WO2006088962A9 - Replikin peptides and uses thereof - Google Patents
Replikin peptides and uses thereofInfo
- Publication number
- WO2006088962A9 WO2006088962A9 PCT/US2006/005343 US2006005343W WO2006088962A9 WO 2006088962 A9 WO2006088962 A9 WO 2006088962A9 US 2006005343 W US2006005343 W US 2006005343W WO 2006088962 A9 WO2006088962 A9 WO 2006088962A9
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- WIPO (PCT)
- Prior art keywords
- replikin
- lysine
- virus
- peptide
- replikins
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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
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- 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
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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
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
- 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.
- 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 H1N2 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 or other vaccines. The following are examples of deduced UTOPEs and UVAXs:
- 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 0.5g NaH2P04 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.
- 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.
- the other entries in the dialog box allow some of the match criteria to be adjusted.
- the values given are those originally requested.
- 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 "
- 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 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.
- 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.
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06735145A EP1859063A4 (en) | 2005-02-16 | 2006-02-16 | Replikin peptides and uses thereof |
AU2006214332A AU2006214332B2 (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 |
NZ560415A NZ560415A (en) | 2005-02-16 | 2006-02-16 | Systems and method for identifying replikin scaffolds and uses of said replikin scaffolds |
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 |
Applications Claiming Priority (4)
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US65308305P | 2005-02-16 | 2005-02-16 | |
US60/653,083 | 2005-02-16 | ||
USPCT/US2005/014443 | 2005-04-28 | ||
PCT/US2005/014443 WO2005104754A2 (en) | 2004-04-28 | 2005-04-28 | System and method for identifying complex patterns of amino acids |
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WO2006088962A2 WO2006088962A2 (en) | 2006-08-24 |
WO2006088962A9 true WO2006088962A9 (en) | 2006-11-16 |
WO2006088962A3 WO2006088962A3 (en) | 2008-01-24 |
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EP (1) | EP1859063A4 (en) |
JP (1) | JP2008539164A (en) |
AU (1) | AU2006214332B2 (en) |
CA (1) | CA2598381A1 (en) |
IL (1) | IL185308A0 (en) |
NZ (1) | NZ560415A (en) |
SG (1) | SG160327A1 (en) |
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Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
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US7420028B2 (en) | 2001-03-27 | 2008-09-02 | 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 |
AU2007319669A1 (en) * | 2006-05-30 | 2008-05-22 | Elenore S. Bogoch | Replikin peptides and uses thereof |
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 |
US20090017052A1 (en) * | 2007-01-18 | 2009-01-15 | Samuel Bogoch | Methods of determining lethality of pathogens and malignancies involving replikin peak genes |
WO2008156914A2 (en) * | 2007-05-30 | 2008-12-24 | Samuel Bogoch | Synthetic replikin peptides against pathogenic infection of invertebrates in aquaculture |
CA2720465A1 (en) * | 2008-04-23 | 2009-10-29 | Samuel Bogoch | Methods and compounds for mitigating pathogenic outbreaks using replikin count cycles |
WO2010017514A2 (en) * | 2008-08-08 | 2010-02-11 | Samuel Bogoch | Methods of predicting cancer lethality using replikin counts |
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 |
EP2421546A1 (en) * | 2009-04-23 | 2012-02-29 | Samuel Bogoch | Replikin-sequences for preventing/treating influenza and determining infectivity/lethality |
WO2013013075A2 (en) | 2011-07-20 | 2013-01-24 | 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 |
Family Cites Families (6)
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EP0221748A3 (en) | 1985-11-01 | 1988-10-12 | Samuel Dr. Bogoch | A process for the production of human antibodies |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
US6242578B1 (en) | 1994-02-17 | 2001-06-05 | Samuel Bogoch | Aglyco products and methods of use |
US7420028B2 (en) * | 2001-03-27 | 2008-09-02 | Samuel Bogoch | Replikins and methods of identifying replikin-containing sequences |
US7442761B2 (en) * | 2003-06-06 | 2008-10-28 | Samuel Bogoch | Replikin peptides and uses thereof |
US11620305B2 (en) | 2021-03-25 | 2023-04-04 | Salesforce.Com, Inc | Ruleset engine for generating homogeneous data sets |
-
2006
- 2006-02-16 SG SG201001000-7A patent/SG160327A1/en unknown
- 2006-02-16 EP EP06735145A patent/EP1859063A4/en not_active Withdrawn
- 2006-02-16 JP JP2007555371A patent/JP2008539164A/en active Pending
- 2006-02-16 CA CA002598381A patent/CA2598381A1/en not_active Abandoned
- 2006-02-16 WO PCT/US2006/005343 patent/WO2006088962A2/en active Search and Examination
- 2006-02-16 NZ NZ560415A patent/NZ560415A/en not_active IP Right Cessation
- 2006-02-16 AU AU2006214332A patent/AU2006214332B2/en not_active Ceased
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AU2006214332A1 (en) | 2006-08-24 |
EP1859063A4 (en) | 2012-02-01 |
AU2006214332B2 (en) | 2012-03-22 |
EP1859063A2 (en) | 2007-11-28 |
NZ560415A (en) | 2010-05-28 |
IL185308A0 (en) | 2008-02-09 |
WO2006088962A2 (en) | 2006-08-24 |
WO2006088962A3 (en) | 2008-01-24 |
JP2008539164A (en) | 2008-11-13 |
CA2598381A1 (en) | 2006-08-24 |
SG160327A1 (en) | 2010-04-29 |
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