim $extent] ] " "] { set exists ($word) 1 } } regsub -all { [Λ- [ :alnum: ] @%\ (\)_+=/] +} $string { } string set any 0 foreach word [concat [split [string tolower [string trim $string] ] " ' unknown] { set any [info exists exists ($word) ] if {$any} break
} if {!$any} {set PAGE (%accept) 0; break}
} if {$PAGE(%accept) } { foreach {kind descr string} $additionalCriteria { if { [string equal $kind =] } continue set PAGE (%accept) 1 catch {unset exists} foreach {p extent} [array get PAGE $descr] { regsub -all { [Λ- [ :alnum :]_]+} $extent { } extent foreach word [split [string tolower [string trim $extent] ] " "] { set exists ($word) 1 } regsub -all { [A- [:alnum:]_] +} $string { } string foreach word [split [string tolower [string trim $string] ] " "] { if $kind[info exists exists ($word) ] { set PAGE (%accept) 0; break
} } if {$PAGE(%accept) } break
} set ch [open . cache/$accessionnumber w] foreach {p v} [array get PAGE] { puts $ch " [list $p] [list $v] "
} close $ch if {$PAGE(%accept) } { reportStep [pass] incr pass } else { fail incr fail } update aasequenceHistoryReport replikinReport close $failing status "Completed." 1 1 proc reportstep {r} { upvar 1 PAGE PAGE upvar 1 mindate mindate upvar 1 maxdate maxdate upvar 1 nrep nrep upvar 1 sumrep sutnrep upvar 1 sumsqrep sumsqrep upvar 1 reference reference if {[[string equal $PAGE(%year) unknown]} { if { ! [info exists mindate] || $PAGE (%year) <$mindate} {set mindate $PAGE(%year) } if { ! [info exists maxdate] jj $PAGE(%year) >$raaxdate} {set maxdate $PAGE(%year) }
} if { I [info exists nrep ($PAGE (%year) ) ] } { set nrep($PAGE (%year) ) 0 set sumrep ($PAGE (%year) ) 0.0 set sumsqrep ($PAGE (%year) ) 0.0 set reference ($PAGE (%year) ) ""
} incr nrep($PAGE (%year) ) 1 set sumrep ($PAGE (%year) ) [expr {$sumrep($PAGE (%year) ) + $r}] set sumsqrep ($PAGE (%year) ) [expr {$sumsqrep ($PAGE (%year) ) + $r*$r}] append reference ($PAGE (%year) ) "\n$PAGE(%link) <a href='pass/$PAGE(%accession) .htm'> [llength $PAGE(%result) ] </a>" } proc aasequenceHistoryReport {} { global replikinRef file mkdir replikin-ref foreach fn [glob -nocomplain "replikin-ref/*"] { if {[catch {file delete $fn} rs] } {puts $rs} foreach aasequence [lsort [array names replikinRef *.fid]] { set f [open $replikinRef ($aasequence) wj set aasequence [lindex [split $aasequence .] 0] log "Writing sequence history: $aasequence" puts $f "<htmlxheadxtitle>Replikin History</titlex/headχbody>
<Hl>Replikins, Inc.</Hl>
<H2>Sequence History by Year</H2>
<H2>$aasequence</H2>
<p>All occurences of the sequence by year:</p>
<dl> foreach x [lsort [array names replikinRef $aasequence : *] ] { puts $f "<dt> [lindex [split $x : ] 1] </dtxdd> [join $replikinRef ($x) { , } ] . </dd>" puts $f " </dl; close $f
} proc replikinReport { } { upvar 1 term term upvar 1 mindate mindate upvar 1 maxdate maxdate upvar 1 nrep nrep upvar 1 sumrep sumrep upvar 1 sumsqrep sumsqrep upvar 1 reference reference if { [info exists mindate] } { catch {file delete -force $term.htm} log "Writing report $tertn.htm" set stats [open $term.htm w] puts $stats "<htmlxheadxtitle>Replikin Analysis</titlex/headxbody> <Hl>Replikins, Inc.</Hl> <H2>Replikin Count by Year</H2> <H3> [string map {+ { }} $term]</H3> <TABLE> <TR>
<TH align=' center' valign= ' top1 >Year</TH>
<TH align= ' center ' valign= ' top ' >PubMed Accession Number-Replikin Count</TH>
<TH align-:' center' valign= ' top' >No . of Isolates per year</TH>
<TH align=' center' valign=' top' >Mean Replikin Count per year</TH> </TR> set Y {} if {[info exists nrep (unknown) ]} {lappend Y unknown} for {set y $mindate} {$y<=$maxdate} {incr y} {lappend Y $y} foreach y $Y { if { [info exists nrep ($y) ] } { set mean [expr { $sumrep ($y) /$nrep ($y) } ]
# var = (sum (x - m)A2)/(n-l)
# = (sum (xA2 - 2xm + mA2))/(n-l)
# = (sum xΛ2 - 2m sum x + mA2 sum l)/(n-l)
# = (sumsq - 2*m*sum + n mΛ2)/(n-l) if {$nrep($y)==l} { set sd 0.0 } else { if {[catch {expr {sqrt (abs ( ($sumsqrep($y) + $nrep($y) *$mean*$mean - 2*$mean*$sumrep($y))/($nrep($y) -1) ) ) }} sd] } { set sd 0.0 } puts $stats "<TR>
<TD align= ' center ' valign=' top' >$y</TD> <TD align='left' valign=' top' >$reference ($y) </TD> <TD align=' right' valign=' top' >$nrep($y) </TD> <TD align= 'right' valign=' top' > [format %,lf $mean] </TD> <TD align= ' right ' valign=' top' > [format %.lf $sd] </TD> </TR>" } else { puts $stats "<TRxTD align=' center' valign= ' top' >$y</TD><TDx/TD><TD></TD><TD></TD><TD></TDx/TR>"
} } puts $stats "</TABLE>" close $stats if {$: preference (openHTML) } { log "open location \"file: //127.0.0.1 [file join [pwd] $term.htm] \" " exec osascript -e "open location \"file: //127.0.0.1 [file join [pwd] $term.htm]\" } }
#####################################################################
# #
# MATCH #
# # ##################################################################### #
# Check the sequence data against the primary match criteria: #
# Discover a subsequence h...k...k, k...h...k, or k...k...h such that
# (1) The distance between ks is in the range kmin..kmax.
# (2) The distance between an h and the farthest k is in the range kmin+1..hmax.
# (3) The fraction of k in the subsequence is percent or larger. #
# The sequence is searched for all possible subsequences that match,
# and all these subsequences are returned. set kmin 6; set kmax 10 set hmax 50 set percent 6 proc match {sequence} { global kmin kmax global hmax global percent
set pos 0 set L {} array set F { } foreach e [regexp -all -indices -inline k $sequence] { for { set i l} { $i< [1 length $L] } { incr i } { set kO [lindex $L [expr { $i-l} ] ]
# rule 1. for {set j $i; set wideenough 0} { ! $wideenough && $j<[llength $L] } {incr j } { set kl [lindex $L $j] if {$kl-$kθ<$kmin} continue if {$kl-$kθ>$kmax} break
# rule 2. set offset [expr $kl-$hmax] if {$offset<0} {set offset θ} while 1 { set h [string first h $sequence $offset] if {$h<0 Il $h>$kθ+$hmax} break if {$h<$kθ} { set b $h
} else { set b $kθ
} if {$h>$kl} { set e $h } else { set e $kl }
# rule 3. set subsequence [string range $sequence $b $e] set nk [regexp -all k $subsequence] if {double ($nk) /double ( [string length $subsequence] ) *100>=$percent} { set "F($b $e) " 1 } incr offset }
} lsort -integer -index 0 [array names F]
}
#####################################################################
# #
# PASS #
# # #####################################################################
#
# Handle a passing page. The sequence is formatted on its own
# formatted html page . #
# Some attempt is made to color code separate matched subsequences
# and overlaps. Perhaps less clear than intended, but it does look
# pretty, doesn't it? proc pass { } { global replikinRef upvar 1 PAGE PAGE file mkdir pass set passing [open pass/$PAGE (%accession) .htm w] puts $passing {<htmlxheadxtitle>Replikin Analysis</titlex/headxbody>} puts $passing {<Hl>Replikins, Inc.</Hl>} puts $passing {<H2>Replikin Analysis</H2>} puts $passing "<dl>" puts $passing "<dt>PubMed Code; <dd>$PAGE (%link) " puts $passing "<dt>Description: <dd>$PAGE (%definition) " puts $passing "<dt>Isolated:<dd>$PAGE (%year) " if {[info exists PAGE (%country) ] } {puts $passing "in $PAGE (fccountry) " } if {[[string equal $PAGE(%source) unknown]} {puts $passing "<dt>Source:<dd>$PAGE (%sourσe) "} if {! [string equal $PAGE(%strain) unknown]} {puts $passing "<dt>Strain: <dd>$PAGE (%strain) " } " <dt>Serotype : <dd>$PAGE (%serotype) " } puts $passing " </dl>" set n 0 array set where {Amino-terminal {} Mid-molecule {} Carboxy-terminal {}} foreach ch [split $PAGE(%sequence) ""] { set C{$n) " <b>$ch</b><SUP> [expr {$n+l}] </SUP>" set S($n) {} set B($n) {} set E($n) {} incr n
} set s 0 foreach pq $PAGE(%result) { foreach {p q} $pq break if {$p<=$n/3} { lappend where (Amino-terminal) $p $q } elseif {$p<= (2*$n) /3} { lappend where (Mid-molecule) $p $q } else { lappend where (Carboxy-terminal) $p $q } set B($p) [linsert $B($p) 0 $s] lappend E($q) $s for {set i $p} {$i<=$q} {incr i} { foreach t $S($i) { set G($s,$t) 1 set G($t,$s) 1 } lappend S($i) $s
} set G($s) {} incr s
} set W {} for {set i 0} {$i<$s} {incr i} { lappend W [list $i [llength [array names G $i,*]]]
} foreach w [lsort -index 1 -integer $W] { set i [lindex $w 0] array unset A * foreach ij [array names G $i,*] { set j [lindex [split $ij , ] end] if {[info exist K($j)]} {set A($K($j)) 1} for {set k 0} {$k<$s} {incr k} { if { ! [info exists A($k)]} break set K($i) [expr {$k%4}]
) array set colour {
0 #000000
1 #00A000
2 #0000C0
3 #0080A0
4 #C00000
5 #80A000
6 #A000A0
7 #404OAO
8 #C08000
9 ttcocooo
10 #00A080
11 #608060
12 #P08000
13 #C0A040
14 #A04080
15 #808080 for {set i 0} {$i<$n} {incr i} { set NS 0 foreach j $S($i) { set NS [expr {$NS | (l«$K($j) ) }] set S($i) $colour($NS) puts $passing "<p>" set string { } for {set i 0} {$i<$n} {incr i} {
#foreach j $B($i) {lappend string "S=It;" $colour ( [expr {l<<$K($j ) }] ) } lappend string $C($i) $S($i)
#foreach j $E($i) {lappend string "&gt;" $colour ( [expr {l<<$K($j) }] ) } set wascol #000000 puts -nonewline $passing "<font color=' #000000 '>" set i 1 foreach {ch col} $string { if { ! [string equal $col $wascol] } { puts $passing "</font>" puts -nonewline $passing "<font color=' $col' >"
} set wascol $col puts -nonewline $passing " $ch" incr i
} puts $passing "</fontx/p><dl>" set aatag 1 foreach w {Amino-terminal Mid-molecule Carboxy-terminal} { puts $passing <dt>$w</dtχdd> if { [llength $where($w) ] } { foreach {p q} $where($w) { set aasequence [string range $PAGE (%sequence) $p $q] if { ! [info exists replikinRef ($aasequence. fid) ]} { if { ! [info exists replikinRef (+fid) ]} { set replikinRef (+fid) 1
} set replikinRef ($aasequence. fid) replikin-ref/ [format %05d $replikinRef (+fid)] .htm incr replikinRef (+fid)
} lappend replikinRef ($aasequence: $PAGE (%year) ) \
"<a href=' .. /pass/$PAGE(%accession) ,htm#$aatag' >$PAGE (%accession) position [expr {$p+l}] </a>" puts $passing "<pxa name= ' $aatag' xa href=.. /$replikinRef ($aasequence. fid) >" for {set i $p} {$i<=$q} {incr i} { puts $passing $C($i)
} puts $passing "</ax/ax/p>" incr aatag
} puts $passing </dd> } else { puts $passing "Zero replikins . "
) ' puts $passing </dl> set replikincount [expr {double (100* [llength $PAGE (%result) ]) /double ($n) }] puts $passing "<p>Replikin Count = Number of Replikins per 100 amino acids = [llength $PAGE(%result)] /$n =
[format %.lf $replikincount] </p>" close $passing return $replikincount
#####################################################################
# #
# FAIL #
# # ##################################################################### #
# Add the sequence to the failing page. proc fail { } { upvar 1 PAGE PAGE global failing kmax if { ! [info exists failing] } { set failing [open fail. htm w] puts $failing "<p>$PAGE (%link) " set d $kmax set i 0 foreach e [split $PAGE (%sequence) ""] { switch $e { h { set j [string last k $PAGE(%sequence) $i] if {$J<0} { set j "" } else { set j <sub> [expr {$i-$j }] </sub> } set k [string first k $PAGE(%sequence) $i] if {$k<0} { set k "" } else { set k <sub> [expr {$k-$i}] </sub>
} incr d if {$d<=$kmax} { puts -nonewline $failing " <sup>$d</supxi>$j$e$k</i>" } else { puts -nonewline $failing " <i>$j$e$k</i>" } k { puts -nonewline $failing " <b>$e</b>" set d 0
} default { incr d if {$d<=$kmax} { puts -nonewline $failing " <sup>$d</sup>$e" } else { puts -nonewline $failing " $e" }
} incr i
} puts $failing ""
#####################################################################
# #
# SEARCH CONTROL GUI #
# # ##################################################################### wm title . {Genome Explorer) . configure -menu .menubar menu .menubar menu .menubar , file -tearoff 0
.menubar add cascade -label File -menu .menubar . file
.menubar . file add command -label "Show Spy" -command {activitylog} .menubar .file add command -label Quit -command {exit 0} -accel Command-Q bind . <Command-q> {exit 0} menu .menubar. edit -tearoff 0 .menubar add cascade -label Edit -menu .menubar . edit
.menubar. edit add command -label Cut -command {event generate %W <<Cut>>} -accel Command-X
.menubar .edit add command -label Copy -command {event generate %W <<Copy>>} -accel Command-C
.menubar. edit add command -label Paste -command {event generate %W <<Paste>>} -accel Command-V
.menubar .edit add command -label Clear -command {event generate %W <<Delete>>} menu .menubar.help -tearoff 0 .menubar add cascade -menu .menubar .help
.menubar.help add command -label "Genome Explorer Help" -command { exec open $Contents/Resources/Documents/Instructions . rtfd
.menubar .help add command -label Manifest.rtf -command { } wtn resizable . 1 0 bind . <Command-k> {console show} bind . <Command-l> {console hide} pack [frame .query] -side top -fill x pack [menubutton . query. db -textvariable db -menu . query. db.m] -side left menu .query. db.m -tearoff 0
.query. db.m add command -label www.ncbi.nlm.nih.gov -command {set db NCBI} .query. db.m add command -label www.flu.lanl.gov -command {set db LANL} pack [label .query. label -text "query: "] -side left pack [frame . query.padd -width 20] -side right pack [entry .query. text -textvariable query] -side left -fill x -expand 1 pack [frame .k] -side top -fill x pack [label .k.l -text "k distance: "] -side left pack [entry .k.min -textvariable kmin -width 5] -side left pack [label .k.2 -text "min "] -side left pack [entry .k.max -textvariable kmax -width 5] -side left pack [label .k.3 -text "max"] -side left pack [frame .h] -side top -fill x pack [label .h.l -text "h distance: "] -side left pack [entry .h.max -textvariable hmax -width 5] -side left pack [label ,h.3 -text "max"] -side left pack [frame .percent] -side top -fill x pack [label .percent .label -text "percent: %"] -side left pack [entry .percent .text -textvariable percent -width 5] -side left pack [frame .count] -fill x -side top pack [label . count.1 -text "pass: "] -side left pack [label .count.2 -textvariable pass -width 5] -side left pack [label .count.3 -textvariable passpercent -width 6] -side left pack [label .count.4 -text " fail: "] -side left pack [label .count.5 -textvariable fail -width 5] -side left pack [label . count .6 -textvariable failpercent -width 6] -side left pack [label .count.7 -text " total: "] -side left pack [label .count.8 -textvariable seen -width 5] -side left pack [canvas .status -height 20 -bd 0 -background white] -side top -fill x trace variable pass w changed trace variable fail w changed proc changed {namel name2 op} { global pass passpercent global fail failpercent global seen if {$seen==θ} { set passpercent "" set failpercent "" } else { set passpercent ( [expr { (100*$pass) /$seen}] %) set failpercent ([expr { (100*$fail) /$seen}J %)
}
.status create rectangle 0 0 0 19 -fill red -tags bar .status create text 3 19 -anchor sw -fill black -tags text proc status {message i n} { if { [string length $message] && ! [string match +* $message] } { log $message
} if { [string match +* $message] } {set message [string range $message 1 end] }
.status dchars text 0 end
. status insert text 0 $message if {$n==0} {
.status coords bar 0 0 0 19 } else {
.status coords bar 0 0 [expr { ( [winfo width .status] *$i) /$n}] 19 update } pack [frame .button] -side top -fill x pack [button .button. quit -text Quit -command { exit 0
}] -side left pack [button .button. search -text Search -command { .button. search configure -state disabled log "Seaching... "
GenomalEnquirer $db $query [collectCritera]
.button. stop configure -state disabled
.button. search configure -state normal }] -side right pack [button .button. stop -text Stop -command { set stop 1 status "Stopping..." 0 0 }] -side right
#####################################################################
# #
# ADDITIONAL CRITERIA #
# #
#
# Additional criteria can be added to the search. These are
# substrings that must be included or excluded from the sequence
# data description, either in any description or in specific
# description. These criteria are checked in order: the first that
# fails, fails everything. If all pass, the page passes. #
# It might seem you would want a way to do nested includes
# and excludes , rather than conj oining of them . However after doing
# boolean algebra, it turns out that would be same result. #
# This is encoded with timers so that if anything is changed, a
# pass is made through the cached data after a few seconds . image create photo praised -data {
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ACwAAAAAHgAeAAAI/wABCBw40MCFFDqOQImShAcKCgIISpwIIIGHH3H4cPmyZcsXMI30CPlAgOJECz8G WcI0psKDlw9s0FKEqRERDCYFDhDBBpOkRZLCTCCYQ5efRYsw5VFhϋsAKQpaQAhVKEIdRqZIiuaB4gtBP rFQHWj OqFVKlFhIzyIkqdepQsVfbQvozYuABI5j alg36VuBYvYsgdYkg0IQj SID5Vo2rVxESAAaW5E0c li/jtoUObαΗgCJOlz6AtaULTF0COX4tCg5Y0CQgNQFpiy44NpkgBgjA4UZktO8uePkO8QGDQoLjxBgsC SFTg4LhxFqZCZblyIKdliTJkoVpjpfrl7zFk3fiC0/07eFi9sGBBYP56jFa+gtwxk0aN/ft0lEjsMObN /ftlXILLLi+I4owOOSSoYDWzlCTWNs8oKOEOrLjCgSB42KHhhnbsϋUVpONRyBocbnkHKIwBIgRhlIF4m lRzIlACAD4oA5tZiZLUVyCe5CCSBG4WwiCNgbVDTw0A7MFJjW4rBlSNScwAzikRMUBIkWC0+aUcqxkgw UR2b5NikZTm2oQo2IJiUCC14BDkmAFYFsogfcxSjTAnW0WGLJ3YUQkYTϋwT6BCK9nNHGKdyU4uVUHQy zCqgHMPMMsskI00svGgTzA3tCRSCGK8Q08wl2VgDjTCZzJBTQAA7
} image create photo xraised -data {
R01GODlhHgAeAOYAAP///9bWlt7e3ufn5+/v7/f39/f37///997elvf35///797ezufnlv//5/f33ufn zv//3vf31u/vzufnxv//lvf3zu/vxv//zvf3xv//xufn7+/v9/f3/9bW3t7e5+fn987O3tbW597e78bG lufn/97e99bW78bG3s7O57291rWlztbW987O77293q2tztbW/6Wlxs7O98bG77W13s7O/8bG972975yc xpSUva2t3sbG/6Wllr2995yczq2t56W13r29/7W19Syclq2t7SSUzqW157Wl/5yc3q2t94SEvZSUlpyc 54SExpSU3oyMlpyc74SEzpSU54yM3nt7xoSElnt7zoSE3nt71gAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACwAAAAAHgAeAAAH/4AAgoOEEBkWEwsL DxIRDYSQkYIHFxMoQJg6mEA6LAwQkpIQEzWcOqeop5kCCqGDFSgdNamOqCAhNQmuGDUIFAWltaogGQor QAaSETIBFILApqlAHRgHAAosQAWQDSi+hAUxOafEhAomOhyDBwggzpAb4sMZluAtL4MONTUkkvGYHTJI EtKEhggA7DgF+SCpgA5q9QgJoUJDhwwAEGadWijJAIaBTipqGhCBFkdXEimmAsFgnA4eJVAKmigSlYwO woDEdNUjJCOaPkK4VMVQUg4rNVMVQZETRIVQGuT9XOLBJZAAA12FG3pkAK2rWVFuTUVDCIFoYGUOGovK hQJUaZMj 7VjRUCqSEQDQxYVERArMujpoOCEAQMPDsIT6VtTZsBSMQTFkbIikG050Qi200NEwiACPk4Iq p/o7aAaVKCkgiQDCYzIAOV93triC5IakSzw+wK7Fg8RsJEnUSRoRZEhBYaObRMGxzZWHHOCSlqKBZIoK tQAKpBCChBON74ufTLlBGPskEC6IQKnCngmOEc01BQIAOW==
} pack [frame .0] -side bottom -fill x pack [button .O.more -image praised -bd 0 -highlightthickness 0 -command { addCriteria pack [label .0. version -text $versionDate -anchor s -font {Helvetica 9}] -fill x -side bottom pack [label .O.text -text "More criteria..."] -side left proc triggerSearch { } { global timedSearchToken if { [info exists timedSearchToken] } {after cancel $timedSearchToken} set timedSearchToken [after 5000 timedSearch]
} proc triggerLogChanged {k} { global timedLogToken if {[info exists timedLogToken ( $k) ]} {after cancel $timedLogToken($k) } set timedLogToken($k) [after 5000 "logToken $k"]
} proc timedSearch { } { global timedSearchToken timedLogToken query db stop if {[string equal [.button. search cget -state] disabled]} { set stop 1 after 500 timedSearch } else { foreach k [array names timedLogToken] {logToken $k}
■unset -nocomplain timedSearchToken
.button. stop configure -state normal
.button. search configure -state disabled
GenomalEnquirer $db $query [collectCritera]
.button. stop configure -state disabled
.button. search configure -state normal } proc logToken {k} { global timedLogToken criteria if {[info exists timedLogToken ($k) ]} { after cancel $timedLogToken($k) unset timedLogToken($k)
} log "Changed criteria $k words to ' $criteria ($k. words) '" } array set criteria {count 0} proc addCriteria { } { global criteria incr criteria (count) set k $criteria (count) log "Adding criteria $k" frame . $k pack [button .$k.more -image xraised -bd 0 -highlightthickness 0 -command " log {Deleting criteria $k} destroy . $k array unset criteria $k.* "] -side left pack [menubutton .$k. elude -textvariable criteria ($k.cludelabel) -menu .$k. elude. m] - side left menu . $k. elude. m -tearoff 0
. $k. elude. m add command -label include -command " set criteria ($k. cludelabel) include set criteria ($k. elude) {1} log {Change criteria $k cludelabel to 'include'} triggerSearch
. $k. elude.tn add command -label exclude -command " set criteria ($k. cludelabel) exclude set criteria ($k. elude) {} log {Change criteria $k cludelabel to 'exclude'} triggerSearch
. $k. elude.m add command -label "must have" -command " set criteria ($k. cludelabel) {must have} set criteria ($k. elude) = log {Change criteria $k cludelabel to 'must have'} triggerSearch set criteria ($k. cludelabel) exclude set criteria ($k. elude) {} pack [entry ,$k. words -textvariable criteria ($k. words)] -side left -fill x -expand 1 set criteria ($k. words) "" pack [menubutton . $k, extent -textvariable criteria ($k.extentlabel) -menu . $k . extent .m] -side left menu .$k. extent.m -tearoff 0
.$k. extent.m add command -label "in all lines" -command " set criteria($k.extentlabel) {in all lines} set criteria ($k. extent) * log {change criteria $k extentlabel to '*'} triggerSearch set criteria ($k. extentlabel) {in all lines} set criteria($k. extent) *
.$k. extent.m add command -label "in definition" -command " set criteria ($k. extentlabel) {in definition} set criteria ($k. extent) %definition log {Change criteria $k extentlabel to ' %definition' } triggerSearch
,$k. extent.m add command -label "in source" -command " set criteria ($k. extentlabel) {in source} set criteria ($k. extent) %source log {Change criteria $k extentlabel to '%source'} triggerSearch
.$k. extent.m add command -label "in strain" -command " set criteria ($k. extentlabel) {in strain} set criteria ($k. extent) %strain log {Change criteria $k extentlabel to '%strain'} triggerSearch
.$k. extent. m add command -label "in serotype" -command " set criteria ($k. extentlabel) {in serotype} set criteria ($k. extent) %serotype log {Change criteria $k extentlabel to '%serotype'} triggerSearch
.$k. extent.m add command -label "in publications" -command " set criteria ($k. extentlabel) {in publications} set criteria ($k. extent) *.pub.* log {Change criteria $k extentlabel to '*.pub.*'} triggerSearch
. $k. extent.m add command -label "in annotations" -command " set criteria ($k. extentlabel) {in annotations} set criteria ($k. extent) *.annot.* log {Change criteria $k extentlabel to '*.annot.*'} triggerSearch
. $k. extent .m add command -label "in keywords" -command " set criteria ($k. extentlabel) {in keywords} set criteria ($k. extent) *. keywords.* log {Change criteria $k extentlabel to '*. keywords .*'} triggerSearch pack forget .0 pack . $k .0 -side top -fill x focus ,$k. words
} proc collectCritera {} { global criteria set sort { } set collected { } foreach w [winfo children . ] { if { [string match {.[123456789]*} $w] } { lappend sort [lindex [split $w .] 1] } } foreach i [lsort -unique -integer $sort] { lappend collected $criteria($i. elude) $criteria($i. extent) $criteria($i. words)
} return $collected # #
# ACTIVITY LOG #
# # ##################################################################### #
# Log user activity and http responses . This is intended to spy
# on the program user, (hence the menu to show this is called Spy) ,
# but nonmaliciously. The intent is to retain information to help
# naive users to use the program. Nothing is transmitted from the
# user's machine without her knowledge. proc activitylog { } { global logFile if {! [winfo exists .log] } { set re [catch { destroy . log destroy . logmenu toplevel . log -menu . logmenu wm title .log {Activity Log} menu . logmenu menu .logmenu. file -tearoff 0
.logmenu add cascade -label File -menu .logmenu. file
. logmenu . file add command -label Quit -command {exit 0} -accel Command-Q bind .log <Command-q> {exit o) menu . logmenu . edit -tearoff 0 .logmenu add cascade -label Edit -menu .logmenu. edit
. logmenu. edit add command -label Cut -command {event generate %W <<Cut>>} - accel Command-X
. logmenu . edit add command -label Copy -command {event generate %W <<Copy>>} -accel Command-C
. logmenu . edit add command -label Paste -command {event generate %W <<Paste>>} -accel Command-V
. logmenu . edit add command -label Clear -command {event generate %W <<Delete>>} menu . logmenu . help -tearoff 0
. logmenu add cascade -menu . logmenu . help
. logmenu.help add command -label "Genome Explorer Help" -command { exec open $Contents/Resources/Documents/Instructions .rtfd
}
.logmenu.help add command -label Manifest.rtf -command { exec open $Contents/Resources/Documents/Manifest .rtf
} wm geometry .log 500x400 wm resizable .log 1 1 bind .log <Command-k> {console show} bind .log <Command-l> {console hide} place [ frame .log.t
] -x 0 -relwidth 1.0 -relheight 1 -height -35 pack [ scrollbar .log.t.y -orient vertical -command ".log.t.t yview" ] -side right -fill y pack [ text .log.t.t -yscrollcommand ".log.t.y set" ] -side top -fill both -expand 1 catch { set ch [open $logFile r] .log.t.t insert 1.0 [read $ch]
} catch { close $ch } place [ button .log. clear -text "Remove Log" -command { if { [info exists log] } { close $log unset log
} file delete $logFile destroy . log
}
] -x 25 -width 150 -rely 1.0 -y -30 -height 25 place [ ] -relx 1. 0 -x -175 -width 150 -rely l . o -y -30 -height 25
} rs] if {$rc} {log $rs} } wm deiconify .log raise .log
} proc emailOmyxLog { } { global pr mail -from support@omyx . com -to wyrmwif@rawbw . com -host smtp . rawbw . com \
"Subj ect : Transmittal of Genome-Explorer Log\n\n [ . log . t . t get 1. 0 end] "
} log "Start Genome-Explorer $versionDate"
# Send a mail message with SMTP. #
# mail [-from I -sender userθhost] [-to | -receiver user@host]
# [-host emailMailer] [-port smtpport]
# [-mI —] message #
# The sender, receiver, and message must be specified. The
# default smtp host is the sender's host, and the default port
# is the smtp port . #
# The message includes any headers and the body. No headers,
# even From: and To:, are set by this proc. proc mail {args} { set host { } set message { } set port 25 set receiver { } set sender { } while { [llength $args]>0} { set p [lindex $args 0] ; set args [Irange $args 1 end] switch -glob -- $p {
-[FfSs]* {set sender [lindex $args O]; set args [Irange $args 1 end]} -[TtRr]* {lappend receiver [lindex $args O]; set args [Irange $args 1 end] -[Hh]* {set host [lindex $args O]; set args [Irange $args 1 end]} -[Pp]* {set port [lindex $args O]; set args [Irange $args 1 end]} -[Mm]* {set message [lindex $args O]; set args [Irange $args 1 end]}
-* {error "unknown option mail option: $p"} __ i if {[llength $args]!=l} { error "too many parameters: only the one message is expected."
} set message [lindex $args 0] ; set args [Irange $args 1 end]
} default { if {[llength $args]!=θ} { error "too many parameters: only the one message is expected." set message $p
set receiver [join $receiver ,] set R { } ; set r " " ; set s 0 foreach c [split $receiver ""] { switch -glob $s:$c { 0:< { set r " " ; set s m
0:, { set r [string trim $r] if { [string length $r] } {lappend R $r}
0:* { append r $c
0:( { }
[1-9]*: ( { incr s
}
[1-9]*:) { deer s
}
[1-9]* {
} m: > { set r [string trim $r] if { [string length $r] } {lappend R $r} set s e } m:* { append r $c β:, { set s 0 }
} set r [string trim $r] if { [string length $r] } {lappend R $r} if {[string equal $host ""] && [regexp {@( [Λ>] +)(>?)} $sender all hostl] } { set host $hostl } set channel [socket $host $port] fconfigure $channel -translation {crlf crlf} smtpCommandResponse $channel -none puts $channel "HELO $host" while 1 { flush $channel set response [smtpCommandResponse $channel -none] if { [string compare [string range $response 0 2] 250] == 0} {break}
} smtpCommandResponse $channel "MAIL FROM: <$sender>" foreach r $R { smtpCommandResponse $channel "RCPT T0:<$r>"
} smtpCommandResponse $channel "DATA" puts $channel "Date: [clock format [clock seconds] -format {%e %b %y %H:%M %Z}J " puts $channel "From: $sender" puts $channel "To: $receiver" foreach line [split $message \n] { if {[string match .* $line] } {set line .$line} puts $channel $line
} smtpCommandResponse $channel " . " smtpCommandResponse $channel "QUIT" close $channel return } proc smtpCommandResponse {channel command} { if { ! [string equal $command -none] } { puts $channel $command; flush $channel
} set line {} set re [gets $channel line] if { $rc < 0 } { close $channel error "Connection broken. " switch -glob -- $line { 1* - 2* - 3* { return $line
}
4* - 5* { close $channel; error $line close $channel; error "SMTP: unrecognised response: $line" } } proc bgerror args { set ei $ : : errorlnfo set message [join $args " "] puts $message\n$ei status $message 0 1 log $ei
.button. stop configure -state disabled
.button. search configure -state normal }
#####################################################################
# #
# FINAL LOOK AND FEEL #
# # ##################################################################### catch {console hide} bind . <Control-t> { set db NCBI set query {influenza H5N1 2003}
.button. stop configure -state normal
.button. search configure -state disabled
GenomalEnquirer $db $query [collectCritera]
.button. stop configure -state disabled
.button. search configure -state normal } proc tkAboutDialog { } { set re [catch { destroy .about catch {image delete aboutlmage} toplevel . about wm geometry .about +50+50 wm overrideredirect . about 1 pack [canvas .about. image -width 576 -height 160 -highlightthickness 0] image create photo aboutlmage -data {
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}
.about. image create image 0 0 -image aboutlmage -anchor nw
.about. image create text 288 160 -anchor s -font {Helvetica 12} -text $ : :versionDate bind .about <Button> {destroy .about; image delete aboutlmage} bind .about <Key> {destroy .about; image delete aboutlmage} } rs] if {$rc} {puts "about dialog: $rs"} x> — msii uciiυus iυr runiiiug UIE vrcuuπie Ji/xpiυrer /vμμiiciuiuu
(1) Move the application Genome (looks like the PubMed double helix) to wherever you want it, (or run it off the CD).
(2) Run the application (double click the icon).
(3) It will present a dialog box with database (NCBI or LANL) and a query: [ ] on the top. Select the database and enter the same string in [ ] as you would for the PubMed query page 'Search [ Protein ] for [ ]' (NCBI database), the Search for: [ ] in the Influenza Sequence Database query page (LANL database).
NCBI fetching ncbi.gif "
LANL fetching lanl.gif "
(4) Hit the Search button.
The application will now contact the database through your internet connection and fetch the query results. It will then download each protein page to access the amino acid sequence and description.
After fetching all the pages, they will be parsed from the database form to a more convenient local form. parsing.gif "
Finally the fetched and parsed pages are searched for the matching peptide subsequences. Passing sequences will be written to as a separate htm page for each protein to the desktop folder 'pass'; all failing proteins will be written to the single desktop htm page 'fail1. searching.gif "
The other entries in the dialog box allow some of the match criteria to be adjusted. The values given are those originally requested.
The parse and searching are done off-line: an internet connection is not necessary for these phases.
As the proteins are checked, their identifiers will be shown at the bottom of the dialog box, and a red progress bar will move across the screen, 'total' counts the total number of proteins checked so far in this query; 'pass' and 'fail' count the number so far passing or failing the criteria. The progress bar will be progressed separately for fetch, parse, and search phases.
(5) To stop an active search, hit the Stop button. The progress text will change to 'stopping' then 'stopped'. \υj i u L|uιι, mi me wuii I/U LLUI I.
(7) The pass results can be viewed by openning the desktop 'pass' folder, and then openning the htm pages in your browser.
NCBI report ncbi.gif "
LANL report lanl.gif "
Each Replikin sequence in the Replikin Analysis page is linked to a history of everywhere that specific sequence is found in the current query. The entries in the Replikin History are linked back to the entries in the Replikin Analysis. sequence-history.gif "
(8) The fail results can be viewed by openning the desktop 'fail. htm' in your browser. If no additional criteria, the search is a full text search of the database. completed.gif "
Additional criteria can be added to the search with the + icon at the bottom. Exclude criteria exclude those pages that have the given string, while include criteria includes them. Exclude and include can be intermingled to refine the search results, include exclude.gif "
The "must have" criteria indicates strings that must occur in the fields. must have.gif "
The search will automatically restart after editting additional criteria, or the search can be restarted by hitting the Search button. If additional criteria are editted while search is in progress, the search is restarted with the new criteria. Also the search will try to use cached results: as long as the database and query: strings are left unchanged, only the additional criteria are changed, the fetch and parse phases are skipped. An internet connection is not necessary to refine a previously fetched search.
A few preferences can be set with the application preferences preferences.gif "
The Working directory is where all the files and cache are written. The default is the desktop.
Save fetched pages is intended for debugging. It saves intermediate query pages to the working directory.
Purge query cache removes all pages from the hidden cache. This happens automatically if the database or query string are changed. Open results in browser will open the Replikin report in the default browser when it is completed.
Appendix C - "Genome Explorer" Screen Snapshots.
Figure imgf000143_0001
Appendix C - "Genome Explorer" Screen Snapshots.
In) ft]
Figure imgf000144_0001
Appendix C - "Genome Explorer" Screen Snapshots.
Figure imgf000145_0001
Appendix C — "Genome Explorer" Screen Snapshots.
Figure imgf000146_0001
Appendix C - "Genome Explorer" Screen Snapshots.
Figure imgf000147_0001
Appendix C - "Genome Explorer" Screen Snapshots.
Figure imgf000148_0001
Figure imgf000149_0001
Appendix C - "Genome Explorer" Screen Snapshots.
Figure imgf000150_0001
Figure imgf000151_0001
Appendix C - "Genome Explorer" Screen Snapshots.
Figure imgf000152_0001
Appendix C - "Genome Explorer" Screen Snapshots.
Figure imgf000153_0001
ppendix D - "Dr. Peptide"Tcl Application with complex amino acid pattern recognizer.
DR PEPTIDE #####################################################################
# #
# Copyright (C) 2003 # b##y# S##am##u#e#l# B##o#g#o##ch## a#n#d## E#l#e#n#o#r#e#B##o#g#o#c#h################################# All rights reserved. main open-application reopen main open-document.tcl.path main open-document.peptide path main top mbox main top remove main quit set q [query tracker path db term fsm criteria] $q stop report path term set arrname [manual path] manual-okay arrname set w [window...] file-menu {label command...} geometry <geometry> resizable {horizontal vertical} title <title> window <w> $window quit $window zoom w h x y $window configure w h x y $window configurerequest w h x y $S(space) : :status message i n $S(space) : :count seen pass fail $S(space) : :completed peptide new Ι open path manualchecklist arrname title set Contents [file dir [file dir [lindex $tcl_libPath 0]]] set env (PATH) $env (PATH) :$Contents/Resources/bin lappend env (TCLLIBPATH) $Contents/Resources/lib set versionDate 2003-07-25 set openning 1 set opendocs { } set bootlog { } proc capture dcript { set r c [catch {uplevel 1 $script} rs] if {$rc} {bgerror $rs} } proc log {message} { if {$ : :opening} { lappend : :bootlog "starting: $message" } else { log : :tell $message } } log "Dr-Peptide $ : :versionDate."
WINDOW DRESSING #####################################################################
# #
# #
# # ##################################################################### D-01 # Generic window decoration: create a toplevel, set up its
# window frame decorations, and initial behaviour. global unique; set unique 0 proc window {window args} { global unique; incr unique set window [string map "%U $unique" $window] array set S [list \ window $window \ title Untitled \ menubar $window.menubar \ space [string range $window 1 end] \ file-menu { } \ ] array set S $args if { ! [winfo exists $window] } { toplevel $window } wm title $window $S (title) if {[info exists S (geometry)] && ! [string match 1x1* $S (geometry) ]} { wm geometry $window $S (geometry) } if {[info exists S (resizable) ]} { wm resizable $window [lindex $S (resizable) 0] [lindex $S (resizable) 1] } wm protocol $window WM_DELETE_WINDOW "catch {$S (space) :: quit} " $window configure -menu [menu $S (menubar) ] menu $S (menubar) . file -tearoff 0 $S (menubar) add cascade -label File -menu $S (menubar) .file foreach {label command accel binding} [string map [list %W $window %S $S (space)] $S (file-menu)] { if {[string equal $label -] } {
$S (menubar) . file add separator } elseif { [string length $accel] } {
$S (menubar) . file add command -label $label -command $command -accel $accel } else {
$S (menubar) . file add command -label $label -command $command } if { [string length $binding] } { bind $window <$binding> $command }
$S (menubar) .file add command -label Quit -command main: : quit -accel Command-Q bind $window <Command-q> main: :quit menu $S (menubar) .edit -tearoff 0 $S (menubar) add cascade -label Edit -menu $S (menubar) .edit
$S (menubar) .edit add command -label Cut -command {event generate %W <<Cut>>) -accel Command-X
$S (menubar) .edit add command -label Copy -command {event generate %W <<Copy>>} - accel Command-C
$S (menubar) .edit add command -label Paste -command {event generate %W <<Paste>>} - accel Command-V
$S (menubar) .edit add command -label Clear -command {event generate %W <<Delete>>} menu $S (menubar) .help -tearoff 0 $S (menubar) add cascade -menu $S (menubar) .help
$S (menubar) .help add command -label "Dr Peptide Help" -command { exec open $Contents/Resources/Documents/lnstructions .rtfd
}
$S (menubar) .help add command -label Manifest.rtf -command { exec open $Contents/Resources/Documents/Manifest .rtf
} bind $window <Configure> " if {\ [string equal %W $window\] } {
$S (space) : : configure %w %h %x %y } bind $window <ConfigureRequest> " if {\ [string equal %W $window\] } { if {\ [string equal %d Zoomed\] } {
$S (space) : : zoom %w %h %x %y } else {
$S (space) : : configurerequest %w %h %x %y } namespace eval $S (space) " variable S array set S [list [array get S] ] namespace eval $S (space) { proc configure {w h x y} {} proc configurerequest {w h x y) {} proc quit {} {variable S; destroy $S (window) ; return 1} proc show {} (variable S; wm deiconify $S (window) } proc zoom {w h x y} {}
} list $S (space) $window
#####################################################################
# #
# MAIN #
# # ##################################################################### #
# Operating system interface to handle open events . proc : : tk: :mac: :OpenApplication {} {} proc : : tk: :mac: :ReopenApplication {} { capture {main: : open-application 1} proc : :tk: :mac: : OpenDocument {args} { capture { foreach document $args { if {$: :openning} { lappend : :opendocs $document } else { main: : open-document $document }
catch {console hide} window . \ create 0 \ menubar . menubar \ title 11Dr Peptide" \ resizeable {θ 0} \ space main \ file-menu {
{New} {peptide new} {Command-N} {Command-n}
{Open...} {peptide open-ask} {Command-O} {Command-o}
{Show Log} {log: :show} {} {}
} pack [ message .opensesame -text "Initialising Dr Peptide. \n\n$versionDate" ] -padx Ii -pady .5i update after idle { log "Initialisation completed."
.opensesame configure -text "Use the New or Open... menu commands to get a new query window. \n\n$versionDate" set openning 0 foreach message $bootlog { log:: tell $message
} main: : open-application 0 foreach document $opendocs { main: : open-document $document
} log "All windows σpenned." namespace eval main { proc queries { } { set q {} foreach w [winfo children . ] { if { [string match .peptide* $w] } { lappend q $w } } set q
} proc open-application { reopen} { if { ! $reopen} {prefs : : open-prefs } set windows [queries] if { [llength $windows] } {
[string range [lindex $windows end] 1 end] : : show } elseif { $reopen} { peptide new } proc open-document {document} { log "Openning $document" if { [string match "/*" $document] } { set path $document } else { set path [exec osascript -e "get POSIX path of file \ " $document\ " " ]
} regsub {/$} $path {} path switch -- [file extension $path] { .tcl { set upgrade [tk_raessageBox -message "Install upgrade: [file tail $path]?" -type yesno -title "Install Upgrade"] if { [string equal $upgrade yes] } { log "upgrade: [file tail $path] " file copy -force $path $ : : Contents/Resources/Scripts/ [file tail $path] main quit }
.peptide { peptide open $path
} default { tk_messageBox -icon error -message "Not a peptide query folder: $path" -type ok -title "Error"
} } proc quit { } { foreach window [queries] { if { [catch { [ [string range $window 1 end] : :quit] } rs] } { set rs 1
} if {!$rs} {return}
} prefs : : save-immediate log: :quit exit } proc bgerror args { set ei $ : : errorlnfo set message [join $args " "] puts stderr \n$message\n \n$ei log $ei } proc : :tk: :mac: : ShowPreferences {} { capture {prefs: :show}
} proc tkAboutDialog { } { capture { destroy . about catch {image delete aboutlmage} toplevel . about wm geometry .about +50+50 wm overrideredirect .about 1 pack [canvas .about. image -width 576 -height 160 -highlightthickness 0] image create photo aboutlmage -data {
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}
. about . image create image 0 0 -image aboutlmage -anchor nw
.about. image create text 288 160 -anchor s -font {Helvetica 12} -text $ : :versionDate bind .about <Button> {destroy .about,- image delete aboutlmage} bind .about <Key> {destroy .about; image delete aboutlmage}
#####################################################################
# #
# .PEPTIDE WINDOW
# # ##################################################################### #
# <path>
# <path>/config
# criteria include | exclude | must-have %descriptor | * string
# db NCBI I LANL
# geometry =<w>x<h>+<x>+<y>
# modified o|i
# path <path>
# query active query
# seqpattern +<aminos> | -<aminos> | : <mingap> : <maxgap> ...
# term query term
# title <title>
# zoom =<w>x<h>+<x>+<y>
# zoomed o|i
Tr # Create the peptide search window. This fills out the peptide
# search window and creates the behaviour for it . if {[catch {load [lindex $env(TCLHIBPATH) end] /ncbiasn.dylib} rs] } { log "load ncbiasn.dylib failed: $rs" uplevel #0 source "SContents/Resources/Scripts/asn-parser . tcl" log "sourced asn-parser.tcl" } else { log "loaded ncbiasn.dylib" } package require http proc peptide {op {path .}} { switch $op { new { set k [clock seconds] while { [file exists /tmp/$k.peptide] } {incr k} array set S [list path /tmp/$k.peptide title "Untitled Query"]
} open-ask { set S (path) [tk_chooseDirectory -message {Select the folder which is the peptide query. }] if {! [string length $S (path) ] } return set S (title) [file root [file tail $S (path) ] ]
} open { set path [file root $path] .peptide if {![file exists $path] } {bell; return} array set S [list path $path title [file root [file tail $path] ] ] } array set S { file-menu {
{New} {peptide new} {Command-N} {Command-n} {Open...} {peptide open-ask} {Command-O} {Command-o}
{Close} {%S::quit} {Command-w} {Command-w} - - {} {} {Save} {%S::save} {Command-S} {Command-s}
{Save as...} {%S: :save-as} {Command-Shift-S} {Command-S}
{Revert} {%S::revert} {} {}
- - {} {}
{Query} {%S::guery} {Command-?} {Command-question} {Manually Check} {%S::manual} {Command-M} {Command-m} {Report} {%S::report} {} {}
- - {} {}
{Show Log} {log::show} {} {} db NCBI term { } seqpattern { } criteria {} focus { }
} if {[file exists $S (path) / .peptide-configure] } { set path $S (path) set f [open $S (path) / .peptide-configure] array set S [read $f] set S (path) $path close $f } else { array set S { zoomed 0 }
} array set S {seqnum 1 critnum 1 modified 0} foreach {space w} [eval window .peptide%U [array get S]] break wm withdraw . log "Open peptide query $S (path) in $w." pack [frame $w.aminoacids] -side right -fill y -expand 1 foreach code {
A B C D E F G H I K L M N P Q R S T V W Y Z } name {
Ala Asx Cys Asp GIu Phe GIy His lie Lys Leu Met Asn Pro GIn Arg Ser Thr VaI Trp Tyr GIx
} { pack [button $w.aminoacids. [string tolower $code] -font {Helvetica 10} -text "$name ($code) " -command "
${space}: : seqpattern-add-acid $code "] -side top -fill x } namespace eval $space { proc show { } { variable S wm deiconify $S (window) } proc save { } { variable S array set Z [array get S] foreach x {file-menu focus modified window space menubar title} { unset -nocomplain Z($x)
} set Z (geometry) [wm geometry $S (window) ] file mkdir $S (path) set f [open $S (path) / .peptide-configure w] log "Save $S (window) to $S (path) / . peptide-configure : db=$Z (db) term=$S (term) seqpattern=$Z (seqpattern) criteria=$Z (criteria) . " foreach {p v} [array get Z] { puts $f " [list $p] [list $v] "
} close $f set S (modified) 0 proc save-as { } { variable S set path [tk_chooseDirectory -mustexist 0 -message {Select the folder which is the peptide query.}] if { I [string length $path] } return if {! [string equal [file extension $path] .peptide]} { set pathl [file root $path] .peptide if { [file exists $path] } { if { [file exists $path/ .peptide-configure] } {
} elseif { [llength [glob -nocomplain "$path/* "] ] } { if { [string equal [ tk_messageBox -parent $S (window) -default no -icon warning -title "Overwrite? " -type yesno \
-message "Overwrite existing [file tail $path] ? " ] no]
} { return
} file rename $path $pathl
} set path $pathl
} file rnkdir $path if { ! [string equal $path $S (path) ] } { set fns [glob -nocomplain " $S (path) /* " ] if { [llength $fns] } {eval file copy -force - - $fns [list $path] } set S (path) $path log "Save as $S (window) to $S (path) . "
} array set S [list path $path title [file root [file tail $S (path) ] ] ] wm title $S (window) $S (title) save
} proc quit { } { variable S if {$S (modified) } { switch [ tk_messageBox -parent $S (window) -default yes -icon question -title "Save?" -type yesnocancel \
-message "Save $S (title) before closing?"
] { yes {$S (space) : :save} cancel {return 0} } log "Close $S (window) ." destroy $S (window) after idle [list namespace delete $S (space)] if {! [llength [main: : queries] ]} {wm deiconify .} return 1
} proc manual { } { variable S manualchecklist $S (space) $S (path) $S (title) } proc revert { } { variable S log "Revert $S (window) from $S (path) . " catch {query-stop} foreach s {seqpattern criteria} { catch {eval destroy [$S (window) .$s.t window names]}
$S (window) . $s . t delete 1.0 end
} if { [file exists $S (path) / .peptide-configure] } { set f [open $S (path) / .peptide-configure] array set S [read $f] close $f
} initialise set S (modified) 0
} proc initialise { } { variable S log "Initialise $S (window) : db=$S(db) term=$S (term) seqpattern=$S (seqpattern) criteria=$S (criteria) ." set w $S (window)
$w.query. db configure -text $S(db) $w. query. text insert 0 $S(term) $w. count.pass configure -text 0 $w. count.passpercent configure -text "" $w. count. fail configure -text 0 $w . count . failpercent configure -text "" $w . count . seen configure -text 0 $w . status dchars text 0 end $w. status coords bar 0 0 0 19 $w . button . search configure -state normal $w.button. stop configure -state disabled foreach piece $S (seqpattern) { seqpattern-add init $piece
} seqpattern-add init bottom foreach {test desc string} $S (criteria) { criteria-add init $test $desc $string
} criteria-add init bottom bottom bottom
} proc zoom {w h x y} { variable S set currgeom [wm geometry $S (window) ] if {$S (zoomed) && [string equal $currgeom $S(zoom)]} { set S (zoomed) 0 wm geometry $S (window) $S (geometry) } else { set S (geometry) $currgeora if {! [regexp {A=? (\d+)x(\d+) [+] (\d+) [+] (\d+)$} $currgeom - w h x y] } { foreach {w h x y} {o 0 0 0} break
} if {$w>[winfo screenwidth $S (window) ]} {set w [winfo screenwldth $S (window) ]} set S(zoom) ${w}x[expr {[winfo screenheight $S (window) ] -50}] +$x+20 set S (zoomed) 1 wm geometry $S (window) $S(zoom) }
} pack [frame $w. query] -side top -fill x pack [menubutton $w. query. db -text $S(db) -menu $w. query .db.m] -side left menu $w . query. db .m -tearoff 0
$w . query . db . m add command -label www.ncbi.nlm.nih.gov -command "${space} :: database NCBI"
$w . query . db . m add command -label www.flu.lanl.gov -command "${space} :: database LANL" pack [label $w. query. label -text "query: "] -side left pack [frame $w. query.pad -width 20] -side right pack [entry $w. query. text -validate key -validatecommand " after idle ${ space} :: term; expr 1 "] -side left -fill x -expand 1 focus $w. query. text namespace eval $space { proc database db { variable S set S(db) $db
$S (window) .query. db configure -text $db set S (modified) 1 proc term { } { variable S set newterm [$S (window) .query. text get] if {! [string equal $S(term) $newterm] } { set S (term) $newterm set S (modified) 1 }
} pack [frame $w. count] -fill x -side top pack [label $w. count.1 -text "pass: "] -side left pack [label $w. count .pass -text 0 -width 5] -side left pack [label $w . count . passpercent -text "" -width 6] -side left pack [label $w. count.2 -text " fail: "] -side left pack [label $w . count . fail -text 0 -width 5] -side left pack [label $w. count . failpercent -text "" -width 6] -side left pack [label $w. count.3 -text " total: "] -side left pack [label $w . count . seen -text 0 -width 5] -side left pack [canvas $w. status -height 20 -bd 0 -background white] -side top -fill x $w. status create rectangle 0 0 0 19 -fill red -tags bar $w. status create text 3 19 -anchor sw -fill black -tags text namespace eval $space { proc status {message i n} { variable S
$S (window) . status dchars text 0 end $S (window) . status insert text 0 $message if {$n==0} {
$S (window) . status coords bar 0 0 0 19 } else {
$S (window) . status coords bar 0 0 [expr { ( [winfo width $S (window) .status] *$i) /$n}] 19
} update
} proc count {seen pass fail} { variable S if {$seen==o} {
$S (window) .count .pass configure -text 0
$S (window) .count.passpercent configure -text ""
$S (window) .count. fail configure -text 0
$S (window) . count . failpercent configure -text ""
$S (window) .count. seen configure -text 0 } else {
$S (window) .count .pass configure -text $pass
$S (window) .count .passpercent configure -text ([expr { (100*$pass) /$seen}] %)
$S (window) . count . fail configure -text $fail
$S (window) .count. failpercent configure -text ([expr { (100*$fail) /$seen}] %)
$S (window) . count . seen configure -text $seen
} update
} proc completed { } { variable S
$S (window) .button. search configure -state normal
$S (window) .button. stop configure -state disabled update } pack [frame $w.button] -side top -fill x pack [button $w.button. search -text Search -state normal -command " ${ space} : : query
"] -side right pack [button $w. button. stop -text Stop -state disabled -command " ${ space} : : query-stop
11 ] -side right bind $w <Command- .> ${ space} :: query-stop bind $w <Control-c> ${space} : :query-stop bind $w <Escape> ${ space} :: query-stop namespace eval $space { proc query { } { variable S
$S (window) .button. search configure -state disabled $S (window) .button. stop configure -state normal query-start } } pack [labelframe $w.seqpattern -text "Peptide Pattern"] -side top -fill both -expand 1 pack [ scrollbar $w. seqpattern.y -orient vertical -command "$w.seqpattern. t yview" ] -side right -fill y pack [ text $w.seqpattern.t \ -width 75 -height 15 \ -state disabled \ -highlightthickness 0 \ -yscrollcommand "$w.segpattern.y set" ] -side top -fill y -expand 1 namespace eval $space { proc windowPositionlnText {s} {
# S = ... t.nnn
# Returns the text index in ...t of where ...t.n is. set w [winfo parent $s] ; set k 0 foreach {key value index} [$w dump -window 1.0 end] { if { [string equal $key window] && [string equal $s $value] } { return [list $k $index]
} incr k
} error "window not found: $s" } proc seqpattern-add {s token} { variable S
$S (window) . seqpattern . t configure -state normal if { [string equal $s init] } { set pos end } else { foreach {i pos} [windowPositionlnText $s] break set S (modified) 1 set S (seqpattern) [linsert $S (seqpattern) $i $token]
} set w $S (window) .seqpattern. t set s $w. $S (seqnum) ; incr S(seqnum)
$w window create $pos -window [frame $s] pack [button $s.insertgap -text "ΛGap" -font {Helvetica 10} -command "
$S (space) :: seqpattern-add $s : "] -side left pack [button $s .insertamino -text "ΛMatch" -font {Helvetica 10} -command "
$S (space) :: seqpattern-add $s + "] -side left if { ! [string equal $token bottom] } { pack [button $s. delete -text Delete -font {Helvetica 10} -command "
$S (space) : : seqpattern-delete $s "] -side left if {[string match :* $token] } { foreach {- mingap maxgap} [split $token :] break if {! [string length $mingap] } {set mingap θ} if {\ [string length $maxgap] } {set maxgap θ} pack [ label $s .mingaplabel -text "A gap of at least " -font {Helvetica 10} ] -side left pack [ entry $s.mingap -width 5 -justify right -validate key -validatecommand after idle $S (space) : : seqpattern-gap $s; expr 1
] -side left -fill x -expand 1 pack [ label $s .maxgaplabel -text " but no more than " -font {Helvetica 10} ] -side left pack [ entry $s.maxgap -width 5 -justify right -validate key -validatecommand after idle $Ξ (space) :: seqpattern-gap $s; expr 1
] -side left -fill x -expand 1 $s.mingap insert 0 $mingap $s.maxgap insert 0 $maxgap pack [ label $s .endgaplabel -text " amino acids." -font {Helvetica 10} ] -side left focus $s. mingap $s.mingap select range 0 end } else { set quantifier [string index $token 0] pack [ label $s.beginaminoslabel -text "One amino acid which is " -font {Helvetica 10}
] -side left pack [ menubutton $s .quantifier -width 9 -menu $s .quantifier .m -font {Helvetica 10}
] -side left menu $s.quantifier.ra -tearoff 0
$s.quantifier.m add command -label "any of" -command " $S (space) : :seqpattern-quantifier $s +
$s. quantifier .m add command -label "none of" -command " $S (space) : :seqpattern-quantifier $s - seqpattern-menu-label $s [string index $token 0] pack [ entry $s. aminos -width 20 -validate key -validateαommand " after idle $S (space) : : seqpattern-aminos $s; expr 1
] -side left -fill x -expand 1 bind $s. aminos <FocusIn> "$S (space) : :seqpattern-focus-in %W" bind $s. aminos <FocusOut> "$S (space) : : seqpattern-focus-out %W" $s. aminos insert 0 [string range $token 1 end] focus $s . aminos }
$S (window) .seqpattern. t configure -state disabled
} proc seqpattern-add-acid code { variable S if { [winfo exists $S (focus)]} { $S (focus) insert end $code
} proc seqpattern-focus-in w { variable S set S (focus) $w
} proc seqpattern-focus-out w { variable S if {[string equal $S (focus) $w] } { set S (focus) ""
} } proc seqpattern-delete {s} { variable S set t $S (window) .seqpattern. t
$t configure -state normal foreach {i pos} [windowPositionlnText $s] break set S (seqpattern) [lreplace $S (seqpattern) $i $i]
$t delete $pos
$t configure -state disabled set S (modified) 1
} proc seqpattern-gap {s} { variable S set i [lindex [windowPositionlnText $s] 0] set mingap [$s.mingap get] set maxgap [$s.maxgap get] set S (seqpattern) [lreplace $S (seqpattern) $i $i : $mingap : $maxgap] set S (modified) 1
} proc seqpattern-quantifier {s quantifier} { variable S set i [lindex [windowPositionlnText $s] 0] set e [lindex $S {seqpattern) $i] seqpattern-menu-label $s $quantifier set aminos [string range $e 1 end] set S (seqpattern) [lreplace $S (seqpattern) $i $i $quantifier$aminos] set S (modified) 1
} proc seqpattern-aminos {s} { variable S set i [lindex [windowPositionlnText $s] 0] set e [lindex $S(seqpattern) $i] set quantifier [string index $e 0] set aminos [$s. aminos get] set S(seqpattern) [lreplace $S (seqpattern) $i $i $quantifier$aminos] set S (modified) 1 } proc seqpattern-menu-label {s token} { if { [string equal $token +] } {
$s. quantifier configure -text "any of"
} else {
pack [labelframe $w. criteria -text "Additional Criteria"] -side top -fill x -expand 0 pack [ scrollbar $w. criteria.y -orient vertical -command "$w. criteria. t yview" ] -side right -fill y pack [ text $w. criteria. t \
-width 75 -height 10 \ -state disabled \ -highlightthickness 0 \ -yscrollcommand "$w. criteria. y set" ] -side top -fill y -expand 0 namespace eval $space { proc criteria-add {s test desc string} { variable S
$S (window) .criteria. t configure -state normal if { [string equal $s init] } { set pos end } else { foreach {i pos} [windowPositionlnText $s] break set i [expr {3*$i}] set S (modified) 1 set S (criteria) [linsert $S (criteria) $i $test $desc $string]
} set w $S (window) .criteria. t set s $w.$S (critnum) ; incr S(critnum)
$w window create $pos -window [frame $s] if { [string equal $string bottom] } { pack [button $s.insertgap -text "New Criteria" -font {Helvetica 10} -command
$S (space) :: criteria-add $s include * {} 11 ] -side left } else { pack [button $s.delete -text Delete -font {Helvetica 10} -command "
$S (space) : : criteria-delete $s "] -side left pack [ menubutton $s.test -width 11 -menu $s.test.m -font {Helvetica 10} ] -side left menu $s.test.m -tearoff 0
$s.test.m add command -label "Include" -command " $S (space) :: criteria-menu $s 0 include
$s.test.m add command -label "Exclude" -command " $S (space) :: criteria-menu $s 0 exclude
$s.test.m add command -label "Must have" -command " $S (space) :: criteria-menu $s 0 must-have pack [ entry $s . string -validate key -validatecommand " after idle $S (space) :; criteria-string $s; expr 1
] -side left -fill x -expand 1 focus $s . string pack [ menubutton $s.desc -width 30 -menu $s.desc.m -font {Helvetica 10} ] -side left menu $s . desc . m -tearoff o
$s . desc .ra add command -label "in any lines . " -command " $S (space) : : criteria-menu $s 1 *
$s . desc .m add command -label "in definition, " -command " $S (space) : : criteria-menu $s 1 %definition
$s.desc.m add command -label "in source." -command " $S (space) :: criteria-menu $s 1 %source
$s.desc.m add command -label "in strain." -command " $S (space) :: criteria-menu $s 1 %strain
$s.desc.m add command -label "in serotype." -command " $S (space) :: criteria-menu $s 1 %serotype
$s.desc.m add command -label "in publications." -command " $S(space) :: criteria-menu $s 1 *.pub.*
$s.desc.m add command -label "in annotations." -command " $S (space) :: criteria-menu $s 1 *.annot.*
$s.desc.m add command -label "in keywords." -command " $S (space) :: criteria-menu $s 1 *. keywords.* criteria-menu-label $s 0 $test criteria-menu-label $s 1 $desc $s. string insert 0 $string
}
$S (window) .criteria. t configure -state disabled
} proc criteria-delete {s} { variable S set t $S (window) . criteria . t
$t configure -state normal foreach (i pos} [windowPositionlnText $s] break set i [expr {3*$i}] set j [expr {$i+2}] set S (criteria) [lreplace $S (criteria) $i $j]
$t delete $pos
$t configure -state disabled set S (modified) 1
} proc criteria-menu {s k what} { variable S set i [lindex [windowPositionlnText $s] 0] set i [expr {3*$i+$k}] criteria-menu-label $s $k $what set S (criteria) [lreplace $S (criteria) $i $i $what] set S (modified) 1
} proc criteria-string {s} { variable S set i [lindex [windowPositionlnText $s] 0] set i [expr {3*$i+2}] set S(criteria) [lreplace $S(criteria) $i $i [$s. string get]] set S (modified) 1 proc criteria-menu-label {s k what) { switch $k$what {
Oinclude {$s.test configure -text "Include"} Oexclude {$s.test configure -text "Exclude"} Omust-have {$s.test configure -text "Must have"} 1* {$s.desc configure -text "in any lines."} l%definition {$s.desc configure -text "in definition."} l%source {$s.desc configure -text "in source."} l%strain {$s.desc configure -text "in strain."} l%serotype {$s.desc configure -text "in serotype."} l*.pub.* {$s.desσ configure -text "in publications."} l*.annot.* {$s.desc configure -text "in annotations."} 1*. keywords.* {$s.desc configure -text "in keywords."} } ${ space} : : initialise pack [label $w.version -text $ .- :versionDate -anchor s -font {Helvetica 9}] -fill x -side bottom
# #
# QUERY #
# #
#
# STATE
# cache: cache/query
# criteria: include) exclude | must-have %descriptor| * string ...
# db: database to search
# failing: fail.htm channel.
# fetching: accessionlink accesslonurl accessionnumber ...
# seqpattern: +<aminos> | -<aminos> | : <mingap> : <maxgap> ...
# offset: next fetching
# path: *.peptide folder path
# retry: fetching pending retry
# stop: stop requested
# term: query text search term
# token: pending http token
# tracker: mailbox to send status to.
# tracker status <message> <i> <n>
# tracker count <seen> <pass> <fail>
# tracker completed
# <path>
# <path>/cache
# <path>/cache/<accession>.prop
# %accession: accession number
# %definition
# %link: accession link
# %pass: if passes all matched criteria (can be set manually)
# %reason: reason why %pass is set as it is
# %seqdata: amino acid sequence
# %serotype
# %source
# %strain
# %subsequences matched-subsequence bounds
# %uri : accession url
# %year
# <...>: ASN entry
# <path>/cache/cache/qαery
# term: query term
# LANL: if LANL searched
# NCBI: if NCBI searched
# Code adapted from GenomeExplorer to query a remote database
# (NCBI or LANL) and parse the returned page. namespace eval $space { proc query-start {} { capture { variable S log "Query $S(term)." file mkdir $S (path) /cache file mkdir $S (path) /pass foreach fn [glob -nocomplain "$S (path) /pass/*. htm"] {file delete $fn} file delete $S (path) /index. htm file delete $S (path) /fail.htm regsub -all {\s+} $S(term) + S(termns) if { [catch { set f [open $S (path) /cache/query r] array set cache [read $f] set cached [expr { [string equal $cache (term) $S (termns) ] && $cache ( $S (db) ) } ] catch { close $f } } rs] } { set cached 0 array set cache [list term $S(termns) $S(db) I] array set S [list \ cache [array get cache] \ fail 0 \ pass 0 \ seen 0 \ ] status " " 0 0 count 0 0 0 if {$cached} { match } else { foreach fn [glob -nocomplain "$S (path) /cache/*"] {file delete $fn} fetch-list-$S(db) 20
proc query-stop {} { capture { variable S catch {http: .-wait $S(token); http: :cleanup $S(token)} log "Query stopped." status "Stopped." 0 0 completed } proc debugging-dump {file text} { if { [pref debuggingMode default 0] } { variable S file mkdir $S (path) /cache set f [open $S (path) /caσhe/$file w] puts $f $text close $f } proc fetch-list-LANL {n} { capture { variable S log "Querying for $n results from LANL." status "Querying for $n results from LANL." 0 0 set S(token) [http: :geturl \ http ; //www. fIu. lanl . gov/search2/resultN.html?search=l&field=ALL&num=$n&hspecies=Any&seg=any &nucorpro=nuc&orderby=dateasc&text=$S (termns) \
-command [list $S (space) : :parse-list-LANL $n] ] } proc fetch-list-NCBI {n} { capture { variable S log "Querying for $n results from NCBI." status "Querying for $n results from NCBI." 0 0 set S (token) [http: :geturl \ http: //www.ncbi .nlm.nih.gov: 80/entrez/query. fcgi?cmd=Search&:db=Protein&term=$S (termns) fcdisp max=$n&doptcmdl=DocSum \
-command [list $S (space) : :parse-list-NCBI $n] ]
proc parse-list-LANL {n token} { capture { variable S unset S (token) set queryPage [ : :http: :data $token] debugging-dump debug-list.htm $queryPage if { [regexp {Your request could not be processed due to a problem} $queryPage] } log "Server busy: retrying in 5 seconds..." status "Server busy: retrying in 5 seconds..." 0 0 after 5000 $S (space) :: fetch-list-LANL $n } elseif {! [regexp {Got <B> ( [0-9] +) </B> hits total} $queryPage all numentries]
I I ! [info exists numentries] | | [string equal $numentries ""] | | $numentries==0
} { log "Nothing matched the query." status "Nothing matched the query. " 0 0 } elseif {$numentries>$n} { fetch-list-LANL $numentries } else { array set S {fetching {} retry {} offset 0} set queryPage [string map {"\"» '} $queryPage] foreach {accessionlink accessionurl accessionnumber} \
[regexp -all -inline {<A HREF=' (view_record.html [?] [Λ '<>]*)' [Λ<>] *>( [Λ<] *) </A>} $queryPage] \
{ set accessionurl "http://www.flu.lanl.gov/search2/$accessionurl" set accessionlink [string map { "HREF='" "HREF= 'http: //www. flu. lanl .gov/search2/" } $accessionlink] lappend S (fetching) $accessionlink $accessionurl $accessionnumber
} fetch-page-LANL
} http :: cleanup $token
} proc parse-list-NCBI {n token} { capture { variable S unset S (token) set queryPage [:: http :: data $token] debugging-dump debug-list.htm $queryPage if { [regexp {Your request could not be processed due to a problem} $queryPage] } log "Server busy: retrying in 5 seconds..." status "Server busy: retrying in 5 seconds..." 0 0 after 5000 $S (space) :: fetch-list-NCBI $n } elseif {! [regexp {items l-[0-9]+ of ([0-9]+)} $queryPage all numentries]
I ! [info exists numentries] || [string equal $numentries ""] || $numentries==0
} { log "Nothing matched the query." status "Nothing matched the query." 0 0 } elseif {$numentries>$n} { fetch-list-NCBI $numentries } else { array set S {fetching {} retry {} offset 0} set queryPage [string map {"\"" '} $queryPage] foreach {accessionlink accessionurl accessionnumber} \
[regexp -all -inline {<a href=' ( [Λ?<>] * [?]cmd=Retrieve[A'<>] *) ' > ( [A<] *)</a>} $queryPage] \
{ lappend S (fetching) $aσcessionlink $accessionurl $accessionnumber
} fetch-page-NCBI
} http: : cleanup $token
proc fetch-page-LANL { } { capture { variable S if { $S (offset) < [llength $S (fetching) ]} { set accessionlink [lindex $S (fetching) $S (offset) ]; incr S (offset) set accessionurl [lindex $S (fetching) $S (offset) ] ; incr S (offset) set accessionnumber [lindex $S (fetching) $S (offset) ]; incr S (offset) set S (token) [http: :geturl $accessionurl \ -command [list $S (space) : :parse-page-LANL $accessionlink $accessionurl $accessioπnumber] ]
} elseif { [llength $S (retry)]} { array set S [list fetching $S (retry) retry { } offset 0] log "Retrying [expr {[llength $S (fetching) ] /3}] entries in 5 seconds..." status "Retrying [expr {[llength $S (fetching) ] /3}] entries in 5 seconds..." 0 0 after 5000 $S (space) :: fetch-page-LANL } else { fetch-complete match
) , ' proc fetch-page-NCBI { } { capture { variable S if { $S (offset) < [llength $S( fetching) ]} { set accessionlink [lindex $S (fetching) $S (offset) ],• incr S (offset) set accessionurl [lindex $S (fetching) $S (offset) ] ; incr S (offset) set accessionnumber [lindex $S (fetching) $S (offset) ] ; incr S (offset) set S(token) [http: :geturl [string map {query. fcgi viewer. fcgi} $accessionurl] &view=asn \
-command [list $S (space) : :parse-page-NCBI $accessionlink $accessionurl $accessionnumber] ]
} elseif {[llength $S (retry)]} { array set S [list fetching $S (retry) retry {} offset 0] log "Retrying [expr {[llength $S (fetching) ] /3 }] entries in 5 seconds..." status "Retrying [expr {[llength $S (fetching) ] /3 }] entries in 5 seconds..." 0 0 after 5000 $S (space) :: fetch-page-NCBI } else { fetch-complete match }
} proc parse-page-LANL {accessionlink accessionurl accessionnumber token} { capture { variable S unset S (token) log "Fetching $accessionnumber" status "Fetching $accessionnumber" [expr {$S (offset) +l}] [llength $S (fetching) ] set page [:: http :: data $token] debugging-dump debug-page.htm $page if { [regexp {try again later} $page] } { lappend S (retry) $aσcessionlink $accessionurl $accessionnumber } else { array set PAGE [list \
%accession $accessionnumber \ %link $accessionlink \ %uri $acσessionurl \ %pass 1 \
%reason "Fetched page from $S(db)." \ ] catch { set f [open $S (path) /cache/$accessionnumber.prop r] array set PAGE [read $f] close $f
} array set coding {
GCT a GCC a GCA a GCG a
TGT b TGC c
GAT d GAC d
GAA e GAG e
TTT f TTC f
GGT g GGC g GGA g GGG g
CAT h CAC h
ATT i ATC i ATA i
AAA k AAG k
TTG 1 TTA 1 CTT 1 CTC k CTA 1 CTG 1
ATG m AAT n AAC n
CCT p CCC p CCA p CCG p
CAA q CAG q
CGT r CGC r CGA r CGG r AGA r AGG r
TCT S TCC S TCA S TCG S AGT S AGC S
ACT t ACC t ACA t ACG t
GTT V GTC V GTA V GTG V
TGG W
TAT y TAC y
TAA * TAG * TGA *
} foreach {all attribute value} \
[regexp -all -inline {<trxtdxspan class="bold">( [Λ<>] +) </spanx/td>\s*<td> ( (? : [Λ<] |<[Λ/] | </ Tt] |</t[Ad] |</td[Λ>] ) *) </td>\s*</tr>) $page] \
{ regsub -all { < [*<>] *> } $value { } value set value [string trim [string map { Scribsp; " " &amp; & &lt ; < &gt ; > &apos ,- ' &quot » \" " } $value] ] regsub -all {\s+} $value { } value set PAGE ( [string map { " " - } $attribute] ) $value switch $attribute {
Strain {set PAGE (%strain) $value} Definition {set PAGE (%definition) $value} Source {set PAGE (%source) $value} "Collection Year" {set PAGE(%year) $value} Serotype {set PAGE(%serotype) $value} "Raw Sequence" { regsub -all {\s+} $value {} value set PAGE(Srseqdata) " " for {set c 0} {$c< [string length $value] } {incr σ 3} { set codon [string range $value $c [expr {$c+2}]] if {[info exists coding ( $codon) ]} { append PAGE (%seqdata) $coding($codon) } else { append PAGE (%seqdata) ? }
}
} file mkdir $S (path) /cache set f [open $S (path) /cache/$accessionnumber . prop w] foreach {p v} [array get PAGE] {puts $f " [list $p] [list $v] " } close $f
} http: : cleanup $token fetch-page-LANL } } proc parse-page-NCBI {accessionlink accessionurl accessionnumber token} { capture { variable S unset S (token) log "Fetching $aσcessionnumber" status "Fetching $accessionnumber" [expr {$S (offset) +1}] [llength $S (fetching) ] set page [:: http ;: data $token] debugging-dump debug-page.htm $page if { [regexp {try again later} $page] } { lappend S (retry) $accessionlink $accessionurl $accessionnumber } else { array set PAGE [list \
%accession $accessionnumber \ %link $accessionlink \ %uri $accessionurl \ %pass 1 \
%reason "Fetched page from $S(db) ." \ ] catch { set f [open $S (path) /cache/$accessionnumber. prop r] array set PAGE [read $f] close $f set starts [string first " <pre>Seq-entry : : = " $page] if { $starts>=0 } { incr starts 5 set stops [string first "</pre>" $page $starts] if {$stops>0} {incr stops -1} else {set stops end} set page [string range $page $starts $stops] array set PAGE [ncbiasn $page]
} set PAGE(%seqdata) {} foreach {p seqdata} [array get PAGE * .seq-data.ncbieaa.*] { set q [string map {inst. seq-data.ncbieaa id. O.ddbj .accession} $p] if {[info exists PAGE($q)] && ! [string equal $PAGE($q) $accessionnumber] } continue regsub -all {\s+} $seqdata {} seqdata lappend PAGE(%seqdata) [string tolower $seqdata] if { [llength $PAGE (%seqdata) ] >θ} { set PAGE(%definition) {} foreach {p v} [array get PAGE * .descr. * .article, title. * .name. >] {lappend PAGE(%definition) $v} set PAGE(%definition) [join $PAGE {%definition) " "] set PAGE(%source) unknown foreach {p v} [array get PAGE *. source.org. taxname. >] { set PAGE(%source) $v
} set PAGE (%strain) unknown foreach {p v} [array get PAGE *. subtype. >] { if { [string equal $v strain] } { set PAGE (%strain) $PAGE ( [string map {. subtype. > .subname.>} $p] )
} if { [string equal $PAGE (%strain) unknown]
&& [regexp { \ ( ( [A 0 ] * (\ ( [Λ 0 ] *\ ) ) ? ) \) \s*$ } $PAGE (%source) - > strain]
} { set PAGE (%strain) $strain
} set slash [string last / $PAGE (%strain) ] if {$slash>=0 && [regexp -start $slash {/ ( [0-9] +) } $PAGE (%strain) - > y] }
{ if {$y<30} {incr y 2000} elseif {$y<100} {incr y 1900} set PAGE(%year) $y } else { set PAGE(%year) unknown
} foreach {p y} [array get PAGE *.year.>] { if {$y<30} {incr y 2000} elseif {$y<100} {incr y 1900} if {$PAGE (%year) eq "unknown" || $PAGE (%year) >$y} {set \PAGE(%year)
$y} if {[regexp {\ ( ( [A 0 ] +) \) \s*$} $PAGE (%strain) -> s] } { set PAGE (%serotype) $s } elseif {[regexp {\ ( ( [Λ () ] +) \) \s*$} $PAGE(%source) -> s] } { set PAGE(%serotype) $s } else { set PAGE (%serotype) unknown } } file mkdir $S (path) /cache set f [open $S (path) /cache/$accessionnumber.prop w] foreach {p vj [array get PAGE] {puts $f "[list $p] [list $v]"} close $f
} http: : cleanup $token fetch-page-NCBI
proc fetch-complete {} { capture { variable S file mkdir $S (path) /cache set f [open $S (path) /cache/query w] foreach {p vj $S (cache) {puts $f "[list $p] [list $v] " } close $f
# This is the new code that traverses the amino sequence data and compares
# against the peptide pattern in the peptide search window. proc match { } { capture { variable S unset -nocomplain S (fetching) unset -nocomplain S (retry) set S (props) [lsort -dictionary [glob -nocomplain "$S (path) /cache/* .prop"] ] log "Matching [llength $S (props)] entries." for {set g o) {$q< [llength $S(props)]} {incr q} { set fn [lindex $S (props) $q] set accessionnumber [file tail [file root $fn] ] status $accessionnumber [expr {$q+l}] [llength $S (props)] set f [open $fn r] array set PAGE [read $f] close $f set results { } foreach sequence $PAGE (%seqdata) { set sequence [string tolower $sequence] array unset F * set machine { } for {set i 0; set j -1} {$i< [string length $sequence] } {incr i; incr j} { lappend machine 0 $i $j 0
} while { [llength $machlne] } { foreach {state 11 ul gap} $machine break; set machine [lrange
$machine 4 end] incr ul if {$state>= [llength $S (seqpattern) ] } { set "F ($11 [expr {$ul-l}])" 1 } elseif { [regexp {*[+] (.*)$} [lindex $S (seqpattern) $state] - aminos] } {" set aminos [string tolower $aminos] if {[string first [string index $sequence $ul] $aminos]>=0} { lappend machine [expr {$state+l}] $11 $ul 0
} } elseif {[regexp {*-(.*)$} [lindex $S (seqpattern) $state] - aminos] set aminos [string tolower $aminos] if {[string first [string index $sequence $ul] $aminos]<0} { lappend machine [expr {$state+l}] $11 $ul 0
} elseif {[regexp {Λ : (\d+) : (\d+) $} [lindex $S (seqpattern) $atate] - mingap maxgap] } { if {$gap<$mingap} { lappend machine $state $11 $ul [expr {$gap+l}] } else { if {$gap<$maxgap} { lappend machine $state $11 $ul [expr {$gap+l}]
} lappend machine [expr {$state+l}] $11 [expr {$ul-l}] 0
} } else { error "bad seqpattern element : [lindex $S (seqpattern) $state] " } } set result [lsort -integer -index 0 [array names F] ] if { [llength $result] } { lappend results [list [llength $result] $result $sequence] }
} if { [llength $results] } { set results [lsort -integer -index 0 $results] set PAGE (%subsequences) [lindex $results end 1] set PAGE (% sequence) [lindex $results end 2]
} else { set PAGE (%subsequences) { } set PAGE (fcsequence) [lindex $PAGE (%seqdata) 0] if { [llength $PAGE(%subsequences) ] } { array set PAGE {%pass 1 %reason "Matched amino acid pattern."} } else { array set PAGE {%pass 0 %reason "Did. not match amino acid pattern."} if {$PAGE(%pass) } { foreach {kind descriptor string} $S (criteria) { if { ! [string equal $kind must-have] } continue catch {unset exists} foreach {p extent} [array get PAGE $descriptor] { regsub -all { [Λ- [ :alnum: ] @%\ (\)_+=/] +} $extent { } extent foreach word [split [string tolower [string trim $extent] 3 " •] { set exists ($word) 1
}
} regsub -all { [Λ- [ :alnum:] @%\ (\)_+=/] +} $string { } string set any 0 foreach word [concat [split [string tolower [string trim $string] ]
11 ] unknown] { set any [info exists exists ($word) ] if {$any} break
} if { ! $any} { array set PAGE [list %pass 0 %reason "Missing must have word:
$string"] break
} if {$PAGE(%pass) } { foreach {kind descriptor string} $S (criteria) { if { [string equal $kind must-have] } continue array set PAGE {%pass 1 %reason "Matched amino acid pattern."} catch {unset exists} foreach {p extent} [array get PAGE $descriptor] { regsub -all { [*- [ :alnum: ]_] +} $extent { } extent foreach word [split [string tolower [string trim $extent] ] " "] set exists ($word) 1
}
} regsub -all { [Λ- [ :alnum: ]_] +} $string { } string foreach word [split [string tolower [string trim $string] ] " "] { if {[string equal $kind exclude] == [info exists exists ($word) 1} { array set PAGE [list %pass 0 treason "$kind word: $word"] break
}
} if {$PAGE(%pass) } break
if {$PAGE(%pass) } { log "Match pass $accessionnumber ([llength $PAGE (%subsequences) ] matches): $PAGE (%reason) "
} else { log "Match fail $accessionnumber : $PAGE (%reason) "
} file mkdir $S (path) /cache set f [open $S (path) /cache/$accessionnumber . prop w] foreach {p vj [array get PAGE] {puts $f " [list $p] [list $v] " } close $f if {$PAGE(%pass) } {incr S(pass)} else {incr S(fail)} incr S (seen); count $S(seen) $S(pass) $S(fail) update
}
:: report $S (path) $S(term) status "Completed." 0 0 completed } } proc report { } { variable S toplevel . reporttnessage wm overrideredirect .reportmessage 1 pack [label . reportmessage .1 -text "Creating pass/fail report."] -padx 20 -pady 10 uuppddaattee
:: report $S (path) $S(term) update destroy . reportmessage
#####################################################################
# #
# REPORT #
# # ##################################################################### #
# Format the search results into set of pages . This reporting is
# adapted the report generation of GenomeExplorer . proc report {path term} { capture { set fns [lsort -dictionary [glob -nocomplain "$path/cache/* .prop"] ] eval file delete [list "$path/fail .htm"] [glob -nocomplain "$path/pass/* .htm"] foreach fn $fns { array unset PAGE * set f [open $fn r] array set PAGE [read $f] close $f if {$PAGE(%pass) } { set passing [open $path/pass/$PAGE (%accession) .htm w] puts $passing "<html><headxtitle>$PAGE (^accession) </titlex/headxbody>" puts $passing "<Hl>$PAGE(%accession) </Hl>" puts $passing "<dl>" puts $passing "<dt>PubMed Code: <dd>$PAGE (%link) " puts $passing "<dt>Description: <dd>$PAGE (%definition) " puts $passing "<dt>Isolated: <dd>$PAGE (%year) " if {([string equal $PAGE (%source) unknown]} {puts $passing "<dt>Source: <dd>$PAGE (%source) "} if {! [string equal $PAGE (%strain) unknown]} {puts $passing "<dt>Strain: <dd>$PAGE (%strain) " } if {! [string equal $PAGE (%serotype) unknown]} {puts $passing "<dt>Serotype: <dd>$PAGE (%serotype) " } puts $passing "</dl>" set n 0 array set where {Amino-terminal {} Mid-molecule {} Carboxy-terminal {}} foreach ch [split $PAGE(%sequence) ""] { set C($n) " <b>$ch</bxSUP> [expr {$n+l}] </SUP>" set S($n) 0 incr n
} foreach pq $PAGE (%subsequences) { foreach {p q} $pq break if {$p<=$n/3} { lappend where (Amino-terminal) $p $q } elseif {$p<= (2*$n) /3 } { lappend where (Mid-molecule) $p $q } else { lappend where (Carboxy-terminal) $p $q
} for {set i $p} {$i<=$q} {incr i} {incr S($i)} set r [llength $PAGE (%subsequences) ] puts $passing "<p>" set wascol black puts -nonewline $passing "<font color= 'black' >" for {set i 0} {$i<$n} {incr i} { if {$S($i)} {set iscol red} else {set iscol black} if { ! [string equal $iscol $wascol] } { puts $passing "</font>" puts -nonewline $passing "<font color=τ $iscol ' >" set wascol $iscol puts -nonewline $passing " $C ($i) "
} puts $passing "</fontx/p><dl>" foreach w {Amino-terminal Mid-molecule Carboxy-terminal} { puts $passing "<dt>$w</dtxdd>" if { [llength $where($w)] } { foreach {p q} $where($w) { puts $passing "<p>" for {set i $p} {$i<=$q} {incr i} { puts -nonewline $passing $C($i)
} puts $passing "</p>"
} puts $passing "</dd>" } else { puts $passing "Zero subsequences." } puts $passing "</dl>" close $passing if {! [string equal $PAGE(%year) unknown]} { if {! [info exists mindate] j| $PAGE (%year) <$mindate} {set mindate
$PAGE (%year) } if { ! [info exists maxdate] || $PAGE (%year) >$maxdate} {set maxdate $PAGE (%year) }
} if { ! [info exists nsubs ($PAGE (%year) ) ] } { set nsubs ($PAGE (%year) ) 0 set sum($PAGE{%year) ) 0.0 set sumsq($PAGE (%year) ) 0.0 set reference ($PAGE(%year) ) ""
} incr nsubs ($PAGE (%year) ) 1 set sum ($PAGE (%year) ) [expr {$sum($PAGE (%year) ) + $r}] set sumsq($PAGE (%year) ) [expr {$sumsq($PAGE (%year) ) + $r*$r}] append reference ($PAGE (%year) ) "\n$PAGE (%link) <a href= 'pass/$PAGE (%accession) .htm' >$r</a>" } else { if { ! [info exists failing] } { set failing [open $path/fail .htm w] puts $failing {<htmlxheadxtitle>Failing Sequences</titlex/headxbody>} puts $failing "<pxb>query: </b> $term"
} puts $failing "<p>$PAGE (%link) : $PAGE (%reason) " set i 1 foreach e [split $PAGE (%sequence) " " ] { puts -nonewline $failing " <b>$e</bxSUP>$i</S0P>" incr i
} puts $failing " "
} catch {close $failing} set stats [open $path/report.htm w] puts $stats "<htmlxheadxtitle>Subsequences Analysis</titlex/headxbody>
<Hl>Subsequences Count by Year</Hl>
<H2>$term</H2>
<TABLE>
<TR>
<TH align= ' 1 center valign='top' >Year</TH>
<TH align=' 1 center valign=' top' >PubMed Accession Number-Subsequences
Count</TH>
<TH align=' 1 center valign=' top' >No. of Isolates per year</TH>
<TH align= ' 1 center valign=' top' >Mean Subsequences Count per year</TH>
<TH align= ( 1 center valign=' top1 >S.D.</TH>
</TR> set Y {} if {[info exists nsubs (unknown) ]} {lappend Y unknown} if { ! [info exists mindate]} {set mindate 2000} if { ! [info exists maxdate]} {set maxdate 2000} for {set y $mindate} {$y<=$maxdate} {incr y} {lappend Y $y} foreach y $Y { if { [info exists nsubs ($y) ] } { set mean [expr {$sum ($y) /$nsubs ($y) }]
# var = (sum (x - m)A2)/(n-l)
# = (sum (x*2 - 2xm + mΛ2))/(n-l)
# = (sum xA2 - 2m sum x + mΛ2 sum l)/(n-l)
# = (sumsq - 2*m*sum + n mΛ2)/(n-l) if {$nsubs($y)-==l} { set sd 0.0 } else { set sd [expr {sqrt ( ($sumsq($y) + $nsubs ($y) *$mean*$mean - 2*$mean*$sum($y) ) / ($nsubs ($y) -1) ) }]
} puts $stats "<TR>
<TD align= ' center ' valign=' top1 >$y</TD> <TD align='left' valign=' top' preference ($y) </TD> <TD align= ' right ' valign=' top1 >$nsubs ($y) </TD> <TD align= ' right ' valign=' top' > [format %.lf $mean] </TD> <TD align= 'right' valign= ' top1 > [format %.lf $sd] </TD> </TR>" } else { puts $stats "<TRxTD align= 'center' valign=' top1 >$y</TD><TD></TD><TD></TD><TD></TD><TD></TDx/TR>" } puts $stats "</TABLE>" close $stats set index [open $path/index. htm w] puts $index "<html><head><title>Index</title></headxbody><dl>" set fns [glob -nocomplain "$path/pass/*"] if { [llength $fnsj } { puts $index "<dt>Pass : <dd>" foreach fn $fns {puts $index "<a href=' pass/ [file tail $fn] ' > [file tail [file root $fn]]</a>"} puts $index "</dd>11 } puts $index "<dt>Report:<ddxa href=' report .htm1 >Subsequences by year</ax/dd>" if {[file exists $path/fail. htm] } { puts $index "<dt>Pail :<ddxa href=' fail .htm' >Failing sequences</ax/dd>" } close $index if { [pref openHTML default 0] } { exec osascript -e "open location \"file://l27.0.0.1 [file join $path/index.htm] \"" } }
#####################################################################
# #
# MANUAL CHECKLIST WINDOW #
# # ##################################################################### #
# The database query started in query-start extracts information
# about the protein, and the match function assigns a pass/fail
# whether the protein matches the pattern. This window allows
# manual editting of the query information presented in the report
# and alterring the pass/fail result. proc manualchecklist {sspace path title} { capture { array set S [list \ file-menu {
{Save and Close} {%S::save} {Command-S} {Command-s} {Cancel and Close} {%S::quit} {Command-W} {Command-w}
} \ title "$title Manual Checklist" \ zoomed 0 \ path $path \ sspace $sspace \ ] foreach {space w} [eval window ,manual%U [array get S]] break namespace eval $space { variable A; array set A {accessions {}} proc load-array {} { variable S variable A set fns [lsort -dictionary [glob -nocomplain "$S (path) /cache/* .prop"] ] foreach fn $fns { array unset PAGE * set f [open $fn r] array set PAGE [read $f] close $f lappend A(accessions) $PAGE (%accession) array set A [list \
$PAGE(%accession) -.pass $PAGE(%pass) \ $PAGE(%accession) : reason $PAGE (%reason) \ $PAGE (%accession) :uri $PAGE(%uri) \ $PAGE(%accession) -.year $PAGE(%year) \ $PAGE(%accession) :passθ $PAGE(%pass) \ $PAGE(%accession) :yearo $PAGE(%year) \ $PAGE(%aσcession) :path $fn \ ] } trace add variable $S (space) : :A write $S (space) •. :manualchanged
} proc manualchanged {arrname subscript op} { variable S variable A foreach {accession what} [split $subscript :] break if { [string equal $what pass] } { set A ($accession: reason) "Manually set."
} } proc manual-okay {} { variable S variable A foreach accession $A (accessions) { if {$A($accession:pass) !=$A($accession:passO) || $A($accession-.year) !=$A($accession-.yearO) } { array unset PAGE * set f [open $A($accession:path) r] array set PAGE [read $f] close $f if {$PAGE(%pass) !=$A($accession:pass) } { if {$A($accession:pass) } { set PAGE(%reason) "Manually set to pass." } else { set PAGE(%reason) "Manually set to fail." } } set PAGE(%pass) $A($accession:pass) set PAGE(%year) $A($accession:year) set f [open $A($accession:path) w] foreach {p v} [array get PAGE] { puts $f " [list $p] [list $v] "
} close $f
} } proc save { } { variable S manual-okay
[set S (sspace) ];: report quit proc quit { } { variable S destroy $S (window) after idle [list namespace delete $S (space)] } proc zoom {w h x y} { variable S set currgeom [wm geometry $S (window) ] if {$S(zoomed) && [string equal $currgeom $S(zoom)]} { set S (zoomed) 0 wm geometry $S (window) $S (geometry) } else { set S (geometry) $currgeom if {! [regexp {Λ=? (\d+)x(\d+) [+] (\d+) [+] (\d+)$) $currgeom - w h x y] } { foreach {w hx y} {θ 0 0 θ} break
} if {$w>[winfo screenwidth $S (window) ]} {set w [winfo screenwidth $S (window) ] } set S (zoom) ${w}x[expr {[winfo screenheight $S (window) ] -50}] +$x+20 set S (zoomed) 1 wm geometry $S (window) $S(zoom)
pack [frame $w. buttons] -side bottom -fill x -expand 1 -pady 5 pack [button $w.buttons. okay -text Okay -command "$ {space} :: save"] -side right -padx 20 pack [button $w.buttons. cancel -text Cancel -command "${space} : :quit"] -side left ${space} : : load-array upvar #0 ${ space} : :A A pack [scrollbar $w.y -orient vertical -command "$w.t yview"] -side right -fill y pack [text $w.t -yscrollcommand "$w.y set" -tabs {.7i .9i 1.11} -width 80] -side top - fill both -expand 1 set num 0 foreach accession $A(accessions) { $w.t insert end "$accession: \t"
$w.t window create end -window [checkbutton $w.t.pass$num -variable ${space} : :A($accession:pass) -text Pass] $w.t insert end "\t Year: "
$w.t window create end -window [entry $w. t .year$num -textvariable ${space} : :A($accession:year) -width 4]
$w.t window create end -window [button $w.t.uri$num -text "URL" -command "exec open $A($accession:uri) "]
$w.t insert end " \t$A($accession: reason) \n" incr num
}
$w.t configure -state disabled
} }
#####################################################################
# #
# LOG #
# # ##################################################################### #
# Spyware to track program/user interaction. window .log \ title {Activity Log} \ file-menu {
{Save as} {log save } {Command-S} {Command-s}
{Email} {log send-email} {} {}
{Close} {log hide} {Command-w} {Command-w}
} \ zoomed 0 wm withdraw . log pack [scrollbar .log.y -orient vertical -command ".log.t yview"] -side right -fill y pack [text .log.t -yscrollcommand ".log.y set"] -side top -fill both -expand 1 wm protocol .log WM_DELETE_WINDOW "log: :hide" namespace eval log { proc save { } { set path [tk_getSaveFile -message "Where to save the log" -title "Save Log"] if { [string length $path] } { set f [open $path w] puts $f [string trim [$S (window) .t get 1.0 end]] $S (window) . t show end close $f } proc quit { } { variable S destroy $S (window) } proc show { } { variable S wm deiconify $S (window) raise $S (window)
} proc hide { } { variable S wm withdraw $S (window) } proc zoom {w h x y} { variable S set currgeom [wm geometry $S (window) ] if {$S (zoomed) && [string equal $currgeom $S(zoom)]} { set S (zoomed) 0 wm geometry $S (window) $S (geometry) } else { set S (geometry) $σurrgeom if {! [regexp {Λ=? (\d+) x (\d+) [+] (\d+) [+] (\d+)$} $currgeom - w h x y] } { foreach {w h x y} {o 0 0 0} break
} if {$w> [winfo screenwidth $S (window) ]} {set w [winfo screenwidth $S (window) ]} set S(zoom) ${w}x[expr {[winfo screenheight $S (window) ] -50}] +$x+20 set S (zoomed) 1 wm geometry $S (window) $S(zoom) } } proc send-email {} { variable S exec mail -s {Log file} wyrmwif@rawbw.com << [string trim [$S (window) .t get 1.0 end]] } proc tell {text} { variable S set message "[clock format [clock seconds] -format %y/%m/%d.%H: %S: %S] .$text" puts stderr $message
$S (window). t insert end $message\n
#####################################################################
# #
# PREFERENCES #
# #
#
# Preference panel . proc pref {var args} { global preference if {! [info exists preference]} prefs: : open-prefs switch [llength $args] {
0 { if {[info exists preference ($var) ]} { return $preference ($var) } else { return " "
}
1 { set preference ($var) [lindex $args 0] prefs save }
2 { if { ! [string equal [lindex $args 0] default] } { error "usage : pref var default default-value"
} if { [info exists preference ($var) ] } { return $pref erence ($var) } else { return [1index $args 1] } default { error "usage : pref var . . . " }
} namespace eval prefs { variable S array set S { path " ~/Library/Preferences/com . omyx . drpepper .prefs . tcl" showing 0 factoryDefaults { debuggingMode 0 outputDirectory -/Desktop openHTML 0
proc open-prefs { } { variable S array set : preference $S (factoryDefaults) catch {source $S (path) } } proc save { } { variable S catch {cancel $S (pending) } set S (pending) [after 5000 prefs :: save-immediate] } proc save-immediate {} { variable S catch {cancel $S (pending)} set f [open $S (path) w] puts $£ "array set ::preference {" foreach {p v) [array get ::preference] { puts $£ " [list $p] [list $v] "
} puts $f "}"
proc show { } { variable S if { !$S(showing) } { global preference preferencel array set preferencel [array get preference] toplevel .preferences wm title .preferences Preferences wm geometry .preferences 400x90+100+100 place [ label .preferences.ldebug -text "Save for debugging:" -anchor e ] -x 0 -y 0 -relwidth 0.45 -height 25 place [ checkbutton . preferences . cdebug -text " " -variable preference1 (debuggingMode) ] -relx .50 -y 0 -relwidth 0.45 -height 25 place [ label .preferences .lopen -text "Open results in browser:" -anchor e ] -x 0 -y 30 -relwidth 0.45 -height 25 place [ checkbutton .preferences .copen -text " " -variable preferencel (openHTML) ] -relx .50 -y 30 -relwidth 0.45 -height 25 place [ button .preferences. cancel -text Cancel -command { destroy .preferences unset -nocomplain preferencel
1
] -x 0 -rely 1.0 -y -35 -width 100 place [ button .preferences. reset -text Reset -command { array set preferencel [array get preference]
] -relx 1.0 -x -210 -rely 1.0 -y -35 -width 100 place [ button .preferences. okay -text Okay -command { array set preference [array get preferencel] file mkdir $preference (outputDirectory) cd $preference (outputDirectory) set ch [open $prefs: :S (path) w] puts $ch "array set preference {" foreach {p v} [array get preference] { puts $ch " [list $p] [list $v] "
} puts $ch " } " close $ch destroy .preferences unset -nocomplain preferencel foreach p [lsort [array names preference] ] { log "preference $p = $preference($p) "
}
] -relx 1.0 -x -100 -rely 1.0 -y -35 -width 100 place [ button .preferences. crap -text " " -command {
] -relx 1.0 -x -100 -rely 1.0 -y -35 -width 100 lower .preferences. crap

Claims

What is claimed is:
1. A method of identifying a Replikin Scaffold in a virus or organism comprising identifying a series of Replikin Scaffold peptides comprising about 16 to about 30 amino acids comprising
(1) a terminal lysine and a lysine immediately adjacent to said terminal lysine;
(2) a terminal histidine and a histidine immediately adjacent to said terminal histidine, (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines.
2. The method of claim 1 further comprising identifying an individual member or plurality of members of said series of Replikin Scaffold peptides.
3. The method of claim 1 further comprising identifying an Exoskeleton Scaffold wherein said series of Replikin Scaffold peptides is identified in a first series of virus, strain of virus, or organism and said Exoskeleton Scaffold is identified in a later-arising virus, strain of virus, or organism as compared to said first series of virus, strain of virus, or organism wherein said Exoskeleton Scaffold comprises an amino acid sequence comprising about the same number of amino acids as said Replikin Scaffold and further comprises (1) a terminal lysine and a lysine immediately adjacent to said terminal lysine, (2) a terminal histidine and a histidine immediately adjacent to said terminal histidine, and (3) no lysine within about 6 to about 10 amino acids from another lysine.
4. The method of identifying said Replikin Scaffold peptide of claim 1 further comprising identifying a second Replikin Scaffold peptide of claim 1, comparing said Replikin Scaffold peptide to said second Replikin Scaffold peptide and if said second Replikin Scaffold peptide is unchanged from said second Replikin Scaffold peptide, choosing either Replikin Scaffold peptide as a vaccine.
5. An isolated or synthesized influenza virus peptide comprising from 7 to about 50 amino acids comprising (1) at least one lysine residue located six to ten residues from a second lysine residue wherein at least one lysine residue is a terminal lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
6. The isolated or synthesized influenza virus peptide of claim 5 wherein the isolated or synthesized influenza virus peptide is present in an emerging strain of influenza virus.
7. An isolated or synthesized influenza virus peptide comprising from 7 to about 50 amino acids comprising (1) at least one lysine residue located six to ten residues from a second lysine residue; (2) at least one histidine residue; (3) at least 6% lysine residues wherein said isolated or synthesized influenza virus peptide is present in an H5N1 strain of influenza virus.
8. The isolated or synthesized influenza virus peptide of claim 7 further comprising a terminal lysine.
9. The isolated or synthesized influenza virus peptide of claim 7 comprising a peptide comprising the amino acid sequence KKNSTYPTIKRSYNNTNQEDLLVLWGIHH.
10. The isolated or synthesized influenza virus peptide of claim 5 comprising about 16 to about 30 amino acids, and
(1) a terminal lysine and a lysine immediately adjacent to said terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to said terminal histidine;
(3) a lysine within about 6 to about 10 amino acids from another lysine; and
(4) at least 6% lysines wherein a homologue of said peptide is present in a plurality of different specimens of viruses, virus strains or organisms wherein said homologues comprise about 16 to about 30 amino acids, and
(5) a terminal lysine and a lysine immediately adjacent to said terminal lysine; (6) a terminal histidine and a histidine immediately adjacent to said terminal histidine; (7) a lysine within about 6 to about 10 amino acids from another lysine; and
(8) at least 6% lysines.
11. A preventive or therapeutic virus vaccine comprising at least one isolated or synthesized peptide of influenza virus comprising (1) at least one lysine residue located about six to about ten residues from a second lysine residue; (2) at least one histidine residue; (3) at least 6% lysine residues.
12. The preventive or therapeutic virus vaccine of claim 11 wherein said isolated or synthesized peptide is present in an emerging strain of influenza virus.
13. The preventive or therapeutic virus vaccine of claim 11 wherein said isolated or synthesized peptide is present in an H5N1 strain of influenza virus.
14. The preventive or therapeutic virus vaccine of claim 11 comprising a peptide comprising the amino acid sequence comprising KKNSTYPTIKRSYNNTNQEDLLVLWGIHH.
15. The preventive or therapeutic virus vaccine of claim 11 further comprising any of SEQ NO:732, SEQ NO:733; SEQ NO:734; SEQ NO:735; SEQ NO:736; SEQ NO:737; SEQ NO:738; SEQ NO:739; SEQ NO:740; SEQ NO:741; SEQ NO:742.
16. The preventive or therapeutic virus vaccine of claim 14 further comprising SEQ ID: 732.
17. The preventive or therapeutic virus vaccine of claim 11 further comprising a pharmaceutically acceptable carrier and/or adjuvant.
18. The preventive or therapeutic virus vaccine of claim 14 further comprising Vaccine V120304U2.
19. A method of stimulating an immune system of a subject to produce antibodies to influenza virus comprising administering an effective amount of at least one isolated or synthesized influenza virus Replikin peptide comprising from 7 to about 50 amino acids comprising (1) at least one lysine residue located six to ten residues from a second lysine residue wherein at least one lysine residue is a terminal lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues.
20. The method of claim 19 further comprising a pharmaceutically acceptable carrier and/or adjuvant and further preventing or treating an influenza infection.
21. The method of claim 19 wherein said isolated or synthesized influenza virus peptide is present in an emerging virus.
22. The method of claim 19 wherein said isolated or synthesized influenza virus peptide is present in an H5N1 strain of influenza virus.
23. The method of claim 19 wherein said isolated or synthesized influenza virus peptide comprises an amino acid sequence comprising KKNSTYPTIKRSYNNTNQEDLLVLWGIHH.
24. A method of making a preventive or therapeutic virus vaccine comprising identifying a Replikin Scaffold comprising a plurality of Replikin Scaffold peptides comprising about 16 to about 30 amino acids and isolating or synthesizing at least one of said Replikin Scaffold peptides as a preventive or therapeutic virus vaccine wherein said Replikin Scaffold peptides comprise:
(1) a terminal lysine and a lysine immediately adjacent to said terminal lysine;
(2) a terminal histidine and a histidine immediately adjacent to said terminal histidine;
(3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines.
25. The method of claim 24 wherein said Replikin Scaffold peptide is present in an influenza virus.
26. A method comprising: applying a plurality of criteria to data representing protein sequences; based on the criteria, identifying an arbitrary sub-sequence within the protein sequences; and outputting the identified sub-sequence to a data file; wherein the criteria include: a set {a} of amino acids to be included in the sub-sequence; a set {b}of amino acids to be excluded from the sub-sequence; and a minimum and a maximum permissible gap between members of sets {a} and {b}.
27. The method of claim 26, wherein the protein sequences are obtained via a network.
28. A machine-readable medium storing computer-executable instructions to perform a method according to claim 26.
29. A method comprising: applying a plurality of criteria to data representing protein sequences; based on the criteria, identifying a sub-sequence within the protein sequences, the identified sub-sequence having a predetermined allowed range of distance between lysine amino acids thereof, and a predetermined allowed range of distance between a histidine amino acid and a farthest lysine acid thereof; and outputting an identified sub-sequence to a data file.
30. The method of claim 29, wherein the protein sequences are obtained via a network.
31. A machine-readable medium storing computer-executable instructions to perform a method according to claim 29.
PCT/US2006/005343 2005-02-16 2006-02-16 Replikin peptides and uses thereof WO2006088962A2 (en)

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AU2006214332A AU2006214332B2 (en) 2005-02-16 2006-02-16 Replikin peptides and uses thereof
NZ560415A NZ560415A (en) 2005-02-16 2006-02-16 Systems and method for identifying replikin scaffolds and uses of said replikin scaffolds
EP06735145A EP1859063A4 (en) 2005-02-16 2006-02-16 Replikin peptides and uses thereof
JP2007555371A JP2008539164A (en) 2005-02-16 2006-02-16 Replikin peptide and use thereof
CA002598381A CA2598381A1 (en) 2005-02-16 2006-02-16 Replikin peptides and uses thereof
IL185308A IL185308A0 (en) 2005-02-16 2007-08-16 Replikin peptides and uses thereof

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US65308305P 2005-02-16 2005-02-16
US60/653,083 2005-02-16
PCT/US2005/014443 WO2005104754A2 (en) 2004-04-28 2005-04-28 System and method for identifying complex patterns of amino acids
USPCT/US2005/014443 2005-04-28

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US7763705B2 (en) 2001-03-27 2010-07-27 Samuel Bogoch Replikins and methods of identifying replikin-containing sequences
US7682619B2 (en) 2006-04-06 2010-03-23 Cornell Research Foundation, Inc. Canine influenza virus
JP2009538630A (en) * 2006-05-30 2009-11-12 ボゴーク，サミュエル Replikin peptides and uses thereof
WO2008060702A3 (en) * 2006-05-30 2008-07-03 Samuel Bogoch Replikin peptides and uses thereof
WO2008060702A2 (en) * 2006-05-30 2008-05-22 Samuel Bogoch Replikin peptides and uses thereof
WO2008140557A3 (en) * 2006-10-24 2009-03-26 Samuel Bogoch A method of predicting influenza outbreaks
US8050871B2 (en) 2006-10-24 2011-11-01 Samuel Bogoch Method of predicting influenza outbreaks by correlating an increase in replikin count in shrimp white spot syndrome virus and/or taura syndrome virus
WO2008143717A2 (en) 2007-01-18 2008-11-27 Samuel Bogoch Methods of determining lethality of pathogens and malignancies involving replikin peak genes
EP2126140A2 (en) * 2007-01-18 2009-12-02 Samuel Bogoch Methods of determining lethality of pathogens and malignancies involving replikin peak genes
EP2126140A4 (en) * 2007-01-18 2012-03-28 Samuel Bogoch Methods of determining lethality of pathogens and malignancies involving replikin peak genes
JP2011516027A (en) * 2007-01-18 2011-05-26 ボゴーク，サミュエル Methods for determining lethality of pathogens and malignant tumors involving the Replikin Peak gene
WO2008156914A3 (en) * 2007-05-30 2009-03-19 Samuel Bogoch Synthetic replikin peptides against pathogenic infection of invertebrates in aquaculture
US9408902B2 (en) 2007-05-30 2016-08-09 Samuel Bogoch Synthetic replikin peptides against pathogenic infection of invertebrates in aquaculture
WO2008156914A2 (en) * 2007-05-30 2008-12-24 Samuel Bogoch Synthetic replikin peptides against pathogenic infection of invertebrates in aquaculture
EP2594578A1 (en) * 2007-05-30 2013-05-22 Samuel Bogoch Replikin peptides directed against pathogenic infections of invertebrates in aquaculture
JP2010528610A (en) * 2007-05-30 2010-08-26 ボゴーク，サミュエル Synthetic Replikin Peptides against Invertebrate Pathogenic Infections in Aquaculture
WO2009132209A3 (en) * 2008-04-23 2010-03-18 Samuel Bogoch Methods and compounds for mitigating pathogenic outbreaks using replikin count cycles
WO2010017514A3 (en) * 2008-08-08 2010-04-01 Samuel Bogoch Methods of predicting cancer lethality using replikin counts
WO2010017514A2 (en) * 2008-08-08 2010-02-11 Samuel Bogoch Methods of predicting cancer lethality using replikin counts
US20100215675A1 (en) * 2009-01-09 2010-08-26 Samuel Bogoch Replikin-based compounds for prevention and treatment of influenza and methods of differentiating infectivity and lethality in influenza
US9233148B2 (en) 2009-01-09 2016-01-12 Samuel Bogoch Replikin-based compounds for prevention and treatment of influenza and methods of differentiating infectivity and lethality in influenza
WO2010123519A1 (en) * 2009-04-23 2010-10-28 Samuel Bogoch Replikin-sequences for preventing/treating influenza and determining infectivity/lethality
US9320784B2 (en) 2009-08-07 2016-04-26 Samuel Bogoch Peptides shared among lethal cancers and therapeutic compositions comprising said peptides
WO2013151662A2 (en) 2012-03-09 2013-10-10 Samuel Bogoch Therapies, vaccines, and predictive methods for infectious salmon anemia virus

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WO2006088962A3 (en) 2008-01-24
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AU2006214332A1 (en) 2006-08-24
NZ560415A (en) 2010-05-28
AU2006214332B2 (en) 2012-03-22
EP1859063A4 (en) 2012-02-01
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