JP2008539164A - Replikin peptide and use thereof - Google Patents

Abstract

  The present invention relates to a novel class of peptides related to rapid replication and high human mortality, and their use in the diagnosis, prevention and treatment of diseases comprising vaccines and treatments of emerging viral diseases, and novel classes of peptides. And a method for identifying related structures.

Description

  The present invention generally relates to two newly discovered peptides that share the use of bioinformatics to retrieve structural features and other biological information to identify amino acids, nucleic acids and common structural features. About classes. Replikin is a newly discovered class of peptides that share structural features and are involved in the rapid replication of viruses and organisms. The Replikin backbone is a subset of the class of Replikin peptides. The exoskeleton scaffold is another newly discovered class of peptides that share structural features and correlate with reduced replication.

  This application claims priority to US Provisional Patent Application No. 60 / 653,083 filed on Feb. 16, 2005, and US Patent Application No. 11/116, filed Apr. 28, 2005. No. 203, which is a continuation-in-part application, which claims priority from US Provisional Patent Application No. 60 / 565,847 filed on April 28, 2004, and was filed on June 4, 2004. US patent application Ser. No. 10 / 860,050, which is a continuation-in-part of U.S. Provisional Patent Application No. 60 / 531,686, filed on Feb. 23, 2003, on Sep. 23, 2003. No. 60 / 504,958 filed and US Patent Application No. 60 / 476,186 filed June 6, 2003 and filed July 8, 2002 Of No. 10 / 189,437 A continuation-in-part application, which is a continuation-in-part of US patent application Ser. No. 10 / 105,232, filed Mar. 26, 2002, which is a US patent filed on Oct. 26, 2001. This is a continuation-in-part of application 09 / 984,057, which includes the priority of US Provisional Application No. 60 / 303,396 filed on July 9, 2001, and on March 27, 2001. Claim priority of filed 60 / 278,761. The previous application is incorporated herein by reference.

  Fast replication is a pathogenic feature in some bacteria, viruses and malignancies, but no chemicals common to fast replication in different organisms have been described. The inventor has discovered Replikin, a family of conserved small protein sequences for rapid replication. Such Replikin provides a new target for developing effective detection methods and therapies. There is a need in the art for methods of identifying amino acid patterns such as Replikin.

Bioinformatics identification of amino acid sequences Identification of amino acid sequences, nucleic acid sequences and other biological structures can be aided by performing bioinformatics. Publicly available databases containing amino acid and nucleic acid sequence information can be searched to identify and define Replikin, Replikin and Exoskeleton scaffolds in representative proteins or protein fragments or genomes or genome fragments.

  Amino acid and protein databases are maintained by various research institutions, such as the National Center for Biotechnology Information (NCBI) at the National Library of Medicine and the influenza sequence database at Los Alamos National Laboratory. These databases are accessible via the Internet through web pages that offer the possibility to search and obtain specific proteins for researchers.

Amino Acid Search Tools As is known in the art, protein and amino acid databases can be searched using a variety of database tools and search engines. Using these publicly available tools, amino acid patterns can be described and located in many different proteins corresponding to many different organisms. Several methods and techniques are available by which amino acid patterns can be described. One common format is the PROSITE pattern. The description of the PROSITE pattern can be constructed according to the following rules:

(1) The standard international pure applied chemical chemistry (IUPAC) single letter code for amino acids is used (see Figure 1).
(2) The symbol “x” is used for positions where any amino acid is accepted.
(3) Ambiguity is indicated by listing the acceptable amino acids for a given position between square brackets “[]”. For example, [ALT] refers to alanine, leucine or threonine.
(4) Ambiguity is also indicated by listing unacceptable amino acids at a given position between the pair of braces “{}”. For example, {AM} refers to any amino acid other than alanine and methionine.
(5) Each element in the pattern is separated from its neighbors by "-".
(6) The repetition of an element of a pattern can be indicated by a numerical value or numerical range between parentheses following that element. Example: x (3) corresponds to xx-x and x (2, 4) corresponds to xx or xx-x or xx-xx.
(7) If the pattern is limited to either the N- or C-terminus of the sequence, the pattern begins with the symbol “<” or ends with the symbol “>”, respectively.
(8) The period ends the pattern.

Examples of PROSITE patterns include:
PA [AC] -xVx (4)-{ED}. This pattern is translated as [alanine or cysteine] -arbitrary-valine-arbitrary-arbitrary-arbitrary- {optional but not glutamic acid or aspartic acid}.

  PA <Ax- [ST] (2) -x (0,1) -V. Must be at the N-terminus of the sequence ("<") This pattern is translated as follows: alanine-optional- "serine or threonine"-"serine or threonine"-(optional or none) -valine.

  Another common format for describing amino acid patterns is the regular expression format well known to computer scientists. In computer science, regular expressions are typically used to describe feature patterns for which finite automata are automatically constructed to recognize tokens in a language. Perhaps the most notable regular expression search tool is the Unix utility grep.

For describing amino acid sequence patterns, a simplified set of regular expression functions are typically employed. The amino acid sequence pattern defined by these simple regular expression rules will be quite similar to the PROSITE pattern both in appearance and result. A regular expression description of an amino acid sequence is made according to the following rules:
(1) Capital letters are used for amino acid residues, and (although not necessary) a “-” is placed between the two amino acids.
(2) “[]” is used for a plurality of amino acid options at a specific position. [LIVM] means that any one amino acid of L, I, V or M can be in that position.
(3) Use “{}” to exclude amino acids. Therefore, {CF} means that C and F must not be in that particular position. In some systems, exclusion features can be identified with a “^” symbol. For example, ^ G means all amino acids except glycine, and [^ ILMV] represents all amino acids except I, L, M, and V.
(4) For positions that can be any amino acid, use “x” or “X”.
(5) For a plurality of positions, “(n)” where n is a number is used. For example, x (3) is the same as “xxx”.
(6) “(n1, n2)” is used for plural or variable positions. Therefore, x (1,4) represents “x” or “xx” or “xxx” or “xxxx”.
(7) To request that the pattern match the N or C terminus, use the symbol “>” at the beginning or end of the pattern. For example, “> MDEL” finds only sequences that start with MDEL. “DEL>” finds only sequences that end with DEL.

  The regular expression “[LIVM] − [VIC] −x (2) −G− [DENQTA] −x− [GAC] −x (2) − [LIVMFY] (4) −x (2) −G” 17 with L, I, V or M in position 1; V, I or C in position 2; optional residues in positions 3 and 4; G in position 5 Represents an amino acid.

である。 Other similar formats are also used. For example, the Basic Local Alignment Search Tool (BLAST) is a well-known system available on the Internet and provides a tool for rapid searching of nucleotide and protein databases. BLAST accepts sequences entered in three formats: FASTA sequence format, NCBI accession number, or GenBank sequence number. However, these formats are simpler in structure than regular expressions or PROSITE patterns. Examples of FASTA format sequences are:

It is.

  Features of the BLAST system include sequence comparison algorithms that are used to search sequence databases for locally aligned regions to detect relationships between sequences that share similar regions. However, BLAST tools are limited with respect to the structure of amino acid sequences that can be discovered and located. For example, BLAST cannot search for sequences having “at least one lysine residue at a position of 6 to 10 amino acid residues from the second lysine residue” required by the Replikin pattern. BLAST also cannot search for amino acid sequences that contain a particular percentage or concentration of a particular amino acid, such as a sequence having “at least 6% lysine residues”.

Necessity of Replikin Search Tool As can be seen from its definition, a Replikin pattern description cannot be represented as a single linear array of amino acids. Thus, PROSITE patterns and regular expressions, both well-suited to describe ordered arrays obtained according to logical configuration operations such as negation, union and concatenation, describe Replikin patterns. Not suitable for.

  In contrast to the linear sequence of amino acids, the Replikin pattern is characterized by amino acid properties that go beyond a simple contiguous order. In particular, the requirement that the Replikin pattern contains at least 6% lysine residues means that the actual placement of lysine residues in the Replikin pattern is relatively unrestricted. Therefore, it is generally impossible to represent a Replikin pattern description using a single PROSITE pattern or a single regular expression.

  Therefore, there is a need in the art for a system and method for scanning a given amino acid sequence and identifying and counting all cases of a Replikin pattern. Similarly, there is a need for systems and methods for searching protein databases and amino acid databases for amino acid sequences that match a Replikin pattern. In addition, there is a need in the art for generalized search tools that allow researchers to locate amino acid sequences of any specified length that include any desired combination of the following features: 1) a first amino acid residue located above the N position and below the M position, away from the second amino acid residue; (2) a third amino acid residue located anywhere in the sequence; and (3) The sequence comprises at least R percent of the fourth amino acid residue. Finally, the shortcomings of the prior art are more apparent in the field of research related to disease prediction and treatment. In order to predict disease outbreaks in advance (such as strain-specific influenza epidemics), and similarly, it has been found to be preserved over time and has been detected so far by conventional methods for protein and amino acid sequence searches There is a great need in the art for a system to enable synthetic vaccines that have not been designed and designed based on amino acid sequences or amino acid motifs.

SUMMARY OF THE INVENTION The present invention provides a method for identifying nucleotide or amino acid sequences comprising Replikin sequences. This method is referred to herein as a three-point recognition method. By using the “three point recognition” method, (1) at least one lysine residue located 6 to 10 amino acid residues from the second lysine residue; (2) at least one histidine residue, and ( 3) Peptides containing 7 to about 50 amino acids, including at least 6% lysine residues (Replikin), and having replication, transformation or redox functions can be identified.

  An aspect of the present invention is a method for identifying a Replikin scaffold in a virus or organism, comprising: (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and the terminal histidine A series of from about 16 to about 30 amino acids, including immediately adjacent histidine, (3) lysine at about 6 to about 10 amino acid residues from other lysines; and (4) at least 6% lysine. A method is provided comprising identifying a Replikin backbone peptide.

  Aspects of the invention include (1) terminal lysine and lysine immediately adjacent to the terminal lysine; (2) terminal histidine and histidine immediately adjacent to the terminal histidine; (3) about 6 to about 10 from other lysines. A method of identifying a Replikin backbone in a virus or organism comprising from about 16 to about 30 amino acids, including lysine at amino acid residues; and (4) at least 6% lysine can be provided.

  An aspect of the present invention is also a method for producing a prophylactic or therapeutic viral vaccine, comprising identifying a Replikin scaffold comprising about 16 to about 30 amino acids, and as a prophylactic or therapeutic viral vaccine Synthesizing a Replikin backbone, wherein the Replikin backbone further comprises: (1) a terminal lysine and a lysine directly adjacent to the terminal lysine; (2) a terminal histidine and a histidine directly adjacent to the terminal histidine; 3) providing a method comprising from about 6 to about 10 amino acid residues from other lysines; and (4) at least 6% lysine. The Replikin backbone can contain influenza virus peptide Replikin. The Replikin backbone further comprises (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and histidine immediately adjacent to the terminal histidine; (3) about 6 to about 10 amino acids from other lysines. And (4) a group of Replikins containing at least 6% lysine.

  An aspect of the invention is a method for identifying an exoskeleton scaffold, wherein the Replikin skeleton is identified in a primary strain of a virus or organism, and the exoskeleton skeleton is a late strain of said virus or organism. Wherein the exoskeleton skeleton comprises the same number of amino acids as the Replikin skeleton, and further comprises (1) two terminal lysines, (2) two terminal histidines, and (3 ) The method comprises an amino acid sequence that does not contain lysine in about 6 to about 10 amino acids from other lysines.

  In an aspect of the invention, an isolated or synthesized influenza virus peptide is provided with at least one lysine residue located from 7 to about 50 amino acids, 6 to 10 residues from the second lysine residue. The In an even further aspect, the peptide is present in an emerging strain of influenza virus, eg, influenza virus strain H5N1.

  In a further aspect of the invention, an isolated or synthesized influenza virus peptide is provided comprising the H5N1 peptide, KKNSTYPTIKRSYNNTNQEDLLLVWGIHH.

  In another aspect of the invention, the isolated or synthesized influenza virus peptide has about 16 to about 30 amino acids; terminal lysine and lysine immediately adjacent to the terminal lysine; terminal histidine and immediately adjacent to the terminal histidine Histidine; lysine at about 6 to about 10 amino acid residues from other lysines; and at least 6% lysine.

  In another aspect of the invention, the prophylactic or therapeutic viral vaccine comprises at least one lysine residue located 6 to 10 residues from the second lysine residue; at least one histidine residue and at least 6% Provided with at least one influenza virus isolated or synthetic peptide with the following lysine residues.

  In yet a further aspect of the invention, a prophylactic or therapeutic viral vaccine comprises the peptide KKNSTYPTIKRSYNNTNQEDLLLVLVGIHH instead having a synthetic UTOPE tail, adjuvant or a combination thereof. In an even further aspect, the prophylactic or therapeutic viral vaccine comprises a pharmaceutically acceptable carrier.

  In a further aspect of the invention, the prophylactic or therapeutic viral vaccine comprises the peptide KKNSTYPTIKRRSYNNTNQEDLLLVLVGIHHKKKKHKKKKKHKKLH.

  In yet another aspect of the invention, a method of stimulating a subject's immune system to produce antibodies against influenza virus comprises (1) at least one located at 6 to 10 amino acid residues from the second lysine residue. At least one isolated or synthesized influenza virus replied comprising 7 to about 50 amino acids, comprising: 2 lysine residues; (2) at least one histidine residue; and (3) at least 6% lysine residues. It is provided comprising administering an effective amount of a quinpeptide.

  In a further aspect, the Replikin peptide administered in the method for stimulating the immune system may further comprise a pharmaceutically acceptable carrier and / or adjuvant and prevent or treat influenza infection. The method of stimulating the immune system may further comprise an isolated or synthesized influenza virus peptide present in the emerging virus or present in the influenza virus H5N1 strain. The method may further comprise administration of the peptide KKNSTYPTIKRSYNNTNQEDLLLVLVGIHHKKKKHKKKKKHKKLH.

  Aspects of the invention also apply a plurality of criteria to data indicative of a protein sequence; based on the criteria, identify any subsequence in the protein sequence; and identify the identified subsequence in a data file Wherein the criterion is: a set of amino acids {a} included in the subsequence; a set of amino acids excluded from the subsequence {b}; and a set {a} and A method can be provided that includes minimum and maximum allowable gaps between members of {b}. In the method, the protein sequence can be obtained via a network. Aspects of the invention can further include machine-readable media storing computer-executed instructions for performing such methods.

  Aspects of the invention further apply a plurality of criteria to the data representing the protein sequence; based on the criteria, identify any subsequence in the protein sequence, the identified subsequence being a predetermined sequence A distance between the lysine amino acids of a given tolerance and a distance between a histidine amino acid of a given tolerance and the furthest lysine; and outputting the identified subsequence to a data file. The protein sequence can be obtained via a network. A machine-readable medium that stores computer execution instructions may perform such methods.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, the term “peptide” or “protein” refers to two or more in which the carboxyl group of one amino acid is integrated with the amino group of the other amino acid. Means an amino acid compound. The term peptide is also used to denote the amino acid sequence encoding such a compound. As used herein, an “isolated” or “synthesized” peptide or biologically active portion thereof is, after purification, cell material from other cells or tissue sources derived from itself or other When substantially free of contaminating peptides, or when chemically synthesized by any method, substantially free of chemical precursors or other chemicals, or synthesized by recombinant gene technology Means a peptide substantially free from contaminating peptides.

As used herein, a Replikin peptide or Replikin protein is
(1) at least one lysine residue located 6 to 10 amino acid residues from the second lysine residue;
(2) at least one histidine residue; and (3) at least 6% lysine residues;
Is an amino acid sequence having 7 to about 50 amino acids.
Similarly, a Replikin sequence is an amino acid sequence that encodes such a peptide or protein.

  As used herein, an “early-occurring” virus or organism is a virus or organism collected from a natural source of the virus or organism prior to the day that another virus or organism specimen was collected. It is a specimen. A “late-onset” virus or organism is a virus or organism specimen collected from a natural source of the virus or organism after the day that another virus or organism specimen is collected.

  As used herein, the term “emerging strain” as used herein refers to the presence of one or more of its protein sequences relative to the concentration of Replikin in other strains of such organisms. Means strains of viruses, bacteria, fungi or other organisms identified as having an increasing concentration of the Replikin sequence. Increasing or increasing concentrations of Replikin occur, for example, in influenza viruses over a period of at least about 6 months, or preferably over a period of at least about 1 year, and most preferably over a period of at least about 3 years. For other organisms, it may be a much shorter period of time.

  As used herein, the term “mutation” refers to this change in structure and properties of an organism caused by amino acid substitutions. In contrast, the term “conservation” as used herein refers to the conservation of a particular amino acid due to a lack of substitution.

  As used herein, “Replikin Count” means the number of Replikins per 100 amino acids in a protein or organism. A higher Replikin Count in the primary strain of virus or organism results in faster replication of the primary virus or organism compared to an early or late generation strain of virus or organism having a second lower Replikin Count. It was discovered that they are correlated.

  As used herein, “Replikin backbone” means a series of conserved Replikin peptides, wherein each Replikin peptide sequence comprises from about 16 to about 30 amino acids, and further: (1 ) Terminal lysine; (2) terminal histidine and histidine immediately adjacent to the terminal histidine; (3) lysine in 6-10 amino acid residues from other lysines; and (4) about 6% lysine. The “Replikin backbone” peptide may comprise an additional lysine immediately adjacent to the terminal lysine. “Replikin skeleton” also means an individual member or members of a series of “Replikin skeletons”.

Identification of Replikins Identification of a new family of small peptides involved in the fast replication phenomenon, referred to herein as Replikins, provides targets for the development of therapies, including pathogen detection in samples and vaccine development . In general, knowledge and identification of this peptide family allows the development of effective therapies and vaccines for any organism with Replikin. The identification of this peptide family also provides for virus detection and viral vaccine development.

  For example, identification of this peptide family provides detection of influenza viruses and provides new targets and vaccines for influenza treatment, such as treatment and vaccines for influenza H5N1. Further examples provided by detection of the peptide family include detection of infectious disease Replikins, cancer immune Replikins and structural protein Replikins.

  Fast replication is a pathogenic feature in certain bacteria, viruses and malignancies, but to date no chemical reactions common to fast replication in different organisms have been described. We discovered a conserved small protein sequence family involved in fast replication and named it Replikin. Such Replikin provides a new target for developing effective detection methods and therapies. The first Replikin discovered was a glioma Replikin identified in a polymorphic brain glioblastoma (glioma) cell protein called mariignin.

  Marignonin hydrolysis and mass spectrometry revealed a novel 16-mer peptide sequence containing glioma Replikin. This Replikin was not found in the database for normal healthy human genomes and therefore appeared to be derived from some source outside the body.

  We have devised an algorithm to search for glioma Replikin or its homologues. Homologues were not common within more than 4000 protein sequences, but were surprisingly found within all tumor viruses and within algae, plant, fungus, virus and bacterial replication proteins.

  We have identified that 1) both Replikin concentration (number of Replikins per 100 amino acids) and Replikin composition correlate with the functional phenomenon of fast replication. These relationships provide a functional basis for the determination that Replikin is quantitatively and qualitatively related to the replication rate.

  The first functional basis for the role of Replikin for rapid replication was discovered by the applicant in glioma replication. The fact that glioma mariignin is enriched 10-fold compared to a 5-fold increase in cell number and membrane protein concentration in fast replication of glioma cells suggests an essential relationship of Replikin to replication. Abundant anti-malignin antibodies are produced when synthesized in vitro with glioma Replikin and administered to rabbits as a synthetic vaccine. This rigorously validates the antigenicity of the serum anti-malignin antibody (AMAS) test and provides the first potential synthetic cancer vaccine and other in vivo Replikin vaccine prototypes. Passive augmentation and synthesis of immunity with anti-malignin antibodies by demonstrating this innate immune response to cancer Replikin in preference to cell types and this innate immune relationship to replication based on the shared specificity of cancer Replikin and fast replication An active increase with Replikin vaccine is now possible.

  The relationship between the presence of anti-AG antibodies and survival in patients was shown in a study of 8,090 serum specimens from cancer patients. Similar to the increased incidence of cancer within the population, it has been demonstrated that the concentration of anti-malignin antibody increases with age and further increases 2-3 fold in early malignancies regardless of cell type. In vitro, the anti-malignin antibody is cytotoxic to the cancer in picograms (femtomole) per cancer cell, and the concentration of anti-malignin antibody in vivo is quantitatively related to the survival rate of cancer patients. As shown in glioma cells, the concentration of Replikin is now increased in cancer stages that have only been transformed to a malignant state where the cells do not die and remain in sedation or dormancy. It can be distinguished from a more active life-threatening replication state characterized by Furthermore, the fact that glioma glycoprotein 10B shows a 50% reduction in carbohydrate residues when compared to normal 10B may find clues to the viral etiology of cancer. This reduction has been linked to viral entry in other cases, and therefore evidence of viral attachment for delivery of viral Replikin to 10B of glial cells as a step in transformation to a malignant state. possible.

  Our work on influenza virus hemagglutinin protein sequences and influenza epidemiology over the past 100 years provided a second functional basis for the relationship of Replikin to rapid replication. So far, only serological hemagglutinin and antibody classification has been described in influenza, but no strain-specific conserved peptide sequences have been described so far. Furthermore, no changes in the concentration and composition of any strain-specific peptide sequences correlated with epidemiologically proven regional epidemics or rapid replication have been described. A 4-10 fold increase in the concentration of strain-specific influenza Replikin in one of each of the four major strains, influenza B, (A) H1H1, (A) H2N2 and (A) H3N2 From 1902 to 2001, it has been shown to be related to regional epidemics of influenza caused specifically by each strain.

  We have since found that these increased concentrations are due to the emergence of a new strain-specific Replikin composition in addition to the reappearance of at least one specific Replikin composition one year up to 64 years after its extinction showed that. Previously, strain-specific chemical structures that allow us to predict strains that would dominate the upcoming influenza epidemic, or to devise an annual mixture of all virus strains for vaccines, are completely known. It wasn't. The most recent H3N2 Replikin concentration (1997-2000), the largest in H3N2 history, and the high mortality H3N2 pandemic in 1968 and the two high mortality worlds in 1975 and 1977 The reappearance of a specific Replikin composition that was last seen in an epidemic but was absent for 20-25 years may be a warning of an upcoming regional epidemic. This high conservation of the Replikin structure that allows the same structure to persist for 100 years or reappear after 1 to 64 years of absence is due to the random substitution of amino acids in influenza proteins. What was previously thought to be an attributed change represents a higher probability of the change being attributed to the Replikin's organized preservation process.

  Replikin conservation is not unique to influenza viruses, for example, it has been observed in foot-and-mouth disease virus, type O and HIV tat and also in wheat.

  A third functional basis for the role of Replikin in fast replication is seen in the increase in fast replication within HIV. Replikin concentration has been shown to be related to high speed replication in HIV. We found that the Replikin concentration in the slow growing low titer strain of HIV (NS1, “Bru”) that predominates in early infection is the rapidly growing high titer of HIV (which predominates in late HIV infection). It was found to be one sixth of the stock (SI, “Lai”).

  A further example demonstrates the relationship of Replikin to high speed replication. For example, in the “replicating protein” of the tomato leaf curl geminivirus that disrupts the tomato harvest, the first 161 amino acids, a sequence that has been shown to bind to DNA, contains five Replikins. It was shown that. In malaria, which is legendary for fast replication when tens of thousands of trypanosomes are released from the liver at a rate of tens of thousands of trypanosomes, a large number of novel replikins at concentrations of up to 36 overlapping replikins per 100 amino acids. An almost “flame-like” Replikin structure was discovered.

  The preservation of either structure is crucial to whether the structure provides a stable and unchanging target to attack, destroy or stimulate. If a structure is somehow tied to the basic living mechanism of a living body, the structure tends to be preserved. The fluctuating structure provides an invariant target, which is excellent for avoiding attackers such as antibodies that are generated specifically against the previous structure and thus ineffective against the modified form. It is a strategy. This strategy is used for influenza viruses and the like, and thus prior vaccines may now be completely ineffective against pathogenic viruses.

Replikin as a stable target for therapy Both bacteria and HIV have both Replikin and non-Replikin amino acids. In HIV, for example, drug resistance has recently increased by 9% to 13% due to mutations, ie amino acid substitutions that are not essential for the determination of the Replikin structure. (See detailed analysis of HIV TAT protein discussed herein). In bacteria, the development of “resistant strains” is due to a similar mechanism. However, we have found that the Replikin structure is not mutated or altered to the same extent as non-Replikin amino acids (see also the discussion of Replikin's foot-and-mouth disease virus conservation discussed herein). Also see the discussion of coronavirus Replikin conservation discussed herein). Replikin structures, unlike non-Replikin structures, are conserved and thus provide new constitutive targets for therapy.

  Certain structures that are overly closely related to survival function do not appear to be constantly changing. Because the basic component of the Replikin structure is histidine (h), which is known to frequently bind to the energy source and the metal group within the oxidoreductase with a high probability of being required for replication, and As this histidine structure remains constant, it remains an even more attractive destruction or stimulation target.

  From a proteomic point of view, by constructing the template itself based on the newly determined glioma peptide sequence, the inventors have associated conserved structures and special functions, in this case replication. I came to discover a wide variety of protein types. Examples of increased Replikin levels associated with the pathogenicity of one disease are influenza, HIV, cancer and tomato leaf curl virus. This newly recognized species structure has been implicated in various in vivo rapid replication phenomena such as influenza, yeast, algae, plants, gemini tomato leaf blight virus, HIV and cancer.

  Replikin concentrations and compositions provide a new quantitative method to detect and control the replication process that is central to the viability and dominance of each biological population. As demonstrated with anti-malignin antibodies for gliomas and related cancer Replikins, the sharing of immunological specificity by the diverse members of this type uses a similar recognition language for B cells and their product antibodies. This suggests that Replikin can be recognized.

  Examples of peptide sequences as cancer Replikin peptide sequences or those containing Replikin or homologues of the glioma peptide kagvaflhkk are lung, brain, liver, soft tissue, salivary gland, nasopharynx, esophagus, stomach, colon, rectum, gallbladder Can be found in cancers such as, but not limited to, breast, prostate, uterus, cervix, bladder, eye, melanoma morphology, lymphoma, leukemia and kidney.

  Replikin is a 1 Treatment and control; and 3) provide for enhanced growth rate of algal and plant foods.

  The first Replikin sequence to be identified was found in Marignonin, a brain cancer protein that was demonstrated to be 10-fold enriched during fast anaerobic replication of glioblastoma multiforme (glioma) cells. Cancer cell Replikin. (FIG. 2). Mariignin is the 10 KDa portion of the 250 KDa glycoprotein 10B isolated in vivo and in vitro from the membranes of pluripotent glioblastoma (glioma) cells. The hydrolysis and mass spectrometry of mariignin, referred to herein as glioma Replikin, is a 16-mer peptide sequence ykagvaflhkknide (SEQ ID NO: 4) containing the shorter peptide kagvaflhkk (SEQ ID NO: 1) (both in normal human genome) Then it was apparently absent).

  Abundant anti-malignin antibodies were produced when 16-mer glioma Replikin was injected into a rabbit as a synthetic vaccine. (Bogoch et al., Detection and prevention of cancer. 26 (Appendix 1): 402 (2002)). The concentration of anti-malignin antibody in serum in vivo has been shown to be quantitatively related to the survival rate of cancer patients (Bogoch et al. Biofluid Peptide, 31: 739-747 (1984). Anti-malignin antibodies have been shown to be cytotoxic to cancer cells at a concentration of picograms (femtomole) per cancer cell (Bogoch et al., Cancer Detection and Prevention. 26 (Appendix 1): 402 (2002).

  Studies conducted by the inventors have shown that glioma Replikin does not appear in a normal healthy human genome. As a result, the search for the origin of the Replikin sequence and possible homologues was undertaken by analysis of the published sequences of various organisms.

  By using a 16-mer glioma Replikin sequence as a template and constructing a recognition proteome system to visually scan the amino acid sequences of multiple different biological proteins, a new type of peptide, Replikin, has been identified. The present invention provides methods for identifying nucleotide or amino acid sequences that include a Replikin sequence. This method is referred to herein as a three-point recognition method. The three-point recognition method consists of (1) at least one lysine residue located 6-10 residues from the second lysine residue, (2) at least one histidine residue, and (3) at least 6% A peptide of 7 to about 50 amino acids (Replikin) containing These peptides or proteins constitute a new class of peptides among species including algae, yeasts, fungi, amoeba, bacteria, plants, viruses and cancer proteins with replication, transformation or redox functions. Replikin peptides have been found to be concentrated in larger “replication” and “transformation” proteins (referred to by the investigators as such, see Table 2) and cancer cell proteins. . No sequence has been found to be identical to the marignin 16-mer peptide.

  The present invention relates to (1) at least one first lysine located at either end of an isolated or synthesized peptide, (2) 6 to 10 residues from the first lysine residue. To identify a nucleotide or amino acid sequence comprising a Replikin sequence comprising from 7 to about 50 amino acids comprising a second lysine located, (3) at least one histidine; and (4) at least 6% lysine Provide a method. In another aspect of the invention, the isolated or synthesized peptide is an influenza virus peptide. In yet another aspect of the invention, the isolated or synthesized peptide is an H5N1 influenza virus peptide.

Table 2
Various in vivo Replikin Examples-Prototype: Glioma Replikin ^* KAGVAFLHKK (SEQ ID NO: 1)

  The identification of the amino acid sequence as Replikin or containing Replikin, ie as a homologue of the glioma peptide kagvaflhkk, requires that the following three requirements be met. According to the three-point recognition system, the sequence is located within the amino acid sequence of 7 to about 50 residues within the following three elements: (1) 6 to 10 residues from the other lysine residue At least one lysine residue; (2) at least one histidine residue; and (3) at least 6% lysine residues. A typical non-limiting Replikin contains a terminal lysine.

  A database was searched for protein sequences containing Replikin using the National Library of Medicine keyword “PubMed” descriptor. Over 4000 protein sequences were visually examined for homologues. The sequences of all individual proteins within each group of PubMed classified proteins were visually scanned for peptides meeting the three requirements described above. Rare occurrences of homologues have been designated as being associated with malignant transformation or replication, such as among the “viral peptides” (1.5%) (N = 953) and “brain peptides” and “neuropeptides” Among other non-peptides (8.5% combined) (N = 845) was observed. Surprisingly, however, homologues were identified at a much higher frequency among large “replicating proteins” identified as having proven function in replication within bacteria, algae and viruses. Even more surprising, Replikin homologs were found to be 100% (N = 250) of “Tumor virus”, 97% (N = 401) of “Oncoprotein”, and 85% of “transformed virus” (N = 248). These results suggest that there are shared characteristics in the etiology of cancer, regardless of cell type, and that it is more pathogenic from carcinogenesis, ie, from a transformed state, whether it is dormant. This suggests a role for the virus in the transformation of cells to a highly active replicating state.

  A homologue of an amino acid sequence such as kagvaflhkk as defined by the three-point recognition method is adenovirus, lentivirus, a-virus, retrovirus, andeno related virus, human immunodeficiency virus, encephalitis virus, influenza virus, corn streak Virus, herpes virus, bovine herpes virus, feline immunodeficiency virus, foot-and-mouth disease virus, smallpox virus, rous sarcoma virus, neuroblastoma RAS virus oncogene, polyoma virus, Sindbis, human papilloma virus, bone marrow monocyte It has been found among viruses or viral peptides such as, but not limited to, sexual tumor virus, murine acute leukemia, T cell lymphotrophic virus and tomato defoliation virus.

  Furthermore, homologues of the amino acid sequence kagbafhkk are present in known types of coronaviruses that are members of the coat virus family that replicate in the cytoplasm of the host cell. In addition, homologues of the amino acid sequence kagvatlhkk are present in the recently identified class of coronaviruses responsible for severe acute respiratory syndrome or SARS. Replikin is located within the entire nucleocapsid protein sequence of SARS coronavirus. In addition, the location of Replikin is present among other members of the coronavirus type, and more particularly within the nucleocapsid protein sequences from these coronaviruses.

  Replikin is Acetobacter, Achromobacter, Actinomyces, Aerobacter, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Chainia, Clostridium, Corynebacterium, Erwinia, Escheria, Lebsiella, Lactobacillus, Haemophilus, ethyl, , Nocardia, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Staphylococcus, Streptocossus, Streptomyces, Streptosporangium, Streptovirticillium, Vibrio peptide and Xanthomas (but not limited to). Replikins are Penicillium, Diseula, Ophiostoma novoulim, Mycophycophta, Phytophthora infestans, Absidia, Aspergillus, Candida, Cephalosporium, Fusarium, Hansenula, Mucor, Paecilomyces, Pichia, Rhizopus, and Is not). Replikin is present in yeasts such as, but not limited to, Saccharomyces, Cryptococcus, including Cryptococcusneoformas, Schizosaccharomyces, and Oryza. Replikins are Caldophera, Isolepisprolifera, Chondrus, Gracilaria, Gelidium, Caulerpa, Laurencia, Cladophexa, Sargassum, Penicillos, Halimeda, Laminaria, Fucus, Ascophyllum, Undari, Rhodymenia, Macrocystis, ocfelas Is not present). Replikin is present in amoeba such as (but not limited to) Entamoeba (including Entamoeba invadens), Amoebidae, Acanthamoeba and Naegleria. Replikin is present in plants such as, but not limited to, Arabidopsis, wheat, rice and corn.

Auxiliary Identification To be able to classify the Replikin subtypes, (a) cancer cell proteins (eg transforming protein P21B (K-RAS 2B) lung, , Based on structural basis such as the common occurrence of adjacent di- and polylysine in SEQ ID NO: 89) and other adjacent diamino acids in TOLL-like receptors, or (b) as seen, for example, in Table 2 Additional or “auxiliary identification” based on functional evidence showing ATPase, tyrosine kinase or redox activity can be added.

Functional derivatives A “functional derivative” of a Replikin described herein is a fragment, variant, analog or chemistry of a Replikin that retains at least a portion of the immunological cross-reactivity with an antibody specific for Replikin. Derivative. Replikin peptide fragments refer to any subset of the molecule. Variant peptides can be made, for example, by direct chemical synthesis using methods well known in the art. By Replikin analog is meant a non-native protein that is substantially similar to either the whole protein or a fragment thereof. Chemical derivatives of Replikin usually contain additional chemical moieties that are not part of the peptide or peptide fragment.

Replikin and replication As seen in FIG. 2, during anaerobic respiration where the rate of cell replication is increased, mariignin is enriched. That is, malignin is not only found to increase in direct proportion to the increase in cell number and total membrane protein, but it starts at 3% at rest and reaches 30% of total membrane protein, resulting in a 10-fold concentration. Is also enriched. This obvious demonstration of a significant increase in Replikin concentration associated with glioma cell replication suggests and is consistent with the presence of Replikin identified by three-point recognition in various organisms. For example, Replikin is a "Saccharomyces cerevisiae replication-binding protein" (SEQ ID NO: 2) (hsikrelgiifdk); "Maize streak virus replication-related protein A" (SEQ ID NO: 8) (kyivcareehk) and (SEQ ID NO: 9) (kekkpskdeimrdisish); "Replication-related protein of Staphylococcus aureus" (SEQ ID NO: 10) (kkektthnk); "DNA replication protein of bovine herpesvirus 4" (SEQ ID NO: 11) (hkinitngqk); and "Mealgrid herpesvirus 1 replication binding protein" (SEQ ID NO: 12) ) (Hkdlylllmk). Previous studies on tomato leaf blight Gemini virus have shown that regulation of viral accumulation involves binding of amino acids 1-160 of the viral "replication protein" to leaf DNA and other replication protein molecules during viral replication It is shown that. Analysis of this sequence showed that amino acids 1-135 of this “replicating protein” contained high Replikin counts (concentrations) such as 20.7 (see section on tomato curb geminivirus) ).

  Table 2 shows that Replikin-containing proteins are also frequently associated with redox function and protein synthesis or elongation and cell replication. Replikin is all associated with metal-based redox function, enrichment of Replikin-containing glioma malignin concentrations during anaerobic replication, and cytotoxicity of antimalignin at low concentrations (picograms / cell) (FIGS. 4C-4F). Is associated with a central respiratory survival function and suggests that it has been found to be less subject to mutations characteristic of non-Replikin amino acids.

Replikin in influenza outbreaks Of particular interest is that at least one Replikin per 100 amino acids is found to be present in the hemagglutinin protein of almost all of the individual strains of influenza virus tested. It was observed. For each year for which amino acid sequence data is available (1902-2001), the hemagglutinin protein of each of the four dominant strains of influenza virus, influenza B, H1N1, H2N2 and H3N2 isolates. The Replikin sequences observed to occur in are shown in Tables 3, 4, 5 and 6.

  It has been discovered that the concentration and type, ie the observed Replikin composition, is related to the occurrence of influenza pandemics and regional epidemics. The concentration of Replikin in influenza virus is the hemagglutinin amino acid published in the National Medical Library “PubMed” database for the world's isolated influenza strains from human and animal accumulation over the last century, ie 1900-2001 The sequence was examined by visual scanning. These Replikin concentrations (number of Replikins per 100 amino acids, average value +/− SD) were then plotted for each strain.

  Replikin concentrations have been found to be directly related to the occurrence of influenza pandemics and regional epidemics. The concentration of Replikin is found in influenza B hemagglutinin and influenza A strain, H1N1, as shown in FIG. 7, and the concentration of Replikin is two other common influenza virus A strains shown in FIG. , H2N2 and H3N2 (H2N2 and H3N2). The data in FIG. 8 also shows a new emerging strain of influenza virus, as evidenced by the component Replikin (H3N2 (R)).

  Each influenza A strain is due to a pandemic in 1918, 1957 and 1968, respectively. The data in FIGS. 7 and 8 show that there is at least one Replikin per 100 amino acids in each of the influenza hemagglutinin proteins of all four common influenza virus isolates tested. Suggest a function for Replikin in maintaining the survival level of replication. In the 1990s, during the decline of the H3N2 strain, Replikin was not present in many isolates of H3N2, but a high concentration of new Replikin was found in the H3N2 isolate, which is H3N2 (R) Specify the occurrence of stocks. See Tables 3, 4, 5 and 6.

１．インフルエンザＢ型は、ヒトにおけるいかなる世界的流行の原因でもなかった。
２．年号の略号：例えば「４３」＝１９４３年、「０１」＝２００１年。
３．一定の与えられたレプリキンが現われた最初の年は、そのレプリキンが発見された一連の年の始めに示されている。
４．重複するレプリキン配列は別に列挙される。
５．下線の付された、１９７７年の地域的流行の前２年以内の、複数年にわたり不在であったレプリキンの復帰は、合計レプリキン濃度（レプリキン計数＝１００個のアミノ酸残基あたりのレプリキン数）の増加と相関関係をもつ。図７参照。

1. Influenza B was not the cause of any pandemic in humans.
2. Abbreviation of year: for example, “43” = 1943, “01” = 2001.
3. The first year in which a given Replikin appeared was indicated at the beginning of the series of years in which the Replikin was discovered.
4). Overlapping Replikin sequences are listed separately.
5. Reversion of Replikins that were absent for multiple years within 2 years prior to the 1977 regional epidemic, underlined, is the total Replikin concentration (Replikin Count = Replikin Count per 100 amino acid residues). Correlate with increase. See FIG.

１．インフルエンザＨ１Ｎ１型は、１９１８年のヒトにおける世界的流行の地球規模の分布の原因であった。
２．年号の略号：例えば「９６」＝１９９６年。
３．一定の与えられたレプリキンが現われた最初の年は、この研究作業においてはそのレプリキンが発見された一連の年の始めに示されている。
４．重複するレプリキン配列は別に列挙される。
５．（下線の付された）例えば１９１８年及び１９７７年の地域的流行年に、新しいレプリキン構造の数の増加が起こり、このことは合計レプリキン濃度（１００個のアミノ酸残基あたりのレプリキン数）の増加と相関関係をもつ。図７参照。

1. Influenza H1N1 was responsible for the global distribution of the pandemic in 1918 in humans.
2. Abbreviation of year: “96” = 1996.
3. The first year in which a given Replikin appeared was indicated at the beginning of the series of years in which the Replikin was discovered in this research work.
4). Overlapping Replikin sequences are listed separately.
5. For example, in the 1918 and 1977 regional epidemic years (underlined), an increase in the number of new Replikin structures occurred, which increased the total Replikin concentration (number of Replikins per 100 amino acid residues). And have a correlation. See FIG.

１．インフルエンザＨ２Ｎ２型は、１９５７年のヒトにおける世界的流行（地球規模分布）の原因であった。
２．年号の略号：例えば「５８」＝１９５８年。
３．一定の与えられたレプリキンが現われた最初の年は、この研究作業においてはそのレプリキンが発見された一連の年の始めに示されている。
４．重複するレプリキン配列は別に列挙される。
５．（下線の付された）例えば１９５７年及び１９６５年の地域的流行の年に、新しいレプリキン構造の数の増加が起こり、このことは合計レプリキン濃度（１００個のアミノ酸残基あたりのレプリキン数）の増加と相関関係をもつ。図８参照。

1. Influenza H2N2 was responsible for the 1957 human epidemic (global distribution).
2. Abbreviation of year: for example, “58” = 1958.
3. The first year in which a given Replikin appeared was indicated at the beginning of the series of years in which the Replikin was discovered in this research work.
4). Overlapping Replikin sequences are listed separately.
5. In the year of the regional epidemic, eg 1957 and 1965 (underlined), an increase in the number of new Replikin structures occurred, which is the total Replikin concentration (number of Replikins per 100 amino acid residues) Correlate with increase. See FIG.

１．インフルエンザＨ３Ｎ２型は、１９６８年のヒトにおける世界的流行（地球規模分布）の原因であった。
２．年号の略号：例えば「７７」＝１９７７年。
３．一定の与えられたレプリキンが現われた最初の年は、そのレプリキンが発見された一連の年の始めに示されている。
４．重複するレプリキン配列は別に列挙される。
５．（下線の付された）例えば１９７５年といった年において、新しいレプリキン構造の数の増加が起こり、このことは、合計レプリキン濃度（１００個のアミノ酸残基あたりのレプリキン数）の増加と相関関係をもつ。図８参照。

1. Influenza H3N2 was responsible for the 1968 human epidemic (global distribution).
2. Abbreviation of year: “77” = 1977, for example.
3. The first year in which a given Replikin appeared was indicated at the beginning of the series of years in which the Replikin was discovered.
4). Overlapping Replikin sequences are listed separately.
5. In years such as 1975 (underlined), an increase in the number of new Replikin structures occurred, which correlates with an increase in total Replikin concentration (number of Replikins per 100 amino acid residues). . See FIG.

  7 and 8, it can be seen that some characteristics of Replikin concentration are common to all four influenza virus strains. First, the concentration is periodic over the years, with a single cycle of rising and falling occurring over a period of 2 to 30 years. This rise and fall is consistent with the known known enhancement and attenuation of individual influenza virus strains by hemagglutinin and neuraminidase classification. Secondly, the peak Replikin concentration of each influenza virus strain already shown to be responsible for the pandemic was observed to be specifically and individually related to each year of the pandemic for three years. It was. For example, for the 1918 pandemic that was shown to be caused by influenza virus strain H1N1, the peak concentration of Replikin in H1N1 occurred independently (P1); For the 1957 pandemic shown, peak concentrations of Replikin in H2N2 occurred (P2); and for the 1968 pandemic that H3N2 was emerging and shown to be responsible for, H3N2 A peak concentration of Replikin was generated (P3). Third, in each year immediately following each of the three global epidemics mentioned above, the specific Replikin concentration is significantly reduced, probably reflecting the widely distributed immunity generated in each case. . Thus, this decline after the global epidemic is specific to H1N1 immediately after the global epidemic (P1) it was responsible for, and not a general characteristic of all strains at that time. Increasing Replikin concentrations in influenza B simultaneously with decreasing Replikin concentrations in H1N1, such as 1951 EB1 and 1976 EB2, both linked to the influenza B pandemic, both of which have the highest mortality Repeatedly occurred. (Stuart-Harris et al., Edward Arnold Ltd. (1985). Fourth, secondary peak concentrations beyond the primary peak increase in concentration occurred 15 years after each of the three global epidemics. This secondary peak was accompanied by one global epidemic: H1N1 “regional epidemic” year (E1) 15 years after the 1918 global epidemic; E2) 8 years after the 1957 global epidemic; and H3N2 “regional epidemic” year (E3) occurred 7 years after the 1968 global epidemic, these secondary peak concentrations of certain Replikins are: Fifth, the peak of the specific Replikin concentration of each strain often appears to be associated with a decrease in the Replikin concentration of one or both other strains, Suggests competition among stocks against Sixth, there is a clear overall trend in which the Replikin concentration of each strain decays over a period of 35 years (H2N2) to 60 years (influenza B), which is a general trend for influenza vaccines. The effect of the vaccine cannot be attributed to the fact that it was evident in the case of influenza B from 1940 to 1964 prior to use, and in the case of influenza B, the Replikin's recovery from decay was after 1965 As can be seen, Replikin concentrations declined again between 1997 and 2000 (Figure 7), which correlates with a low incidence of influenza B in recent case isolates. Combined with the reappearance and epidemic of the H1N1 strain, the H1N1 Replikin concentration peaked between 1978 and 1979, after which Simultaneously with the N1 pandemic, it peaked in 1996 (Fig. 7) H1N1 Replikin concentrations likewise declined between 1997 and 2000, and the presence of H1N1 strains was obtained during these years. For H2N2 Replikin, there was no recovery from the 35 year decay (Figure 8), which correlates with the absence of H2N2 in recent isolates. For many isolates, the Replikin concentration dropped to zero between 1996 and 2000, while the other H3N2 isolates showed a significant sharp increase in Replikin concentration. It shows the emergence of a sub-strain of H3N2, called H3N2 (R).

  Figures 7 and 8 demonstrate that a gradual increase of 1 to 3 years is frequently observed before the Replikin concentration reaches a peak. This incremental increase precedes the outbreak of regional epidemics that occur simultaneously with the Replikin Peak. Thus, a gradual increase in the concentration of a particular strain is a signal that this particular strain is the most likely candidate to cause a regional or global epidemic.

  Currently, the Replikin concentration of the H3N2 (R) strain of influenza virus is increasing (FIG. 8, 1997-2000). Three similar leading peak increases in H3N2 Replikin concentrations were observed in the 1968 H3N2-based global epidemic (FIG. 8) and the 1972 and 1975 H3N2-based regional epidemics (FIG. 8). It can be seen that this occurred at Each of these global and regional epidemics was associated with excessive mortality (Ailing et al., Am. J. Epidemiol., 113 (1): 30-43 (1981)). Thus, the rapid increase in the concentration of H3N2 (R) subspecies of H3N2 Replikin in 1997-2000 statistically represents an early warning of an upcoming serious regional or global epidemic. A regional epidemic of H3N2 occurred in Russia in 2000 (Figure 8, E4), and the December 2001 CDC report shows that H3N2 is currently the most frequently isolated strain of influenza virus worldwide. It is said that there is. (Morbidity and Mortality Weekly Report (MMWR), Center for Disease Control; 50 (48); 1084-68 (December 7, 2001).

  New Replikins are emerging in each case of influenza virus global or regional epidemics. There is no observation for two of the same Replikins in a given hemagglutinin within a given isolate. It is not known to what extent the new Replikin emerging represents a transfer or mutation from another animal or avian pool. In some cases, one or more of the original Replikin structures are preserved each year, while new Replikins are emerging at the same time. For example, in influenza virus type B hemagglutinin, 5 Replikins are permanently stored between 1919 and 2001, while 26 Replikins come and go in the same period (partially Relapsed after several years of absence). The disappearance of certain Replikin structures and revival many years later suggest that Replikin returns from another viral host pool rather than through a new mutation.

  In the case of the H1N1 Replikin, the two Replikins present within the P1 peak associated with the 1918 pandemic were not present in the 1933 recovered E1 peak, which contained 12 new Replikins. Thus, permanently stored Replikin is the best option for a vaccine, either alone or in combination. However, even the recently appearing Replikins that accompany the 1-year concentration increase often persist and increase after 1 year, resulting in concentration peaks and regional epidemics, thus an early warning for vaccination with synthetic Replikin (See, eg, H1N1, early 1990s, FIG. 7, as well as, eg, H5N1, 1995-2002, FIG. 11. “Replikin Count” (per 100 amino acids). PRK number) means Replikin concentration).

  The data in FIGS. 7, 8, 11 and 15 demonstrate a direct relationship between the presence and concentration of specific Replikins within the influenza protein sequence and the occurrence of influenza pandemic and regional epidemics. Thus, analysis of influenza virus hemagglutinin protein sequences for Replikin presence and concentration provides predictive material for influenza pandemic and / or regional epidemics and targets for influenza vaccine formulation. With reference to this data, strain-specific chemical structures are known that allow for the prediction of strains that would predominate in the upcoming influenza epidemic and to devise that year mixture of all virus strains for vaccines. It is noteworthy that there was no.

  Similar to the discovery of increased strain-specific Replikin counts within the influenza group 1 to 3 years before the occurrence of strain-specific regional epidemics, increased Replikin counts for coronavirus nucleocapsid proteins have also been identified. Replikin counts for coronavirus nucleocapsid proteins were increased as follows. 3.1 (± 1.8) in 1999; 3.9 (± 1.2) in 2000; 3.9 (± 1.3) in 2001; and 5.1 (± 3. 6). This previous increase in pandemic corroborates the discovery that coronavirus is responsible for the current (2003) SARS pandemic. (See Table 7).

  Thus, by monitoring Replikin structure and Replikin enumeration, synthetic strain-specific for 1917-1918 goose Replikin and its modified associated Replikin as observed in both influenza and coronavirus strains A means of developing prophylactic vaccination and antibody therapy is provided.

  FIG. 10 depicts an automated Replikin analysis of nucleocapsid coronavirus proteins for which protein sequences are available for isolates collected from 1962 to 2003. Each individual protein is represented by an accession number and analyzed for the presence of Replikin. The Replikin Count (Replikin Count per 100 amino acids) is automatically calculated as part of an automated Replikin analysis. For each year, an average (± standard deviation (SD)) Replikin count is automatically calculated for all Replikin counts for that year. This early replication increase alert prior to the regional epidemic of a specific protein (nucleocapsid protein) in a specific virus strain (coronavirus) is an increase seen in influenza virus strains prior to the regional epidemic and pandemic of influenza (Figs. 7, 8, 11 and 15). It can be seen that the Replikin Count rose from 1999 to 2002 consistently with SARS coronavirus peptide D, which was emerging at the end of 2002 and lasted until 2003. FIG. 9 provides a graph of Replikin Counts for multiple virus strains, including coronavirus nucleocapsid Replikin from 1917 to 2002.

The origin of SARS and H3N2-Fujian influenza virus Replikin SARS virus, which dates back to the 1918 pandemic Replikin, is still unknown. We have found that a certain SARS virus peptide caused more than 20 million deaths through homologous peptides in several strains of influenza virus isolates since 2002, and the worldwide influenza of 1918 Report evidence that it can be traced back to a single sequence within the epidemic strain.

  By quantitative analysis of the primary protein sequences of influenza viruses and other microorganisms recorded throughout the last century, we have identified lysines that are not related to the type of organism (eg, Table 1) but rather to the phenomenon of rapid replication itself and regional epidemics. And discovered a new type of peptide structure rich in histidine and named it Replikin. We have discovered a new class of peptide structures with an obligatory algorithm of at least 2 lysine 6-10 residue separations in 7-50 amino acids, lysine concentrations over 6%, 1 histidine. Because these peptides are associated with fast replication events and regional epidemics, we named them Replikin. We have found a quantitative correlation between strain-specific Replikin concentration in the hemagglutinin protein (Replikin Count = number of Replikins per 100 amino acids) and influenza regional and global epidemics (FIG. 7). To date, no correlation between the chemical nature of strain-specific viral proteins and the regional epidemic of influenza has been reported. Conservation, condensation and enrichment of Replikin structures were also found in influenza (in Table 7a), HIV and malaria. The detection of Replikin in SARS coronavirus, in addition to its possible evolutionary tracking, allowed the synthesis of small SARS antigens for vaccines.

  We show the quantitative correlation of strain-specific Replikin concentrations (counts) in influenza hemagglutinin protein between the regional epidemic of influenza and the three global epidemics of the last century in 1918, 1957 and 1968. discovered. A similar course was observed for each of these three global epidemics. That is, the strain-specific high Replikin counts followed immediately followed by a decline, followed by an increase in “rebound” accompanied by regional epidemics. Similarly, an increase in warning counts from 1 to 3 years preceded most regional epidemics.

  We have a replikin in hemagglutinin of an influenza virus isolated from a goose in 1917 (we named it goose replikin), which is 2 kg, kkg (t / s) It was discovered that it appeared in the H1N1 strain of influenza, the cause of the 1918 pandemic, the following year with only one substitution. Table 7a shows that influenza 1917 goose Replikin (GR) was basically conserved for 85 years at this time, despite numerous small substitutions and overt translocations to other influenza strains. . We report that the 1917 influenza GR was affected by H1N1 (1918 pandemic), H2N2 (1957-58 pandemic), H3N2 (1968 pandemic, 2000 China and Russia in 2000) It was found that it showed a clear mobility among multiple influenza strains that appeared in the epidemic, the 2003 Fujian strain regional epidemic) and the H5N1 (1997 regional epidemic in China). In 1997, the structure was restored to exactly its 1918 structure kkgssypklsksyvnnnkgkevlvlwgvhh in H1N2.

  SARS coronavirus first appeared during the 2002-2003 influenza epidemic. The double origin in 2002 of SARS Replikins from influenza GR and coronavirus Replikin (or some unknown shared precursor) is suggested by the following events, all occurring in 2002: 1) The first condensation in 85 years is seen at 29-28 amino acids within the GR-H1N2 Replikin sequence (Table 7a) (similar condensation is 29-27 amino acids within Fujian H3N2 in the current regional epidemic [Table 7a]); 2) Replikin count of GR-H1H2 showed significant decline consistent with GR migrating out of H1H2; 3) Replikin count of coronavirus nucleocapsid protein was significantly increased And 4) SARS coronavirus in 2002-2003 with Replikin containing the following motifs Appeared: “kkg” and “k-k” seen earlier in GR1918 and GR-H1N2 2001; “k-kk”, “kk” and “kl” seen in influenza GR-H1N2 2001; “Kk” found in bronchitis coronavirus Replikin; and “kk-kk-k”, “kk”, “kk”, “kl” and “kk” found in Replikin with porcine infectious diarrhea kt "(Table 7a) (SARS is believed to have first appeared in humans as infectious pneumonia that was ejected in a densely populated apartment house with a strong support of fecal contamination and then carried by a ventilator fan) .

  The high Replikin Count Peak, including the presence of the 1917 Goose Replikin (Figure 7), which has continued to increase in recent years, is now approaching the 1917 Replikin Count in H1N2 (Table 7a), leading to high mortality. Coming may already have begun since the current carriers of the short Replikin derivatives of goose Replikin shown in Tables 7 and 7a as accompanying are SARS virus and H3N2-Fujian virus Can be a global epidemic warning.

  Since goose Replikin has a history of at least 85 years involving most and all of influenza A and SARS A strains, it and its components are conserved vaccine candidates for pan strain protection. A condensed short SARS replikin that is 7 to 21 amino acids long and enriched with% of lysine and histidine compared to the goose replikin is more than that of the 29 amino acid goose replikin (2.5%) Occurs with high SARS mortality (10-55%). Here, short Replikins mixed with long Replikins in SARS can be responsible for high mortality. This also applies to other biological Replikins such as Ebola and smallpox virus and Bacillus anthracis (Table 7a). These short SARS replikins showed surprising homology with other living short replikins such as smallpox, anthrax and ebola associated with an even higher untreated mortality rate.

  Short synthetic vaccines are produced much more rapidly (daily rather than monthly) and are much cheaper, but the immunology produced by multiple epitopes of thousands of unwanted proteins across all current whole virus vaccines Side effects associated with mechanical interference and contamination should be avoided. For influenza, in all cases current whole virus vaccines are not effective in more than half of the elderly. But is Short Replikin sufficiently immunogenic? The short glioma Replikin “kagvaflhkk” has proved to be the basis for the successful acquisition of synthetic anti-polymorphic glioblastoma and anti-bronchiogenic cancer vaccines. It produced cytotoxic anti-malignin antibodies that were cytotoxic to cancer cells in picograms / cell and were quantitatively related to the survival rate of cancer patients. To prepare for SARS attacks that are likely to recur because of the spike seen in the 200 coronavirus nucleocapsid Replikin count, we have four SARS found in nucleocapsid, spike and coat protein. A short Replikin was synthesized. We have found that these synthetic short SARS Replikins also produce abundant specific antibodies when injected into rabbits. For example, a 21 amino acid SARS nucleocapsid Replikin antibody binds at a dilution greater than 1 in 204,800. Previous attempts by other researchers to use various small peptides to achieve a strong immune response without the undesirable side effects obtained with thousands of proteins or nucleic acids or whole protein as in smallpox have failed. Thus, the ability of small synthetic Replikin antigens to achieve a strong immune response is significant for the efficacy of these SARS vaccines.

  We investigated the relationship between Replikin structure in influenza and SARS viruses and increased mortality and obtained the results as shown in Table 7. A high mortality relationship to short or condensed Replikin sequences is found in the high mortality in vivo shown in Section B of Table 7 in influenza and viruses and bacteria other than SARS, malaria and cancer. Replikin for rapid replication rather than any cell type or infectious organism, in addition to widespread basic Replikin structures in a wide range of virus, bacteria, malaria and cancer organisms where replication is critical for transmission and pathogenicity The following homologous sequences were observed to support the concept of integrating the relationship between and the Replikin structure. “K” at positions 1 and 2; an alignment of “k” to be presented to DNA, RNA or other receptors or ligands for uptake or to stimulate rapid replication; “double k” and “ Frequency of multiple k's; most condensed shortening associated with living organisms, cancer cells and genes with various highest mortality rates such as smallpox virus, anthrax virus, rous sarcoma virus and glioblastoma multiforme (glioma) Frequency of triplets “kkg”, “hek”, “hdk” and “hkk” in Replikin, c-src in colon and breast cancer and c-yes in melanoma and colon cancer and frequency of “g” at position 3 Please note that. Note also the nearly identical Replikin structures for the two recently emerging high mortality viruses in Australia and Southeast Asia, Nipab and Handrah viruses. These two viruses have been reported to have similar or identical antibodies formed against them, but so far for this similar antibody, the two nearly identical Replikins Along with our findings, there is no known structural basis for this.

  Table 7 also shows the relationship of the five SARS Replikins of 2003 we discovered to both the 1917 influenza goose Replikin and two coronaviruses, avian bronchitis coronavirus and swine infectious diarrhea virus. Yes. The first 2003 human SARS Replikin in Table 7 shows some sequence homology to influenza virus goose 1917 and human 1918 Replikin through the intermediate structure of influenza H1H2 in 2002 (eg, positions 1, 18 and 19 See Replikin “k”). It can be seen that the 1917 goose Replikin sequence has been largely conserved in Table 7 despite numerous substitutions in amino acids that are not critical to the definition of Replikin up to 1999 (substitutions are shown in italics). At this time, it was found that the original 29-amino acid 1917 Replikin sequence had recovered almost exactly to its 1917-1918 structure within the 2001H1N2 Replikin. However, the 2002H1N2 influenza Replikin has been shortened from 29 to 28 amino acids, and the “shift to the left” of the amino acids kevl (i / v) wg (v / i) hh is evident.

  In 2003, one Replikin was further shortened (or consolidated) to the 21 amino acid Replikin of the first listed 2003 human SARS virus. The% of k for the 2003 SARS Replikin is currently 38.1% (8/21) compared to 20.7% for the Goose Replikin and the pandemic Replikin in 1918 humans. Compared to the influenza 29 amino acid Replikin, the three SARS Replikins were found to be further shortened (or consolidated) to 19, 11 and 9 amino acid long sequences, respectively. In the SARS9 amino acid sequence shown, the% of k is 44.4% (4/9). With the shortening of SARS Replikin, SARS human mortality has increased to 10% in young people and 55.5% in older adults compared to 2.5% in the 1918 influenza pandemic.

  The amino acid sequences are shown in Table 7 to highlight the degree of conservation and homology over 85 years (1917-2002) of influenza Replikins whose evidence was first observed in 1917 goose Replikin. No such conservation has ever been observed. Table 7 also shows that the Replikin in the 2003 human SARS virus has homology to the 1917 Goose Replikin and the influenza Replikin that first appeared as a pandemic influenza Replikin in 1918 humans, Avian bronchitis virus Replikin (eg, ends with “k”, “h” at positions 1 and 2) and coronavirus acute diarrhea virus Replikin (eg, “k” at positions 1 and 11, “h” at the end of Replikin) Are shown to exhibit some sequence homology to both. This evidence of the relationship of both influenza and coronaviruses to Replikin is that the SARS virus has occurred in Hong Kong, as has some recent influenza regional epidemics and earlier pandemic, and SARS viruses are nucleocapsids, spikes and other Part of the protein, including the protein, is advantageous because it has been classified as a new coronavirus because of its structure. Some epidemiological evidence also appeared to be the first in humans as SARS was contagious pneumonia airborne by a blower fan in a dense Hong Kong apartment house that was a strong support of fecal contamination Relevant in terms.

Composition of Replikins in Influenza Virus B Strains: Of a total of 26 Replikins identified in this strain (Table 3), the following 10 Replikins were all influenza B types investigated in 1940-2001 Is present in isolates. Overlapping Replikin sequences are listed separately. To demonstrate homology consistent with “three-point recognition”, lysine and histidine are bolded.

  Tables 3 and 4 show that influenza B hemagglutinin appears to have much greater stability of the Replikin structure compared to H1N1 Prikin. Influenza B has never been responsible for any pandemic and does not appear to have an animal or avian reservoir (Stuart-Harris et al., Edward Arnold Ltd., London (1985)) .

Replikin in Influenza in Time Influenza H1N Replikin; Among all the H1N1 degrading strains for which sequences are available from 1918 when this strain first appeared and caused the global epidemic of that year to 2000, 1 There is only one Replikin “hp (v / i) tigcpkyv− (r / k) (s / t) (t / a) k” (Table 4). ("(V / i)" indicates that amino acids v or i are in the same position in different years). Although H1N1 contains only one surviving Replikin, H1N1 appears to be more prolific than influenza B. There are 95 different Replikin structures on H1N1 in 82 years, while there are only 31 different Replikins in 62 years of influenza B isolates (Table 4). An increase in the number of new Replikin structures occurs in the year of regional epidemics (Tables 3, 4, 5 and 6) and correlates with an increase in total Replikin concentration (Figures 7, 8, 11 and 15).

Influenza H2N2 Replikin: Influenza H2N2 was responsible for the 1957 human epidemic. Three of the 20 Replikins identified in the strain for 1957 were conserved in each of the H2N2 isolates that were available for testing on PubMed until 1995 (Table 5). ).

  However, in contrast to H1N1, only 13 additional Replikins were found in H2N2, which began in 1961. This small number of new Replikin occurrences correlates with the appearance of H2N2 and the decline in H2N2 Replikin concentration in isolates over many years (FIG. 8).

  Influenza H3N2 was the cause of the 1968 pandemic in humans. The five Replikins that appeared in 1968 disappeared after 1977 but reappeared in 1990 (Table 6). The only Replikin structure that survived for 22 years was hcd (g / q) f (q / r) nekwdlf (v / i) er (s / t) k, which first appeared in 1977 and persisted until 1998. The emergence of twelve new H3N2 Replikins in mid-1990 (Table 6) shows a simultaneous increase in Replikin concentration (Figure 8) and the spread of H3N2 strains in recent isolates and all of these isolates Correlates with the simultaneous disappearance of Replikin (FIG. 8), suggesting the emergence of a new substrain H3N2 (R). The current regional epidemic of November-December 2003 of a new strain of H3N2 (Fujian type) is the first prediction made in US provisional application US60 / 303,396 dated July 9, 2001. Have confirmed.

  7, 8, 11 and 15 are due to the reappearance of at least one Replikin 1-59 years after the disappearance of the regional and global epidemic of influenza. It is shown to correlate with an increase in the concentration of Replikin in influenza virus. Similarly, there is an emerging new strain-specific Replikin composition in strain A alone (see also Tables 4-6, as well as the increase in the number of new Replikins prior to the regional epidemic for H5N1 in FIGS. 11 and 15). ). Increases in Replikin concentrations due to repeated individual Replikins within a single protein do not appear to occur in influenza viruses, but are seen in other organisms.

  Changes in the activity of different influenza strains are themselves thought to be related to the sequence change of influenza hemagglutinin, a product of substitution brought about by one of the following two poorly understood processes: Yes. I) consecutive antigenic mutations that may be due to the accumulation of a series of point mutations within the hemagglutinin molecule, or ii) gene changes occur between human and non-human host viruses due to the very large changes It is an antigenic discontinuous mutation, which is assumed to be. First of all, the data show that changes in the activity of different influenza strains are not associated with increased non-specific Replikin concentrations, but rather the replication associated with increased strain-specific Replikin concentrations and regional epidemics. It is based on or related to a strain-specific increase in In addition, the data was examined for possible insights about which sequence changes are due to “continuous mutations” or “discontinuous mutations” and which are due to conservation, storage in the reservoir and reappearance. The data show that a regional epidemic-related increase in Replikin concentration is not due to duplication of existing Replikins per hemagglutinin, but a recurrence of at least one Replikin composition from 1 to up to 59 years after its disappearance. The emergence and A strain alone indicates that it is due to the emergence of a new strain-specific Replikin composition (Tables 3-6). Thus, the increase in Replikin concentration in the 1951 and 1977 influenza B regional epidemics was not linked to the emergence of new Replikin compositions in the regional pandemic year, but appeared in the preceding year and subsequently disappeared Only associated with the reappearance of the composition (Table 3). In contrast, in strain A, in addition to the reappearance of the previously extinguished virus Replikin, a new composition appears (eg, six earlier in H1N1 in the year of the 1996 regional epidemic In addition to the reappearance of Replikin, 10 new compositions have emerged). Since only A strain, not influenza B, has access to non-human animals and avian reservoirs, the completely new composition is probably not similar to the new composition except for the basic requirement of “3-point recognition” Rather than from a possible existing human Replikin mutation, but rather from a non-human host reservoir (Table 2-5). The fact that H1N1 is more prolific than type B and the fact that the pandemic was only generated by three A strains and not by type B strains is both a new Replikin composition from the non-human virus reservoir It can also be a function of the ability of the human A strain to receive.

  Some Replikins appear and disappear for only one year and have not reappeared to date (Tables 3-6). Other Replikins disappear from 1 year up to 81 years, at which point the same Replikin sequence reappears. The major Replikin “k” and “h” amino acids and the spaces between them represent the constitutive presence of a particular Replikin over the years, as shown in Tables 2 and 3-6 for the following strain-specific Replikins: Conserved (for influenza B 10, single H1N1 Replikin and H3N2 single Replikin and reappearance of the same Replikin after absence). Despite significant exchange or substitution activity of other amino acids both inside and outside the Replikin structure in the rest of the hemagglutinin sequence, influenza Replikin histidine (h) is never exchanged It appears that lysine (k) is rarely exchanged. An example of this conservation is the H1N1 Replikin “hp (v / i) tigcpkyv (r / k) (s / t) (t / a) k” (SEQ ID NO: 135), which is constant between 1918 and 2000, H3N2 Replikin “hcd (g / q) f (q, r) nekwdlf (v / i) er (s / t) k” (SEQ ID NO: 277), which is constant between 1975 and 1998, and in 1975 “Hqn (s / e) (e / q) g (t / s) g (q / y) aad (l / q) kstq (a /) that first appeared, disappeared for 25 years, and then reappeared in 2000 n) a (i / l) d (q / g) I (n / t) (g / n) k, (l / v) n (r / s) vi (e / c) k "(SEQ ID NO: 276) ). While many amino acids have been substituted, the basic Replikin structure of two lysines, 1 histidine, and a minimum of 6% lysine separated by 6 to 10 residues of about 50 amino acids or less was conserved.

  Completely random substitutions, both the survival of these H1N1 and H3N2 Replikins, the persistence of the 10 Replikin structures in influenza B from 1902-2001, and the 1918 18-mer Replikin after 74 years of absence It does not appear to be possible to reappear in 1993. Rather than random types of substitution, homeostasis suggests an orderly and controlled process, or at least their protection in such a way that the main Replikin is fixed or bound in some way. Suggests (possibly lysine bound to nucleic acid and possibly histidine bound to respiratory oxidoreductase). The mechanism that controls this preservation is currently unknown.

H5N1 Influenza Conservation of Replikin Skeleton The recent pandemic of H5N1 “bird flu” with high mortality in some countries may represent the first stage of an influenza pandemic that has expired. A recent report states that in the first possibility of human-to-human transmission of H5N1, “the sequencing of the viral gene does not change anything in the hemagglutinin receptor binding site or other important features of the virus. , Suggesting that the sequences of all eight viral gene fragments are closely aggregated with other H5N1 sequences from recent poultry isolates in Thailand. Strain development analysis suggested that H5N1 is not a novel mutant, due to the absence of “reassembly with human influenza virus”. However, we now report three recent changes in specific H5N1 protein sequences at sites that have not changed in the last two H5N1 epidemics and in fact have been conserved since 1959.

  To date, there has been no protein chemistry that correlates with viral epidemic and dormancy. We have found that each of the three influenza pandemics of the last century, H1N1, N2N2 and H3N2, is related to fast replication, an increase in the concentration of a particular class of peptides in the virus, called Replikin, in lysine and histidine Retrospectively predicted by abundance and correlated. We have now found a Replikin that is predictive in each of the three H5N1s in 1997, 2001, and 2003-2004 (FIG. 15). Each year they appear in the isolate, Replikins can be counted every 100 amino acids, as in FIG. 15, and their sequences were analyzed and compared as shown in Table 9. Analysis of Replikin can be accomplished manually or, in a preferred aspect of the invention, mechanically by software designed by the inventor to count Replikin concentrations in the available sequence information.

  A graph showing the rapid increase in Replikin pattern in the hemagglutinin protein of the H5N1 strain of influenza before the three “bird flu” pandemics can be seen in FIG. The review in FIG. 15 shows that increasing Replikin concentration (“Replikin Count”) in H5N1 hemagglutinin protein preceded three “avian influenza” epidemics. For example, the increase in Replikin coefficient (mean value +/− SD) in 1995-1997 preceded the Hong Kong H5N1 epidemic (E1) in 1997. The increase in Replikin Count from 1999 to 2001 preceded the 2001 epidemic (E2). And the increase in Replikin Count from 2002 to 2004 preceded the 2004 epidemic (E3). The decline in 1999 was caused by the mass disposal of poultry in response to the E1 epidemic in Hong Kong.

  In addition to the total number of Replikins in the viral protein, the structure of each Replikin over time is beneficial. Table 8 shows the first observed Replikin (Goose Replikin) in geese infected with influenza in 1917. Invariant length, invariant lysine at the amino terminus, and histidine residues at the carboxy terminus have been conserved in different strains in a fixed backbone for decades. Goose Replikin homologs were found in 1917-2006, in strains related to three global epidemics, 1918, 1957, and 1961, respectively, H1N1, H2N2, and H3N2, and H1N2, With further substitution between H7N7, H5N2 and H5N1. Certain substitutions that occurred in the goose replikin tended to be selective rather than optional and were maintained for several years. Thus, despite the general assumption that amino acid substitutions occur arbitrarily, all substitutions in influenza do not appear to be optional in practice. This Replikin storage over decades advantageously allows for the production of synthetic influenza vaccines that can be prepared quickly and inexpensively and can be effective for more than a year.

Thus, the target for synthetic influenza vaccines is the conserved Replikin backbone in influenza viruses. The Replikin skeleton comprises a sequence of about 16 to about 30 amino acids, and (1) terminal lysine (2) terminal histidine and other histidines in the residue immediately adjacent to the terminal histidine (3) at least one At least one lysine in about 6 to about 10 amino acid residues from one other lysine; and (4) at least about 6% lysine in a 16 to about 30 amino acid peptide;
A series of conserved peptides comprising
The Replikin backbone may further comprise an additional lysine immediately adjacent to the terminal lysine. The “Replikin backbone” peptide may comprise an additional lysine immediately adjacent to the terminal lysine. A “Replikin backbone” peptide also means an individual member or members of a series of “Replikin backbone”.

  A non-limiting and preferred synthetic influenza vaccine target may be the Replikin backbone in influenza viruses, which further comprises about 29 amino acids and lysine immediately adjacent to the terminal lysine.

Unfavorable targets for synthetic influenza viruses include a first peptide of about 29 amino acids, and (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine (2) a terminal histidine and a histidine directly adjacent to the terminal histidine And (3) may be an exoskeleton in the first strain of influenza virus that does not contain lysine at 6 to 10 amino acid residues from any other lysine;
Here, an early-occurring specimen of the first strain of virus or other strain comprises a Replikin backbone of about 29 amino acids.

  In the 1997 H5N1 Hong Kong epidemic, human mortality was about 27%. In 2004, about 70% of the 52 people reported to be infected with H5N1 in Asia died. Recently, 9 out of 11 cases died in Vietnam from December 28, 2004 to January 27, 2005. Although the virulence of the virus appears to have increased, it appears that none of the changes considered necessary for further human-to-human transmission have occurred. However, we have recently made recent substitutions in the three H5N1 Replikin amino acid residues at positions 18, 24 and 28 of the goose Replikin skeleton from isolates in Vietnam, Thailand and China in 2004. Found (see Table 1). The substitution at site number 24 did not occur from the emergence of H5N1 in 1959, but together with the last two influenza pandemics caused by other strains that can be associated with more than 2 million deaths, 1957 In H2N2 and 1968 H3N2, and in recent viral regional epidemics caused by H7N7 (see Table 8). Although these are the only hints of potential danger, the data in the elevated Replikin Counts shown in Figure 15 and the replacement combined with such Replikin data and past correlations of the global epidemic have shown that the virus is transmitted between humans. It does not give the same relief as obtained from the phylogenetic analysis that it is not.

  For H5N1 influenza, FIG. 15 shows the rapid in concentration of Replikin per 100 amino acids just prior to the 1997 (shown as E1), 2001 (shown as E2) and 2004 (shown as E3) epidemics. Show rise.

Table 8: Replikin backbones showing ordered substitutions in the conservation of the 89 influenza virus Replikin peptide for rapid replication of the 1917 goose influenza Replikin and the 1918 human pandemic Replikin to 2006 H5N1 “avian influenza” homologues

  Table 8 above provides support for the role of Replikin in regional and global epidemics in humans and birds. In FIG. 8, the history of goose Replikin and its homologues was traced from 1917 to the recent pandemic of avian H5N1 virus. Table 8 shows the conservation of “skeletal” homology of goose Replikin in pathogenic strains of influenza.

  Table 8 shows the history of the presence of goose Replikin and its homologues in the protein structure by year or shorter period in other influenza Replikins. Table 8 further shows the definition of Replikin and the amino acid substitutions and conservation in these homologues of certain amino acids of the Replikin structure that are essential for the function of fast replication provided by Replikin.

  The review in Table 8 shows that if arbitrary amino acid substitutions occur in pathogenic strains of influenza from 1917 to present, certain framework amino acids of goose Replikin are among the strains in which epidemics have occurred. Indicates that it will not be stored for years. However, in contrast to those resulting from optional substitutions, year-to-year influenza pathogenic strains contain amino acids that are consistently conserved at these positions defining Replikin. That is, if a substitution occurs at one of the amino acids defining Replikin, such as lysine or histidine, the Replikin definition is lost. Nevertheless, Replikin sequences have been conserved for over 85 years. Thus, because of certain amino acid conservation over decades, the substitution cannot be said to be completely disordered. The fact that substitutions occur at amino acids that are not required for the definition of Replikin (ie, amino acids other than lysine or histidine) indicates the importance of Replikin in the pathogenicity of the strain.

  Furthermore, it can be noted from Table 8 that when substitution occurs, these occur at certain clearly preferred positions of the Replikin skeleton. Table 8 shows repetitive substitutions at positions 1, 3-24 and 26-27. Furthermore, substitution occurs at these positions, but lysine continues to be present at positions 6-10 amino acids from the second lysine (not substituted in these pathogenic strains).

  As seen in 1957, if there is a substitution at the lysine position during the 29 amino acid extension, even if K at position 11 is shifted to position 10, YP, AY, N (position 15), and This new position was maintained until 2005, with LVLWG to preserve the homology of the Replikin skeleton, with little exception.

  Table 8 shows the integrity of the Replikin skeleton in pathogenic strains of influenza. As mentioned above, denaturation of the Replikin skeleton to an exoskeleton scaffold appears to reduce pathogenicity. Thus, the integrity and conservation of the Replikin backbone is manifested by the fact that there are generally fixed 29 amino acid sequences that begin with two lysines and end with two histidines.

  It is important to note that extra K occurred in the Replikin skeleton of the 2006 strain of H5N1 in China (Anhui). The presence of extra K indicates an increase in Replikin counts in the Replikin skeleton. The 2006 China (Anhui) strain has a Replikin Count of 6.6 (discussed below). The Replikin Count of 6.6 is the highest previously observed for the H5N1 strain and is only comparable to the Replikin Count of influenza strains generated by the 1918 pandemic among all influenza strains. If this first 2006 report is repeated and maintained, this indicates that the 2004 and 2005 counts of 4.5 and 4.0 will increase substantially, respectively A persistent or increasing regional epidemic of H5N1 “bird flu” can be predicted.

  An aspect of the invention is a combination of Replikin structure and function to track the pathogenicity or replication rate of a virus, regional or global epidemic, or to predict the occurrence of a regional or global epidemic. An example of such a combination is the ability of the Replikin algorithm of the present invention to be used to measure increased Replikin counts in influenza strains, such as the 1918 strain and the current H5N1 strain of H5N1. The Replikin Count of the 1918 influenza pandemic and the recent pandemic of “bird flu” demonstrates the predictability of representative aspects made possible according to the present invention.

Relationship of several shrimp vitiligo virus Replikins to influenza fixed skeletal Replikin structure The present invention also established a relationship between pathogenic influenza virus and vitiligo virus in the Replikin skeleton part of the virus. There has been no previous suggestion of the relationship between these two viruses. Although there are widespread substitutions, Applicants' discovery of several short Replikins of Shrimp Vitiligo Syndrome Virus has been found to be particularly length, and important for resin (k) and histidine (h) relative to influenza virus Replikin sequences. Significant homology to the residues (fixed backbone or Replikin backbone) suggests that similar mechanisms of Replikin production are used in both virus groups.

  In addition, without limitation, many species, including swine and birds, are now known to provide animal “reservoirs” for human influenza infection, so now, for example, swine flu and avian flu Marine species, such as shrimp viruses, can be investigated with the benefit of early warning diagnosis of a possible pandemic. Although some influenza virus similarities have been noted between species and the transfer of these viruses between species is known, there is no previous quantification method to assess viral activity. It is therefore impossible to investigate potential hosts for increased activity that can be transferred to different species, thus providing a useful warning. The activity of Replikin in various species can now be constantly monitored for evidence of increased viral replication rates, thereby alerting to epidemics in species that can migrate to other species.

  This data further corroborates Replikin as a new class of peptides, with its own history and the shared function and disease of its host's rapid replication. Vitiligo syndrome virus, with high mortality for its shrimp host, currently has its Replikin investigated as an early form of the virus Replikin or as a similar form, and in both cases avian and animal Replikins such as It can act well as a host for things in influenza viruses. The diagnostic and prophylactic use of the discovery of these Replikins in shrimp is done in influenza and on other organisms containing Replikin.

Conservation of Replikin Structure Also, the foot-and-mouth for which mutations in the viral protein have been well documented throughout the world for decades as to whether the Replikin structure is conserved or subjected to extensive spontaneous mutation It was examined by scanning the protein sequences of various isolates of disease virus (FMDV). The protein sequence of FMDV isolates was visually inspected for the presence of both the entire Replikin and each of the component Replikin amino acid residues observed in a particular Replikin.

  Rather than a wide range of substitutions occur over time as occurs in neighboring amino acids, little or no amino acid containing a Replikin structure is substituted. That is, the Replikin structure is preserved.

  For example, among the FMDVO type O protein VP1, Replikin (SEQ ID NO: 3) “hkqkivapvk” was found to be conserved in 78% of the 236 isolates reported in PubMed, each amino acid being his 95.6%; lys, 91.8%; gln, 92.3%; lys, 84.1%; ile, 90.7%; val, 91.8%; ala, 97.3%; It was found to be stored in individual isolates such as 96.2%; ala, 75.4%; and lys, 88.4%. The high conservation rate suggests the structural and functional stability of the Replikin structure and provides a constant target for therapy.

  Similarly, sequence conservation is different for HIV for that Replikin, such as (SEQ ID NO: 5) “kcfncgkegh” or (SEQ ID NO: 6) “kvylawvpahk” in HIV type I and (SEQ ID NO: 7) “kcwncgkegh” in HIV type II. Found in isolate P (Table 2). Further examples of sequence conservation have been found in HIV tat proteins such as “hclvckqkkglgisygrkk”, where the major lysine and histidine amino acids are conserved (SEQ ID NO: 613) (see Table 9).

  Similarly, sequence conservation was observed in plants such as wheat, such as wheat ubiquitin that activates enzyme E (SEQ ID NOs: 614-616). Replikin in wheat provided even a reliable target for stimulating plant growth as described in. Other preserved examples are the constant presence of mariignin between successive generations over 10 years of tissue culture of glioma cells, and 8,090 human sera from the US, UK, Europe and Asia Found in the homeostasis of the affinity of glioma Replikin for anti-malignin antibodies isolated by immunoadsorption from (eg, FIG. 5 and US Pat. No. 6,242,578 B1).

  Similarly, conservation was observed in Tat (trans-acting factor) protein in HIV isolates. Tat (trans-acting factor) protein is an early RNA binding protein that regulates lentiviral transcription. These proteins are necessary components in the life cycle of all known lentiviruses such as human immunodeficiency virus (HIV). Tat is a transcriptional regulator protein that acts by binding to trans-acting response element (TAR) RNA elements and activates initiation and / or elongation of transcription from the LTR promoter. HIV cannot replicate without tat, but its chemical basis is unknown. Within the 89-102 residue HIV tat protein sequence, we have discovered Replikins that are associated with other in vivo rapid replication. The amino acid sequence of this Replikin is “HCLVCKQKKGLGISYGRKK”. In fact, we have found that this Replikin is present in all HIV tat proteins. Some tat amino acids are frequently substituted with alternating amino acids as shown in Table 9 (small fonts lined under the most frequent amino acids (Table 9), dominant Replikin “HCLVCFQKKGLGISYGRKK”) Save percentage about). These substitutions appeared for most individual amino acids. However, the major lysine and histidine amino acids within the Replikin sequence that define the Replikin structure are 100% conserved in the sequence. Substitutions are common elsewhere in other amino acids, both inside and outside the Replikin, but nothing occurs on these major histidine amino acids.

  As shown in Table 9, lysine is not necessarily substituted within the tat protein amino acid sequence. From the left side of the table, the very first lysine in the sequence immediately adjacent to what is outside the Replikin sequence and the second in the sequence that is inside the Replikin, which is “preliminary” because it is not essential for Replikin formation. Lysine (k) is both frequently substituted. However, the third, fourth and fifth lysines and one histidine in parentheses that together define the Replikin structure are never substituted. Thus, these major amino acid sequences are 100% conserved. As observed in the case of influenza virus Replikin, this selective substitution and selective non-substitution may not occur by chance due to random substitution.

  Conservation of the Replikin structure is a virus with amino acid substitution that is created to avoid antibodies and other “attack”, with the Replikin structure having a specific survival function for HIV viruses that must be protected and preserved. This suggests that it cannot be sacrificed for the “defense” operation. These “defense” functions are equally essential, but cannot “compete” with the virus persistence function of HIV replication.

  In different isolates of HIV, further conservation was observed for that replikin, such as “kcfncgkegh” (SEQ ID NO: 5) or “kvylawvpahk” (SEQ ID NO: 6) in HIV type 1 and “kcwncgkegh” (SEQ ID NO: 7) in HIV type 2. It was. The high conservation rate observed in FMVD and HIV Replikin suggests that the conservation observed within the Replikin of influenza Replikin is a general characteristic of viral Replikin. This preservation makes them constitutive and reliable for either disruption or stimulation by using specific Replikins, such as for influenza, FMVD or HIV vaccines as exemplified for glioma Replikin Become a target.

Similarly, the high conservation rate within Replikin suggests that preservation is a general characteristic of viral Replikin and thus makes Replikin a permanent and reliable target for destruction or stimulation, influenza As provided in the examples found in viruses, including viruses, FMDV and HIV, conservation of Replikin structures occurs in plants. For example, in wheat plants, Replikin is stored, providing a reliable target for stimulation. Examples of preserved Replikins in wheat plant ubiquitin that activate enzyme E include:

  Replikin in wheat ubiquitin activating enzyme E is preserved in a manner similar to that found in the HIVtat protein. As with the HIV tat protein, substitution of amino acids adjacent to the Replikin mutant form within wheat ubiquitin activating enzyme E (designated by “*”) is common. However, the major k and h amino acids that form the Replikin structure are unchanged, whereas only five amino acids (from the first k on the left) are replaced by “non-essential” k.

Anti-Replikin Antibody An anti-Replikin antibody is an antibody against Replikin. Data on anti-Replikin antibodies similarly support the integrity of the Replikin type. The anti-Replikin antibody response has been quantified by immunoadsorption of serum anti-malignin antibody to immobilized malignin (see methods in US Pat. No. 5,866,690). Abundant production of anti-malignin antibodies upon administration to rabbits of synthetic versions of 16-mer peptides derived from malignin, absent carbohydrate or other groups, is that the peptide alone is an epitope, ie this immune response It has been strictly demonstrated to provide a sufficient basis for (FIG. 3). The 16-mer peptide produced both IgM and IgG forms of the antibody. Serum levels of anti-malignin antibodies are found early in the first five years of infection, much earlier than normal adherence to liver cancer that occurs approximately 15-25 years after infection, early in the United States and Asia. It was found to increase in 37% of 79 cases. In connection with both infectious hepatitis and HIV infection, transformed cells can be one form of a safe haven for the virus. That is, extending the life of the cell and avoiding the reclamation of the virus, thus the virus remains inaccessible for antiviral therapy.

  The administration of Replikin is based on the specific influenza virus or Replikin group that has been observed to be the highest in concentration over a given period, as it stimulates the immune system to produce antibodies with cytotoxic effects Peptide vaccines provide protection against certain influenza strains that are most likely to break out in a given influenza epidemic, such as emerging or reviving strains. For example, analyzing influenza virus hemagglutinin amino acid sequences at an annual or biennial pace provides useful data in formulating specifically targeted influenza vaccines for that year. Such analysis can be done by region or at any desired time, thus detecting strains emerging in different regions of the world and prescribing specifically targeted vaccines for each region. Needless to say, you can.

Influenza vaccines, treatment and therapy Currently, vaccine formulations for influenza are changed twice a year at the WHO and CDC international meetings. Vaccine prescription is based on the latest dominant serological evidence of influenza virus strains in a given region of the world. However, prior to the present invention, there was nothing that correlated the occurrence of influenza epidemics or pandemics with changes in influenza virus strain-specific amino acid sequences.

  By observing specific Replikin and its concentration in the influenza virus protein, it provides the first specific quantitative early chemical correlates of influenza pandemic and regional epidemic within a particular region of the world Production and timely administration of specifically tailored influenza vaccines to handle the dominant emerging or reviving strains of influenza viruses are provided. By analyzing the protein sequences of isolates of influenza virus strains such as hemagglutinin protein sequences for the presence, concentration and / or preservation of Replikin, one can predict influenza virus global and regional epidemics . Furthermore, the severity of such influenza outbreaks has been found to be most abundant or shown to be rising for virus isolates over a given period of time, for example about 1 year to about 3 years. Can be significantly reduced by administering an influenza peptide vaccine on the basis of

  Influenza peptide vaccines of the present invention may include a single Replikin peptide sequence, or may include multiple Replikin sequences observed in influenza virus strains. Preferably, the peptide vaccine is based on a Replikin sequence (s) that has been shown to increase in concentration over a given time period and is conserved at least for that time period. However, the vaccine may include Replikin peptide (s) conserved in combination with the new Replikin (s) peptide, otherwise it may be based on the new Replikin peptide sequence. Replikin peptides can be synthesized by any method including chemical synthesis or recombinant gene technology and can include non-Replikin sequences, but peptides based on peptides containing only Replikin sequences are preferred. Preferably, the vaccine composition of the present invention also contains a pharmaceutically acceptable carrier and / or adjuvant.

  Influenza vaccines of the present invention can be used alone or in combination with antiviral agents such as ganciclovir; interferon; interleukins; M2 inhibitors such as amacitadine and rimacytazine; neuraminidase inhibitors such as zanamivir and oseltamivir; Can be administered.

  The influenza vaccine of the present invention can be administered to any animal capable of producing antibodies in an immune response. For example, the influenza vaccine of the present invention can be administered to rabbits, chickens, pigs or humans. Due to the inherent nature of the Replikin sequence, the influenza vaccines of the present invention can be made in various influenza strains or specific influenza strains.

  In a non-limiting aspect according to the present invention, an influenza vaccine is an influenza animal or human strain, such as any of influenza B, (A) H1N1, (A) H2N2 and (A) H3N2, or a virus that may arise in the future. Human variants can be targeted as well as immune responses against the dominant influenza strains in animals such as the recent bird H5N1. Influenza vaccines can further be directed to specific Replikin amino acid sequences in any part of the influenza protein.

  In a non-limiting aspect according to the present invention, the influenza vaccine may comprise the Replikin backbone of the H5N1 virus, such as KKNSTYPTIKRSYNNTNQEDLLVLWGIHH. In a further non-limiting aspect, the influenza vaccine may comprise UTOPE, such as KKKKH or KKKKHKKKKKH. Still further, the vaccine may include additional adjuvants, such as the well-known major limpet hemocyanin having the amino acid sequence KLH. In a further preferred non-limiting aspect, the influenza vaccine may comprise an influenza H5N1 Replikin backbone comprising two UTOPES and an adjuvant sequence, for example KKNSTYPTIKRSYNNTNQEDLLVLWGIHHKKKKHKKKKKHK-KLH (vaccine V120304U2). Aspects of the invention may include the Replikin scaffold shown in Table 8 as one of the avian influenza Replikins previously constructed and labeled “2004, H5N1, Vietnam, highly pathogenic”. With the administration of 100 μg of vaccine V120304U2 peptide injected subcutaneously into rabbits and chickens, antibody responses were observed from dilutions of 1:50 or less without vaccination, from 1: 120,000 to 1: 240,000 or more. Peaks were reached 3 to 4 weeks after dilution vaccination (see Example 7).

Repeated and overlapping Replikin structures Analysis of the primary structure of a tropical Plasmodium malaria antigen located on the merozoid surface and inside the parasite-containing vacuole revealed that the organism, for example, influenza virus, also contains a large number of Replikins . However, there are some differences in the observation of Replikin in tropical malaria parasites and influenza virus isolates. For example, tropical malaria parasites contain several partial Replikins. Another difference seen in tropical malaria parasites is the frequent repetition of individual Replikin structures within a single protein that was not observed with influenza virus. Repeats can occur by (a) sharing lysine residues between Replikins, and (b) repeating portions of a Replikin sequence within another Replikin sequence.

  Another significant difference between Replikin structures observed in influenza virus isolates and tropical Plasmodium is a significant overlap of Replikin structures throughout the malaria protein, for example within the 39 amino acid sequence of SEQ ID NO: 393. There are 9 overlapping Replikins (Replikin concentration = 23.1 / 100 amino acids) and 15 overlapping Replikins (Replikin concentration = 36.6 / 100 amino acids) within 41 amino acids of SEQ ID NO: 467. . Both of these overlapping Replikin structures occur in blood stage trophozoides and schizonts. In contrast, influenza virus Replikin is more scattered throughout the protein, the maximum Replikin concentration is about 7.5 per 100 amino acids (FIG. 7), and tomato leaf curl geminivirus is also It was observed to have overlapping Replikins.

High concentrations of Replikin correlate with rapid replication Tomato leaf blight Geminivirus devastated tomato harvests in China and many other parts of the world. The Replikin has reached a high count due to the overlapping Replikins as exemplified below among the viruses isolated in Japan where the Replikin Count was 20.7.

  The relationship of higher Replikin concentration to fast replication is also confirmed by analysis of HIV isolates. A slow growing low titer strain of HIV (NSI, which predominates in early HIV infection, “Bru”) has a Replikin concentration of 1.1 (+/− 1.6) Replikin per 100 amino acids, A fast growing high titer strain of HIV (S1, “Lai” predominating in late HIV infection) has a Replikin concentration of 6.8 (+/− 2.7) Replikin per 100 amino acid residues Was discovered.

Passive Immunization In another aspect of the invention, an isolated Replikin peptide can be used, for example, to generate antibodies that can be used to provide passive immunity within an individual. Passive immunization against an influenza strain identified as the most probable cause of an upcoming influenza infection by the method of the present invention is achieved by administering to a patient in need an antibody against the Replikin sequence of the identified influenza virus strain. Obtainable. Similarly, passive immunity to malaria can be obtained by administering an antibody against tropical malaria parasite Replikin (s).

  Various procedures known in the art can be used for the production of antibodies against Replikin sequences. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by Fab expression libraries. Antibodies linked to cytotoxic agents can also be generated. It is also possible to administer the antibody in combination with an antiviral agent. In addition, combinations of antibodies against different Replikins can be administered as an antibody cocktail.

  For antibody production, various host animals or plants, including but not limited to rabbits, mice, rats, and larger mammals, are injected with Replikin peptides or combinations thereof. Can be immunized.

  Monoclonal antibodies against Replikin can be prepared by using any technique that provides for the production of antibody molecules. These include the hybridoma technology originally described by Kohler and Milstein (Nature, 1975, 256: 495-497), the human B cell hybridoma technology (Kosbor et al., 1983, Immunology of Tody, 4:72) and the EBV hybridoma technology. (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81: 6851-6855) or other techniques can be used. Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to produce Replikin specific single chain antibodies.

  Particularly useful antibodies of the present invention are those that specifically bind to Replikin sequences contained within influenza virus peptides and / or polypeptides. For example, antibodies to any of the peptides observed to be present in emerging or reviving strains of influenza virus and combinations of such antibodies are useful in the treatment and / or prevention of influenza. Similarly, antibodies to any Replikin present on a malaria antigen and combinations of such antibodies are useful in the prevention and treatment of malaria.

  Antibody fragments containing binding sites for Replikin can be generated by known techniques. For example, such fragments include F (ab ′) 2 fragments that can be produced by pepsin digestion of antibody molecules and Fab fragments that can be generated by reducing disulfide bridges of F (ab ′) 2 fragments. It is not limited. Alternatively, a Fab expression library can be generated (Huse et al., 1989, Science, 246: 1275-1281) to allow fast and easy identification of monoclonal Fab fragments with the desired specificity. it can.

  The fact that the concentration of anti-malignin antibody is increased in human malignant tumors regardless of cancer cell type (see FIG. 5) and thus this antibody binds to malignant cells regardless of cell type is now described herein. Written in most malignant tumors (Figure 1 and Table 2) can be explained by the presence of Replikin structures. Population studies have shown that anti-malignin antibodies increase in concentration in healthy adults with age and increase as the frequency of cancer increases in high-risk families. The additional 2-fold antibody increase that occurs in early malignancies has been independently confirmed by 97% sensitivity in breast cancers 1-10 mm in size. Although it has been shown to preferentially localize in malignant cells in vivo, histochemically, the antibody does not bind to normal cells and is selective for cells transformed in vitro. It binds (FIGS. 4A, B) and is highly cytotoxic to these cells (FIGS. 4C-F). In these examples, the same antibody is bound by multiple cell types, namely cerebral glioma, hematopoietic cells (leukemia) and small cell lung cancer, thus demonstrating the uniformity of the malignant Replikin types.

  Antimalignin does not increase with benign growth, specifically increases only with malignant transformation and replication in the breast in vivo and returns from high to normal at the time of removal of malignant cells (FIG. 5). It has been shown that anti-malignin antibody concentration is quantitatively related to the survival rate of cancer patients, that is, the higher the antibody, the higher the survival rate. Taken together, these results suggest that anti-Replikin antibodies may be part of cell transformation and replication control mechanisms. This increase in immune response can be attributed to active use of synthetic Replikin as a vaccine, or in a similar fashion to specifically target anti-Replikin antibodies or Replikin, such as carbohydrates, lipids, etc. It may be useful to control replication passively by the introduction of non-immune based organic agents.

  In another aspect of the invention, immune sera containing antibodies to one or more Replikins obtained from an individual exposed to one or more Replikins are used to induce passive immunity in another individual or animal Can be used to The immune serum can be administered intravenously to a subject in need of treatment. Passive immunization can also be achieved by injecting a recipient with preformed antibodies to one or more Replikins. Passive immunization can be used to provide immediate protection for individuals exposed to infectious organisms. Administration of immune serum or preformed antibody is routine and a skilled practitioner can readily ascertain the amount of serum or antibody required to achieve the desired effect.

Synthetic Replikin vaccine (active immunity)
Glioma Replikin (SEQ ID NO: 1) “kagvaflhkk” or Hepatitis C Replikin (SEQ ID NO: 18) “hypppkpgivpak” or HIV Replikin eg (SEQ ID NO: 5) “kcfncgkegh” or (SEQ ID NO: 6) Replikin based on “kvylawvpahk” Influenza vaccines based on synthetic Replikin vaccines and preferably Replikin (s) preserved and / or emerging or revived for a given period of time are directed against each virus outside the cell where chemical treatment can then be effective. It can be used to increase antibody concentration to lyse infected cells and release virus. Similarly, malaria vaccines can be used to generate cytotoxic antibodies against malaria, for example, based on Replikins observed in tropical Plasmodium malaria antigens on merozoite surfaces or inside parasite-containing vacuoles. it can. Table 7 shows the relationship between shortening or consolidation of Replikin sequences and mortality caused by organisms containing these Replikins down to as few as 7 amino acids. We have found that this correlation is a general phenomenon regardless of the type of organism. We have also found that progression over time can exist up to a shortened Replikin structure, as in the case of influenza and SARS viruses.

  There is abundant evidence that there is always evolutionary and competitive pressure due to the ever-increasing emergence of “efficacy” in each infectious organism. Based on these observations and by prediction, evolutionary pressure is heading towards Replikin, which becomes shorter with lysine (k) concentrations that continue to rise to as high as 70% as in EEL leukemia. If (Fig. 7), then the theoretical ideal predicted was deduced, including the highest possible% of k (83.3% of k, 5/6 and one mandatory "h") Replikin “see kkkkhk”) results in the shortest possible Replikin or 6 amino acids (2 k separated by 6-10 amino acids) as allowed by the algorithm that defines the Replikin. Therefore, we will pick up what appears to be or should be the next evolutionary stage not yet apparently taken up by the organism itself, and should be used as a general vaccine, Invented the deplied Replikin.

  These Replikins we have deduced have the largest percentage of “k”, and therefore the largest potential binding capacity and by definition the component “h” required for Replikin, for the “h” connection to the redox energy system. Offering potential. These devised Replikins have the lowest probability of being split by the organism due to their short length (proteins are split into lengths of 6-10 amino acids in processing for presentation to and recognition by immune cells) Therefore, they have the highest probability of appearing intact to the immunogenic organ in the body in which they are administered, and because of their high k content, can be generated against short homologous high mortality Replikins There is the highest probability of generating the maximum immune response that can mimic and increase the maximum such response.

Furthermore, we have found that Replikin with a high% of k produces the best antibody response when administered to rabbits. These synthetic peptides we designed are termed universal synthetic epitopes or “UTOPE”, and these UTOPE-based vaccines are termed “UVAX”. UVAX, a deduced synthetic vaccine, can be used as the sole vaccine or as an adjuvant when administered with more specific Replikin vaccines or other vaccines. The following are examples of deduced UTOPE and UVAX.

  In order to elicit the subject's immune system to produce anti-Replikin antibodies, lycognin and / or Replikin peptides can be administered to the subject. Generally, a dosage of 0.5 to about 2 mg, preferably 1 mg of each peptide is administered to a subject to elicit an immune response. Subsequent dosages can be administered if desired.

  The Replikin sequence structure is linked to the replication function. Thus, for example, if a Replikin of the present invention is used to target a Replikin-containing sequence for diagnostic identification purposes, to promote replication or to inhibit or attack replication, The structure-function relationship of Replikin is essential.

  When trying to elicit antibodies that recognize and attach to Replikin fragments and thus cause cell destruction, it is preferable to utilize only specific Replikin structures. Even though larger protein sequences are known in the art as having “replication-related functions”, vaccines using larger proteins have often proved unsuccessful or ineffective.

  Although the inventors do not want to be bound by a single theory, the studies herein suggest that prior art vaccines are ineffective because they are based on the use of larger protein sequences. is doing. Larger protein sequences have invariably one or more epitopes (independent antigen sequences capable of inducing specific antibody formation). Replikin structures usually contain one of these potential epitopes. The presence of other epitopes within larger proteins can interfere with the proper formation of antigens against Replikin by “flooding” the immune system with irrelevant antigen stimuli that can preempt Replikin antigens. For a description of this well-known phenomenon of antigen firstism, where the first peptide epitope presented and recognized by the immune system then prevails, and antibodies against it are made even if other peptide epitopes are presented simultaneously Webster, RG, J. Immunol., 97 (2): 177-183 (1966); and Webster et al., J. Infect. Dis., 134: 48-58, 1976; Klenerman et al. ., Nature 394: 421-422 (1998). This presents a constant Replikin peptide to the immune system first before presenting other epitopes from the body in a way that the Replikin is not preempted and remains in immunological memory in vaccine formation. This is another reason why it is important.

  The formation of antibodies against non-Replikin epitopes may allow binding to cells but does not necessarily lead to cell destruction. The presence of a structural “decoy” on the C-terminus of the malaria protein is due to the fact that the decoy epitope has many lysine residues but no histidine residues, thus interfering with effective anti-Replikin antibody binding. It is another aspect of this ability of the epitope. Thus, the decoy epitope can bind to the anti-Replikin antibody, but keep the antibody away from the respiratory enzyme bound to histidine. Thus, treatment can be most effective in two stages: 1) a protease to hydrolyze the decoy and then 2) an anti-Replikin antibody or other anti-Replikin agonist.

  It is well known in the art that proteins are first hydrolyzed into smaller fragments during the course of antibody production against “foreign” proteins. Usually, fragments containing about 6-10 amino acids are selected for antibody formation. Thus, if protein hydrolysis does not result in a Replikin-containing fragment, anti-Replikin antibodies will not be produced. In this regard, since lysine residues are known to bind to the membrane, it is advantageous that the Replikin contains lysine residues that are located 6-10 amino acids apart. is there.

  Furthermore, the Replikin sequence contains at least one histidine residue. Histidine is frequently involved in binding to the redox center. Thus, antibodies that specifically recognize Replikin sequences are active in picograms per cell and are likely to be found in anti-malignin antibodies, which are probably the most cytotoxic anti-cancer antibodies described so far. The probability of inactivating or destroying cells in which Replikin is located is higher.

  Vaccines directed against specific protein antigens of the disease-causing agent are sufficient to provide protection against the disease (such as the foot-and-mouth disease vaccine developed for the VP1 protein or a large segment thereof) One reason for the lack of effectiveness was that the best antibodies were not produced, that is, there was a high probability that antibodies against Replikin were not produced. Replikin was not produced. That is, any epitope other than Replikin present in a larger protein fragment is larger according to the phenomenon of antigen firstism described above and / or into smaller sequences for processing to produce antibodies. The hydrolysis of the protein sequence is due to the loss of integrity of any Replikin structure present, such as, for example, the Replikin is cleaved in two and / or the histidine residue is lost in the hydrolysis processing. , May interfere. This study suggests that the Replikin sequence should be used as a vaccine and no other epitope should be used in order for an effective vaccine to be produced. For example, the vaccine of the present invention can be generated using any one of the Replikin peptides identified by a three-point recognition system.

  Particularly preferred peptides such as, for example, influenza vaccines, have been demonstrated to be stored over a period of one or more years, preferably more than 3 years and / or Replikin concentrations in other influenza virus strains such as eg emerging strains Included are peptides present in strains of influenza virus that have been shown to have the highest increase in Replikin concentration in relation to. The increase in Replikin preferably occurs over a period of at least about 6 months to 1 year, preferably at least about 2 years or more and most preferably about 3 years or more. Among the preferred Replikin peptides for use in influenza virus vaccines are Replikins that have been observed to “recover” after absence from hemagglutinin amino acid sequences over one or more years.

  The Replikin peptides of the invention are preferably administered to a subject, either alone or in various combinations, by intravenous or intramuscular injection for the purpose of stimulating the subject's immune system to produce antibodies of the peptide. Generally, the peptide dosage is in the range of about 0.1 μg to about 10 mg, preferably about 10 μg to about 1 mg, and most preferably about 50 μg to about 500 μg. A skilled physician can easily determine the dosage and number of doses required to produce an effective immune response.

Quantitative measurement of early response to Replikin vaccine The ability to quantitatively measure early specific antibody response within days or weeks to Replikin vaccine can only measure clinical response after months or years This is a major practical advantage over other vaccines.

Adjuvant Freund's (complete or incomplete) adjuvants, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, gintrophenol and potentially useful humans Depending on the host species, various adjuvants can be used to enhance the immune response, including (but not limited to) adjuvants such as BCG and Corynebacterium parvum. In addition to the use of synthetic UTOPE as the vaccine itself, UTOPE can be used as an adjuvant for other Replikin and non-Replikin vaccines.

Replikin nucleotide sequences Replikin DNA or RNA may have numerous uses for the diagnosis of diseases resulting from infection by viruses, bacteria or other Replikin coding agents. For example, in order to diagnose the presence of a particular organism in a tissue sample or environmental sample, including in situ hybridization assays, for example, in tissue or blood hybridization assays that have undergone biopsy, such as Southern or Northern analysis. Kin nucleotide sequences can be used. The invention also encloses a nucleic acid molecule (sense or antisense) that houses or specifically hybridizes to a particular Replikin present in a particular pathogen in question, and Optionally, kits containing various buffers and / or reagents required for diagnosis are also contemplated.

  Also within the scope of the present invention are oligoribonucleotide sequences, including antisense RNA and DNA molecules, and ribozymes that function to inhibit translation of Replikin or lycognin-containing mRNA. Both antisense RNA and DNA molecules and ribozymes can be prepared by any method known in the art. Antisense molecules can be incorporated into a wide variety of vectors for delivery to a subject. A skilled physician can easily determine the best delivery route, but generally intravenous or intramuscular delivery is routine. The dosage can also be easily confirmed.

  Particularly preferred antisense nucleic acid molecules include (1) at least one lysine located 6 to 10 residues from the second lysine residue; (2) at least one histidine residue; and (3) at least 6% It is complementary to a Replikin sequence contained in mRNA containing, for example, influenza virus polypeptide, containing 7-50 amino acids containing lysine residues. More preferably, it has been demonstrated to be complementary to mRNA encoding Replikin or influenza virus hemagglutinin protein present in the coding strand of the gene and preserved over a period of 6 months to one or more years. An antigen present in one strain of influenza virus that is complementary to the nucleotide sequence encoding the Replikin and / or has been shown to have an increased Replikin concentration relative to the Replikin concentration in other influenza virus strains Sense nucleic acid molecule. The increase in Replikin concentration preferably occurs over a period of at least 6 months, preferably about 1 year, most preferably about 2 years or 3 years or more.

  Similarly, an antisense nucleic acid molecule complementary to mRNA comprises (1) at least one lysine located 6 to 10 residues from the second lysine residue; (2) at least one histidine residue; and (3) Complementary to mRNA encoding bacterial Replikin containing a Replikin sequence of 7-50 amino acids containing at least 6% lysine residues. More preferred are antisense nucleic acid molecules that are complementary to the coding strand of a gene or mRNA encoding a bacterial protein.

Further aspects of Replikin In an aspect of the invention, a method for preventing or treating viral infection comprising administering a prophylactic or therapeutic vaccine to a patient in need thereof is a protein of an emerging strain of virus. Provided comprising at least one isolated Replikin present therein and a pharmaceutically acceptable carrier and / or adjuvant. In a further aspect of the invention, the isolated or synthesized peptide is an influenza virus peptide. In yet a further aspect of the invention, the isolated or synthesized peptide is an H5N1 influenza virus peptide.

The present invention is also a method for producing a prophylactic or therapeutic viral vaccine comprising:
(1) identifying a virus strain as an emerging strain,
(2) selecting at least one Replikin sequence present in the emerging strain as a peptide template for virus vaccine production;
(3) a step of synthesizing a peptide having an amino acid sequence of at least one Replikin sequence selected in step (2); and (4) a therapeutically effective amount of the peptide of step (3) and a pharmaceutically acceptable carrier. And / or combining an adjuvant,
A method comprising:
In a further aspect of the method for producing a prophylactic or therapeutic viral vaccine, the isolated Replikin is derived from influenza virus. In a further aspect, the isolated Replikin is derived from influenza H5N1 virus.

In another aspect, the present invention is a method of identifying an emerging strain of virus for diagnostic, prophylactic or therapeutic purposes:
(1) obtaining at least one isolate of each of a plurality of virus strains;
(2) analyzing the amino acid sequence of at least one isolate of each of the plurality of virus strains for the presence and concentration of Replikin sequences;
(3) the concentration of Replikin sequences in the amino acid sequence of at least one isolate of each strain of the plurality of virus strains, and the concentration of each strain at at least one earlier time point to provide the concentration of Replikin at at least two time points; Comparing the concentration of Replikin sequences observed in the amino acid sequence, wherein said at least one earlier time point is within about 3 years from about 6 months prior to step (1); and (4) Identifying a virus strain having the highest increase in concentration of Replikin sequences at at least two time points;
The method including this is intended.

  In another aspect of the invention, a method for stimulating a subject's immune system to produce an antibody that specifically binds to a Replikin sequence, the method comprising: There is provided a method comprising administering an effective amount of a dose of a composition comprising a quinpeptide. A further aspect of the invention includes at least one peptide present in an emerging strain of an organism when such a new strain emerges. Another aspect of the invention includes at least one peptide present in influenza H5N1.

  The present invention also provides antibodies that specifically bind to Replikins as defined herein, as well as antibody mixtures that contain a plurality of antibodies that specifically bind to Replikins. Another aspect of the present invention provides a composition comprising an antibody or group of antibodies that specifically binds to Replikin, and a pharmaceutically acceptable carrier.

  In one aspect of the invention, other proteins containing viral Replikin sequences, peptides isolated from recombinants, or separated or synthesized, or other methods are provided.

  In another aspect of the invention, a method of stimulating a subject's immune system to produce an antibody that specifically binds to a viral Replikin sequence, the method comprising: There is provided a method comprising administering an effective amount of a dose of a composition comprising a quinpeptide. Another aspect of the present invention includes at least one peptide present in an emerging strain of an organism when such a new strain emerges.

  The present invention also provides an antibody that specifically binds to a viral Replikin as defined herein, as well as an antibody mixture containing a plurality of antibodies that specifically bind to a viral Replikin. Another aspect of the present invention provides a composition comprising an antibody or group of antibodies that specifically binds to a viral Replikin, and a pharmaceutically acceptable carrier.

  The present invention also provides a therapeutic composition comprising one or more isolated Replikin virus peptides and a pharmaceutically acceptable carrier.

In another aspect of the invention, an antisense nucleic acid molecule complementary to a viral Replikin mRNA sequence, wherein the Replikin mRNA sequence is:
(1) at least one lysine residue located 6 to 10 residues from the second lysine residue;
An antisense nucleic acid molecule is provided that exhibits from 7 to about 50 amino acids, comprising (2) at least one histidine residue; and (3) at least 6% lysine residues.

  In yet another aspect of the invention, a method of stimulating a subject's immune system to produce antibodies against a virus, the method comprising: providing an effective amount of at least one viral Replikin peptide to the subject. A method comprising administering is provided.

In another aspect of the invention, a method for selecting viral peptides for inclusion in a prophylactic or therapeutic viral vaccine comprising:
(1) obtaining at least one isolate of each of the plurality of virus strains;
(2) analyzing the amino acid sequence of at least one isolate of each strain of the plurality of virus strains for the presence and concentration of Replikin sequences;
(3) the concentration of Replikin sequences in the amino acid sequence of at least one isolate of each strain of the plurality of virus strains, and the concentration of each strain at at least one earlier time point to provide the concentration of Replikin at at least two time points; Comparing the concentration of Replikin sequences observed in the amino acid sequence, wherein the at least one earlier time point is within about 3 years from about 6 months prior to step (1);
(4) identifying the virus strain having the highest increase in the concentration of Replikin sequences at at least two time points; and (5) the viral peptide identified in step (4) as a peptide for inclusion in a viral vaccine. Selecting at least one Replikin sequence present in the strain of
Is provided.

  In one aspect of the invention, an isolated or synthesized influenza virus peptide comprising a Replikin sequence is provided.

  In another aspect of the invention, a method of stimulating a subject's immune system to produce an antibody that specifically binds an influenza virus Replikin sequence, said method comprising at least one There is provided a method comprising administering an effective amount of a dose of a composition comprising an influenza virus Replikin peptide. A further aspect of the invention includes at least one Replikin peptide present in an emerging strain of influenza virus. Another aspect of the invention includes a composition foreign body comprising at least one influenza H5N1 Replikin peptide.

  The present invention also provides antibodies that specifically bind to an influenza virus Replikin as defined herein, as well as antibody mixtures containing a plurality of antibodies that specifically bind to an influenza virus Replikin. Another aspect of the present invention provides a composition comprising an antibody or group of antibodies that specifically bind to influenza Replikin, and a pharmaceutically acceptable carrier.

The present invention also provides
(1) at least one lysine residue located 6 to 10 residues from the second lysine residue;
Provided is a therapeutic composition comprising one or more isolated influenza virus peptides having (2) at least one histidine residue; and (3) 7 to about 50 amino acids comprising at least 6% lysine residues.

In another aspect of the invention, an antisense nucleic acid molecule complementary to an influenza virus hemagglutinin Replikin mRNA sequence, wherein the Replikin mRNA sequence is:
(1) at least one lysine residue located 6 to 10 residues from the second lysine residue;
An antisense nucleic acid molecule is provided that exhibits from 7 to about 50 amino acids, comprising (2) at least one histidine residue; and (3) at least 6% lysine residues.

In yet another aspect of the invention, a method of stimulating a subject's immune system to produce antibodies against influenza viruses, comprising:
(1) at least one lysine residue located 6 to 10 residues from the second lysine residue;
(2) administering an effective amount of at least one influenza virus Replikin peptide having from 7 to about 50 amino acids, comprising at least one histidine residue; and (3) at least 6% lysine residues. A method of including is provided.

In another aspect of the invention, a method for selecting influenza virus peptides for inclusion in a prophylactic or therapeutic influenza virus vaccine comprising:
(1) obtaining at least one isolate of each of a plurality of influenza virus strains;
(2) analyzing the hemagglutinin amino acid sequence of at least one isolate of each of a plurality of influenza virus strains for the presence and concentration of Replikin sequences;
(3) the concentration of the Replikin sequence in the hemagglutinin amino acid sequence of at least one isolate of each of the plurality of influenza virus strains and at least one earlier time point to provide the concentration of Replikin at at least two time points; Comparing the concentration of Replikin sequences observed in the haemagglutinin amino acid sequence of each strain in said at least one earlier time point within about 6 months to about 3 years prior to step (1) A process;
(4) identifying an influenza virus strain having the highest increase in the concentration of Replikin sequences at at least two time points; and (5) identified in step (4) as a peptide for inclusion in an influenza virus vaccine Selecting at least one Replikin sequence present in a strain of influenza virus peptide;
Is provided.

The present invention is also a method for producing a prophylactic or therapeutic influenza virus vaccine comprising:
(1) identifying an influenza virus strain as an emerging strain,
(2) selecting at least one Replikin sequence present in the emerging strain as a peptide template for influenza virus vaccine production;
(3) a step of synthesizing a peptide having an amino acid sequence of at least one Replikin sequence selected in step (2); and (4) a therapeutically effective amount of the peptide of step (3) and a pharmaceutically acceptable carrier. And / or combining an adjuvant,
A method comprising:

In another aspect, the present invention is a method for identifying emerging strains of influenza virus for diagnostic, prophylactic or therapeutic purposes:
(1) obtaining at least one isolate of each of a plurality of influenza virus strains;
(2) analyzing the hemagglutinin amino acid sequence of at least one isolate of each of a plurality of influenza virus strains for the presence and concentration of Replikin sequences;
(3) the concentration of the Replikin sequence in the hemagglutinin amino acid sequence of at least one isolate of each of the plurality of influenza virus strains and at least one earlier time point to provide the concentration of Replikin at at least two time points; Comparing the concentration of Replikin sequences observed in the haemagglutinin amino acid sequence of each strain in said at least one earlier time point within about 6 months to about 3 years prior to step (1) And (4) identifying an influenza virus strain having the highest increase in the concentration of Replikin sequences at at least two time points;
The method including this is intended.

  In yet another aspect of the invention, prophylactic or therapeutic influenza comprising at least one isolated Replikin present in a hemagglutinin protein of an emerging strain of influenza virus, and a pharmaceutically acceptable carrier and / or adjuvant Viral vaccine is provided.

  The present invention also provides a prophylactic or therapeutic vaccine comprising at least one isolated Replikin present in a hemagglutinin protein of an emerging strain of influenza virus, and a pharmaceutically acceptable carrier and / or adjuvant. There is provided a method for preventing or treating influenza virus infection, comprising administering to a patient in need thereof.

Computer software for identifying Replikin and related structures Identification of Replikin, Replikin and modified exoskeleton structures can be achieved with the aid of bioinformatics.

  Aspects of the present invention are for identifying and / or locating complex patterns of amino acid sequences, such as Replikin patterns, Replikin scaffolds, exoskeleton scaffolds, and other complex patterns in amino acid and nucleic acid sequences. System and method. According to an aspect of the present invention, a technique for facilitating protein database queries is provided. For protein descriptions received in response to the query, aspects of the invention include scanning the received protein descriptions to identify and locate Replikin patterns. According to one aspect, the Replikin pattern is a sequence of 7 to about 50 amino acids, each comprising the following three (3) features that can be recognized according to aspects of the present invention. (1) the sequence has at least one lysine residue located 6-10 amino acid residues from the 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 invention can identify and / or locate complex amino acid sequences having specified length constraints, which sequences further include any combination of the following features: 1) a first amino acid residue located above the N position and less than the M position, away from the second amino acid residue; (2) a third amino acid residue located anywhere in the sequence; and (3) At least R percent of the fourth amino acid residue. According to yet another aspect, the present invention counts the occurrences of identified amino acid sequences and calculates the counted occurrences as raw absolute values or the number of identified amino acid sequences per N amino acids in the protein. Can be reported as any of the ratios. Yet another aspect of the present invention is that the evolution of identified amino acid sequence patterns in a given protein variant can be analyzed over time and also identified amino acid sequence patterns across multiple different proteins. Similarities and differences between examples can also be analyzed over time. As a result of the analysis, yet another aspect of the present invention is that the potential to be conserved over time and between different proteins when components of the identified amino acid sequence pattern are mutated and / or generated Specific amino acid backbone structures can be identified.

  Aspects of the invention refer to the accompanying drawings, wherein like parts are numbered with like reference numerals throughout, and the leftmost digit of each reference number refers to the figure number in which it first appears. However, it will be described.

  FIG. 17 is an advanced block diagram of a computer system incorporating a system and method for identifying Replikin patterns in amino acid sequences, in accordance with aspects of the present invention. As shown in FIG. 17, a computer workstation 610 includes a processor and memory configured to allow a researcher to search a protein database and scan a protein description for a selected amino acid pattern. May be included. To accomplish these functions, the computer workstation 610 may include a protein and amino acid research system 630 that can perform protein searches and amino acid scanning operations in response to instructions from a user / researcher. According to an aspect, the protein and amino acid research system 630 may further include an amino acid sequence scanner 640 that searches and scans the searched protein and amino acid sequences for a particular amino acid pattern, including a Replikin pattern. Protein and amino acid research system 630 can communicate with network interface 620 to obtain protein and amino acid sequences from sources on network 660 that can include the Internet. Alternatively, the protein and amino acid research system 630 can obtain protein and amino acid sequences from the local protein database 650. Furthermore, the protein and amino acid research system 630 may obtain the protein sequence and amino acid sequence directly from other input means such as keyboard input. The protein and amino acid research system 630 can also communicate with the network interface 620 to transmit the results to other computers on the network 660.

Automated Scanning for Replikin Patterns Aspects of the invention may include generalized methods and systems for identifying complex patterns of amino acids within proteins. At any time in the definition of any protein identified or selected by the protein and amino acid research system 630, the user can turn to the aspects of the present invention to search for various complex patterns of amino acids. As an example of one pattern of amino acids, the present invention provides a method for identifying a nucleotide or amino acid sequence comprising a Replikin pattern. FIG. 18 is a simple flowchart illustrating a general method for locating a Replikin pattern in an amino acid sequence according to an embodiment of the invention. The method 700 can begin after the amino acid sequence is obtained. Typically, the amino acid sequence can be represented by alphabetic characters according to the code given in FIG. However, other encoding methods are within the scope of the present invention as well.

Referring to FIG. 18, once an amino acid sequence is obtained, the sequence is searched for a Replikin pattern (710), which has the following characteristics:
(1) the sequence comprises from 7 to about 50 amino acids;
(2) the row comprises at least one lysine residue located at positions 6-10 from the second lysine residue;
(3) the sequence comprises at least one histidine residue; and (4) the sequence comprises an amino acid sequence (or sequence) having at least 6% lysine residues.

  If an amino acid sequence is found to match the Replikin pattern, the sequence can be identified or labeled accordingly (720).

  A given amino acid sequence can include many subsequences or sequences that match a Replikin pattern. Further, the Replikin patterns may overlap each other. Thus, to locate and identify all possible Replikin patterns in an amino acid sequence, the method 700 is iteratively invoked for each subsequence of amino acids contained within the original sequence of amino acids. Can do.

  When the method 700 is iteratively invoked to identify and locate all possible Replikin patterns in an amino acid sequence, aspects of the invention count the number of Replikin patterns obtained be able to. The number of Replikin measurements can be reported as an absolute value. In addition, aspects of the invention can also determine the ratio of the number of Replikins per N amino acids in the sequence. For example, an embodiment can determine that a given protein contains a ratio of 6 Replikins per 100 amino acids. The ratio of Replikin was directly related to the rate at which a given protein replicated, as shown by laboratory experiments and epidemiological evidence. Fast replication of proteins can be an indicator of disease. For example, the presence of a relatively high ratio of Replikin patterns has been linked to influenza epidemics. Similarly, an increase in the number of Replikin patterns observed over time in a protein can be an indicator of future illness caused by the organism from which the protein is derived (eg, FIG. 15). checking). Thus, the ability to detect and count Replikin patterns within an amino acid sequence is a significant benefit of the present invention.

Still referring to FIG. 18, aspects of the invention can utilize method 700 to identify and locate other complex patterns of amino acids that exhibit features similar to Replikin patterns. That is, some aspects of the present invention include: (1) the distance between amino acids, (2) the permissible length of the recognized amino acid sequence, and (3) the percentage or concentration of a particular amino acid. Although exact values can be specified, these exact values may be expressed as variables. Therefore, researchers adopt aspects of the present invention and have the following features:
(1) The sequence comprises amino acids from rmin to rmax;
(2) the sequence comprises at least one lysine residue located from the second lysine residue to kmin to kma amino acid residues;
(3) the sequence comprises at least one histidine residue; and (4) the sequence comprises at least k percent lysine residues, wherein the amino acid sequence in the protein can be identified.

  FIG. 19 is a flowchart illustrating a generalized method 800 for locating multiple Replikin patterns in a given amino acid sequence according to aspects of the present invention. The method 800 begins by locating the first lysine residue in a given sequence (810). The method 800 can then determine whether the second lysine residue is within the position kmin-kmax of the first lysine residue (820). As shown in FIG. 19, kmin and kmax define the distance limit between the first and second lysine residues. For a typical Replikin pattern, kmin will be equal to 6 and kmax will be equal to 10. However, these values can be changed by researchers who focus on finding other similar patterns.

  Once the method 800 has identified two lysine residues that are sufficiently close to each other (820), the method 800 can examine each histidine residue that is within the rmax position of both the first and second lysine residues ( 830). If the method 800 is employed to identify and locate a typical Replikin pattern, typically rmax will be set equal to 50. For each histidine residue that is within the rmax position of the two lysine residues identified in steps (810) and (820), the method 800 uses the first lysine residue, the second lysine residue, and the identified histidine. The shortest sequence of amino acid residues containing residues will be constructed (840). The method 800 then determines whether the shortest column length is within the desired range, that is, whether it includes at least rmin amino acid residues and no more than rmax amino acid residues. (850). Finally, if the identified sequence of amino acids also contains at least k percent lysine residues (860), the sequence will be identified as conforming to the desired Replikin-like pattern (870).

  Referring to FIG. 19, it is clear that the method 800 can identify several Replikin-like patterns from a single given amino acid sequence. This may occur because method 800 can examine more than one histidine residue that is within the rmax position of two identified lysine residues. Each identified histidine residue, together with two lysine residues, may match the desired Replikin-like pattern.

  One aspect of the method illustrated by FIG. 19 is shown in FIG. 20, which includes a procedure for discovering all Replikin patterns present in a given amino acid sequence according to aspects of the present invention. Source code listing. The “match” procedure shown in FIG. 20 is programmed in an interpreted shell language called “Tcl” and recognizes Replikins in a straightforward manner. As known in the art, “Tool Command Language” or Tcl (pronounced “ticle”) is an interpreted scripting language whose roots are in a Unix® command shell. There are additional capabilities that are well adapted to the rapid development of network communications, Internet functions and graphical user interfaces.

Other methods of recognizing Replikin patterns are also included in the teachings of the present invention. For example, the match procedure shown in FIG. 20 could be implemented in Java® or other programming languages such as C or C ⁺⁺ . In addition, other aspects of the Replikin recognition algorithm can identify features of the Replikin pattern in any order, and also recursive procedures, iterative procedures, parallel processing methods, divide-and-conquer methods, or any combination thereof Can be used to search all components of amino acid sequences and subsequences.

Protein Search Engine Returning to FIG. 17, the present invention can include a search engine that accesses and interacts with amino acid and protein databases locally or across a network, such as the Internet, to search for protein definitions. . For example, the protein and amino acid research system 630 can accept protein search criteria from a user, and multiple online amino acid and protein database searches to search for protein definitions that match the supplied search criteria. You can access the engine. The protein database search criteria can include any text string that can form a valid search term in any online protein or amino acid search engine. Typically, these search criteria are associated with text that can be found in a printout that describes each particular protein. For example, if the user provides the search criteria “type A influenza”, aspects of the present invention can forward this text string to multiple Internet protein and amino acid search engines, each of which has the term “A Any protein description found in that database that includes "type influenza" can be returned. Using the amino acid sequence scanner 640, each of the returned protein descriptions can be scanned for the presence of a Replikin pattern.

  A further aspect of the present invention allows the user to select multiple Internet protein search engines and customize the search criteria and protein search capabilities of the present invention for each of the selected online protein search engines. And In addition, aspects of the present invention also provide the user with access to a local protein database 650 or provide, for example, a local filename containing protein definitions or parameters to computer software. It makes it possible to supply a specific protein definition directly by other known methods.

  Other aspects of the invention may include a search engine that accesses and interacts with amino acid and protein databases on the Internet to search for protein definitions or amino acid sequence definitions. After receiving protein or amino acid sequence search criteria from a user, the present invention accesses multiple amino acid and protein database search engines via online access to search for protein definitions or amino acid definitions that match the provided search criteria. can do.

  The initial existing protein search criteria based on an existing definition can include any text string that can form a search term that is valid in any online protein or amino acid search engine. Typically, these search criteria relate to text that can be found in prints that describe each particular protein. For example, if the user supplies the search criteria “type A influenza”, the present invention sends the text string to a plurality of internet protein and amino acid search engines, which then each contain the word “type A influenza”. Returns one of the protein definitions in the database.

  Non-limiting aspects of the present invention, including a protein search engine entitled “Genome Explorer” are included in Appendix A. A Tcl procedure called “GenomalEnquire” can control the macro-level operation of a protein search engine (see “proc GenomalEnquire {detabase term additional Criteria}”). In the procedure GenomalEnquire, a set of specific online protein search engines are You can access and query using the provided protein search terms and further criteria.A further aspect of the invention is to select or exclude multiple Internet protein search engines, and each selected online protein search Allows the user to customize the search criteria and protein search capabilities of the present invention for the engine.In addition, aspects of the present invention also provide access to a local protein database, or a specific definition, eg, a local containing protein definition. Phi Name by, or can be allowed to be subjected directly to the user by other methods known in the art to provide the parameters to the computer software.

  Instructions for operating Genome Explore are included in Appendix B. Screen shots of the Genome Explore application are included in Appendix C.

Replikin Analysis Embodiments of the present invention are not used solely to identify and locate Replikin patterns in amino acid sequences. Examples can also be used to discover and analyze similarities in the structure of Replikin patterns that occur in different proteins, or to analyze different Replikin patterns that occur over time in the same protein. it can. For example, FIG. 21 is a table showing a “fixed scaffold” structure that has been conserved in the Replikin scaffold or “Bird Flu” influenza virus for 87 years from 1917 to 2004. Embodiments of the present invention can collect a large number of Replikin patterns found in a protein, including Replikin patterns found in variants of the same protein. Along with each Replikin pattern, aspects of the invention can also accompany the date that each protein was first identified. To elucidate substantially fixed amino acid structures that are conserved over time in the Replikin pattern and can exist in different proteins as well as variants of the same protein, as directed by researchers Aspects of the invention may include sorting and displaying a plurality of selected Replikin patterns by content, date or other criteria. Furthermore, in order to automatically identify such a fixed amino acid structure when directed by a researcher, aspects of the present invention employ a known pattern analysis method for comparing multiple selected Replikin patterns. May be. As an example, in FIG. 21, the Replikin pattern shown—in this case—starts with a pair of lysine residues (kk) at the amino terminus, ends with a pair of histidine residues (hh) at the carboxyl terminus, and It appears to show a relatively fixed backbone structure of (usually) 29 amino acids containing residues at either 8, 10 or 11 positions. Preservation of this skeletal structure for decades allows synthetic vaccines to be prepared quickly and inexpensively. After the Replikin backbone structure has been identified, to synthesize such a vaccine, researchers can select components of that backbone structure that are stored over time and that are also present in current variants of the protein. . The vaccine can then be prepared based on components selected from the skeletal structure. Because such vaccines are based on a conserved skeletal structure, they are effective for many years and can be developed well before the anticipated outbreak.

  The aspects of the present invention for discovery of Replikin itself and identification and localization of Replikin patterns provide a target for pathogen identification and facilitate anti-pathogen therapy including vaccines. In general, knowledge and identification of the Replikin family of peptides allows the development of effective therapies and vaccines for any organism that has Replikin. In particular, the identification of Replikin provides for the detection of viruses, including influenza viruses, and the development of viral vaccines. In addition, the identification of Replikin provides for the detection of other pathogens such as malaria, anthrax, and smallpox viruses, in addition to enabling the development of treatments and vaccines that target the Replikin structure. Additional examples are provided by the identification of Replikins, including detection of infectious disease Replikins, cancer immune Replikins, and structural protein Replikins.

  Embodiments of the present invention allow important Replikin patterns of amino acids to be recognized, located and analyzed in a manner not found in the prior art. Using the power of the prior art, researchers have been limited to existing techniques for describing amino acid sequences. In fact, the limitations of the prior art are somehow weakening the momentum of research in this field because it has not been possible to identify amino acid sequences that contain non-linear attributes. Until the methods and embodiments of the present invention were developed, descriptions of amino acid sequences included, at best, logical constraints on repeating subsequences and subsequence components. Aspects of the invention allow new classes of amino acids 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 concentrations and distance relationships between specific amino acids. These attributes go beyond simple contiguous order and are therefore not easily described, discovered or located in existing methods known in the art.

  For example, rather than conducting a strict amino acid sequence match (homology) study with other widely used programs such as BLAST, we have identified unique replications for fast replication that define a new class of amino acid groupings. Discovered a quantitative "language". The novel computer program described herein relates to examples of the novel language.

These programs include functions for retrieving electronic data for amino acid subsequences that meet predetermined criteria. The data obtained online can include data defining a particular group of protein sequences.
The criteria are:
i) the appearance of two amino acids in the protein sequence, in this case lysine (K) and histidine (H) at a particular concentration in the sequence
ii) an interval from one of these (K) to the second K in the sequence, and
iii) It can include the concentration of one or more amino acids (eg, K) in a percentage of a specified percentage abnormality.

  Amino acid sequences that meet the above criteria are associated with specific biological functions, such as rapid replication.

  The program includes the ability to identify Replikin subsequences in the genomic sequence. One source of the genomic sequence can publish the genomic sequence obtained from an online, electronic database using search criteria provided by the user. In the context of the present invention, the database may be the NCBI (National Center for Biotechnology Information) or LANL (Los Alamos National Laboratory) database. The program further includes the ability to search for any subsequence (ie not just the Replikin subsequence) based on criteria provided by the user.

  In one aspect, a program entitled “Genome Explorer” can create a user interface that provides user acceleration for search terms. Genome Explorer can apply the search terms to online databases, such as NCBI or LANL databases, to obtain raw sequence data. For example, further data such as literature name, protein source, strain, serotype, and year of discovery for all raw sequences that match the search term can be obtained. Once the raw data is acquired, Genome Explorer can further apply additional search criteria to identify Replikin subsequences in the raw sequence. The search criteria can be specified by the user to implement a relatively strict or relatively relaxed definition that can include a set of matching subsequences reported by the Genome Explorer. Once this identifies the Replikin subsequence, Genome Explorer can compile ongoing statistics and display a progress bar in the user interface. Once Genome Explorer completes its processing, the statistics obtained in the data file can be saved. For example, the data file may be HTML that can be opened in any word processor for inspection of results.

  In another aspect, a program search criterion entitled “Dr. Peptide” can be applied to identify subsequences other than Replikin subsequences. Dr. Peptide makes it possible to search all examples of the sequence hlk... Hlk that are separated by, for example, a maximum of 15 amino acids in a publicly available genome database. These searches allow to create new statistical profiles and new groupings of proteins based on matching criteria. Dr. Peptide can include almost the same functionality as Genome Explorer. For example, like Genome Explorer, Dr. Peptide can be used against online databases such as NCBI or LANL databases to speed up user search terms and obtain raw sequence data via the user interface. The search term can be applied. For example, further data such as literature name, protein source, strain, serotype, and year of discovery for all raw sequences that match the search term can be obtained. Once the raw data is acquired, Dr. Peptide further processes the data to identify any subsequence and in an HTML page format that can be opened with any word processor in the data file, for example. The output can be presented.

  The following is a description of examples of logical sequences that can be included in the Genome Explorer. In the description, “initial server query” means a search criterion applied to one or more network components, eg, server computers, stored electronic data representing protein sequences. The network component can be included in a private network or, for example, in the Internet. The data can exist in a “protein page”, ie, a quantum form of data representing the protein sequence. The letter “k” represents a lysine amino acid and the letter “h” represents a histidine amino acid.

Genome Explorer Logic Sequence Initialize user interface procedures and enter fields for search parameters.
Build a user interface.
Wait for user to specify search parameters.
Search parameters include:
(1) a matching word or phrase in the initial server query to obtain a summary and protein page;
(2) An allowable distance between k expressed as a range of k minimum... K maximum for a subsequence to limit the set.
(3) Tolerance of the distance between h and the furthest k expressed as k min + 1... K max for the subsequence to limit the set.
(4) The allowable fraction of k in the subsequence expressed as x percent or more for the subsequence to limit the set.

Once the search parameters are specified, initialize the output file in HTML format—these are used to display the report.
Compare identified search parameters with previous searches.
If the search parameters are the same, the cached protein page is reused as data input.
If the search parameters are not identical (cached protein pages are not relevant), send a query to the server (NCBI or LANL).
If you do not return all the summaries, send the query requesting all the summaries again.
For each summary, fetch and save the captured protein page.
For each imported protein page,
When derived from NCBI:
Analyze the ASN page.
Extract the found sequence data (seq-data.ncbieaa).
Extract the document name (descr. *. Article.title. *. Name).
Extract the protein source (source.org.taxname).
Extract the subtype.
Pull out the year of discovery.
Derive serotype.
If derived from LANL,
Analyze HTML pages for strain, definition, source, year, serotype, and raw nucleotide sequence.
Nucleotides are converted to amino acids by mapping each three nucleotides in the sequence corresponding to the amino acid.
Save the analyzed value for the protein.
For each analyzed page, update the user interface for progress through the progress bar, and:
For each sequence data found on the page,
Scan amino acid sequence data for each subsequence that matches:
(A) As defined in the parameter (2) derived from the user interface, the distance between k is in the range of k minimum to k maximum.
(B) (a) As defined in the parameter (3) derived from the user interface, the distance between h and the furthest k is in the range of k minimum + 1... K maximum.
(C) As defined in parameter (4) from the user interface above, the fraction of k units in the subsequence is expressed as x percent or more.
Then save the range of each matching subsequence, including the overlap.
Ignore unmatched sequences.
Receives the sequence with the most subsequence matches.
When it receives an array, it lists each array by the year it was discovered.
For an additional set of criteria,
Check additional criteria for other analysis fields.
If it doesn't match, don't accept the page.
If you accept the page,
Add this as a passing page.
Create an HTML page showing the complete sequence and all matched subsequences.
If you do n’t accept the page,
Add this as a reject page.
For each unique matched Replikin sequence,
Create an amino acid history HTML page,
All proteins generated in year order are shown.
Create a statistical HTML page that represents:
For each year
Matched protein and Replikin subsequence numbers are indicated.
Update the user interface to reflect that the operation is complete.
Reinitialize the input field to allow the next set of search parameters specified by the user.

  In view of the foregoing explanation, Genome Explorer is a method comprising applying a plurality of criteria to data representing a protein sequence and identifying a subsequence in the protein sequence based on the criteria, the identification It can be seen that the subsequences performed carry out a method having a predetermined tolerance of the distance between the histidine amino acid and the farthest lysine amino acid. The identified subsequence can be output to a data file.

  The functions of the aspects herein can be provided to various computer platforms that execute program instructions. One such platform 1100 is illustrated in the simplified block diagram of FIG. There, platform 1100 is shown as assumed by processor 1600, which communicates with a number of peripheral devices via 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 can be any of a number of conventional processing systems including a microprocessor, a digital signal processor, and a field programmable logic array. In some applications, it may be advantageous to provide the platform 1100 with multiple processors (not shown). The processor 1160 executes the instructions of the program recorded in the memory subsystem 1110. The memory subsystem 1110 can include any combination of conventional memory circuits including electrical, magnetic, or optical memory systems. As shown in FIG. 22, the memory system can include a read-only memory 1120, a random access memory 1130, and a bulk storage device 1140. The memory subsystem 1110 may not only store program instructions representing the various methods described herein, but may also store data items on which these methods are performed. The network interface subsystem 1170 can provide an external network with an interface including an interface that communicates with a network 1190 including the Internet. The I / O unit 1180 will allow communication with external devices (not shown).

  Several aspects of the invention have been specifically illustrated and described herein. However, it will be understood that modifications and variations of the present invention are encompassed by the teachings of the present invention without departing from the spirit and intended scope of the invention. Furthermore, the teachings of the present invention can be adapted to other sequence recognition problems that have so far been addressed in the identification of specific sequence components using limited continuous linear analysis.

  Using the representative software included in Appendix A, in an unrestricted aspect in accordance with the present invention, the inventor has exceptionally identified the shrimp vitiligo virus nucleocapsid protein to the Replikin investigation to date. It was found to have a high Replikin Count compared to all other viruses and organisms (except malaria). Although Replikin has been shown to be an essential companion for fast replication in fungi, yeast, viruses, bacteria, algae, and cancer cells, the inventor has shown that Replikin is the first in the presence of Replikins in marine organisms other than algae. The proof was provided. And, as with algae, the presence of Replikin is again associated with rapid spread. In shrimps, the vitiligo virus is causing millions of dollars in shrimp recovery, first in countries in the Eastern Hemisphere, and now in countries in the Western Hemisphere. Other examples of Replikin's high mortality marine viral diseases have been demonstrated by us in hemorrhagic diseases in fish, such as carp and salmon, and are probably widespread in marine organisms and diseases.

  The presence of the Replikin repeat of the nucleocapsid protein of shrimp vitiligo syndrome virus (WSSV) reveals a high Replikin count of 103.8 or higher. This viral Replikin Count is, for example, much higher than the influenza virus Replikin Count, usually ranging from 1 or less to a maximum of 5 or 7, and a record of the Replikin Count observed in 111 P. falciparum (malaria) ( It ’s only comparable to the past). Interestingly, the shrimp vitiligo syndrome organism is a virus and the Plasmodium falciparum is trypanosomes, both of which use the main part of their reproductive cycle in red blood cells, shrimp (white spot virus) or human (malaria) Both of these abnormal host cells explode fast replication disease with high mortality of these hosts, and together increase their high Replikin count to such record heights, ie Replikin It seems to use repetition and Replikin duplication.

  As shown in Table 10, examples of Replikin repeats and Replikin duplications were found by the applicant in the above nucleocapsid protein of shrimp vitiligo syndrome virus, as shown below. Using the representative software provided in Appendix A, 497 Replikins were found in vitiligo virus. Of these 497, the Replikins shown in Table 10 below were selected for these short sequences, and as demonstrated throughout this application, high concentrations of lysine seem to be associated with high mortality. is there. Selected sequences are easier to synthesize and less expensive than longer sequences not included in Table 10.

Table 10 shows molecules in the shrimp vitiligo syndrome virus (WSSV) nucleocapsid protein (VP35) gene with a Replikin count of 103.8 (Replikins per 100 amino acids) (497 total Replikins per 479 amino acids). Inner Replikin repeats and Replikin duplications are shown.

  Now that we can identify Replikin using the software described in this application, we synthesize each of these and for antibodies and other inhibition products, as well as shrimp vitiligo syndrome virus, particularly each These can be used as targets for specific synthetic vaccines against repeated Replikins.

  The phenomenon of repetition is well known in protein structure. What is unique and specific in the case is that these are Replikin repeats. Thus, repetition of a particular Replikin sequence increases the Replikin count in a particular molecule and is associated with faster replication, as in ATPase in P. falciparum in malaria, thus It has an apparent survival value for the containing organism, while at the same time it provides an increasing vulnerability, a so-called “weakness”. Replikin repeats are therefore added for attack by higher concentrations per molecule, specific antibodies produced by specific synthetic vaccines produced against these Replikins, and other specific anti-Replikin agents Provide a target site. These new targets could not be used before because they could not be identified.

Complex Amino Acid Analysis A further aspect of the invention includes a protein search engine for the identification of universal amino acid and nucleic acid patterns in an online database. Appendix D is a representative protein search engine for identification of complex amino acid patterns, eg, exoskeleton skeletons. Appendix D is titled “Dr. peptide”. Appendix D is a representative, non-limiting aspect of the present invention and was designed to identify universal amino acid patterns in addition to Replikin patterns.

  The following is a description of one example of a logical sequence that can be included in the Dr. peptide program. In the description, “initial server query” means a search criterion applied to one or more network components, eg, server computers, stored electronic data representing protein sequences. The network component can be included in a private network or, for example, in the Internet. The data can exist in a “protein page”, ie, a quantum form of data representing the protein sequence.

Dr. peptide logical sequence Initializes the user interface procedure and enters fields for the search parameters.
Build a user interface.
Wait for user to identify search parameters including:
(1) a matching word or phrase during the initial server query to obtain a summary and protein page;
(2) A specific set of amino acids that must be included in any subsequence that limits the set.
(3) A specific set of amino acids that must be excluded from any of the subsequences that limit the set.
(4) Minimum m and maximum n sizes for the allowed spacing spacing gaps, applied to set inclusion and exclusion criteria (2) and (3).

Once the search parameters are identified,
Query:
If the saved protein page is not relevant,
Send a query to the server (NCBI or LANL).
If you did not reply with a full summary,
Resend the query requesting a full summary.
For each outline,
Fetch and save the protein page.
For each protein page,
When derived from NCBI:
Analyze the ASN page.
Extract the found sequence data (seq-data.ncbieaa).
Extract the document name (descr. *. Article.title. *. Name).
Extract the protein source (source.org.taxname).
Extract the subtype.
Pull out the year of discovery.
Derive serotype.
If derived from LANL,
Analyze HTML pages for strain, definition, source, year, serotype, and raw nucleotide sequence.
Nucleotides are converted to amino acids by mapping each three nucleotides in the sequence corresponding to the amino acid.
Save the analyzed value for the protein.
For each analyzed page,
For each sequence data found on the page,
Scan the amino acid sequence data for each subsequence matching.
The match pattern is a sequence of separate steps.
(A) As defined in user parameter (2) above, the amino acids in the amino acid sequence data are present in a specific set of amino acids.
(B) As defined in the user parameter (3), the amino acid in the amino acid sequence data does not exist in a specific amino acid set.
(C) As defined in user parameter (4) above, the amino acids in the amino acid sequence data have a spacing gap of m to n amino acids from other amino acids.
The initial subsequence set is all possible end sequences, or the “tail” of the sequence data in the first pattern step,
If the set of subsequences is not empty,
Remove one subsequence and record the distance the evaluation spans in the pattern string.
The amino acids in the current pattern process
-If present in a particular set of amino acids,
If the next amino acid in the subsequence is also present in the set of amino acids,
The extended subsequence and the next pattern step are added to the subsequence.
-If not present in a particular set of amino acids,
If the next amino acid in the subsequence is not one of the set of amino acids,
The extended subsequence and the next pattern step are added to the subsequence.
-Having a gap of any amino acid from m to n
First, decompress each subsequence for each possible length m to n,
Each expanded version of the subsequence is then added to the subsequence set.
If the above pattern is consumed,
The subsequence is a matched subsequence.
Ignore unmatched sequences.
Receives the sequence with the most subsequence matches.
When you receive an array
List each sequence by the year it was discovered.
For an additional set of criteria,
Check additional criteria for other analysis fields.
If it doesn't match, don't accept the page.
If you accept the page,
Add this as a passing page.
Create an HTML page showing the complete sequence and all matched subsequences.
If you do n’t accept the page,
Add this as a reject page.

In view of the foregoing description, Dr. Peptide performs a method that includes applying a plurality of criteria to data representing a protein sequence and identifying any subsequence in the protein sequence based on the criteria. I understand that. The identified subsequence can be output to a data file. The identified subsequence can be output to a data file. The criteria are:
A set of amino acids {a} contained in the subsequence;
A set of amino acids excluded from the subsequence {b}; and a minimum and maximum allowable gap between members of the sets {a} and {b}.

A non-limiting exemplary aspect of the present invention utilizes composite amino acid analysis to analyze Replikin scaffold sequences in early strains of influenza that denature into non-Replikin scaffolds but retain the Replikin scaffold backbone structure. As an example of the use of the exemplary and non-limiting software program in Appendix D to identify generic amino acid patterns, we identified by visual scanning of protein sequences (now with Dr. Peptide software) Discovered what was the Replikin skeleton in early-stage specimens of influenza and what changed in later specimens:
1) A length of 29 amino acids was conserved;
2) The first two amino acid positions (1 and 2), ie KK, were conserved.
3) The last two amino acid positions (28 and 29), ie HH, were conserved.
4) However, K, which is 6-10 amino acids from KK no longer exists (required for the definition of Replikin).

  Thus, the skeleton is no longer a Replikin skeleton, but a so-called exoskeleton skeleton (Scaffold Exoskeleton). The Replikin skeleton is associated with high Replikin counts and the occurrence of epidemics, while the exoskeleton skeleton is associated with viral dormancy and epidemic decline and termination. Thus, the exoskeleton skeleton is a modified structure left as a residue when the Replikin skeleton and certain viral outbreaks are diminished, and therefore appears to be a useful characteristic structure for that purpose. This confirms the new facts and uses of the Replikin backbone as 1) targets for anti-fast replication agents, eg antibodies or small interfering RNA, and 2) the basis of antiviral vaccines. Software according to aspects of the invention can include logic for obtaining and analyzing proteins to identify sequences having the above features 1, 2, 3, and 4. For example, the exoskeleton skeleton can now be detected and measured in any protein sequence by the representative software in Appendix D.

Another non-limiting aspect of the present invention is a method for identifying a Replikin skeleton comprising:
(1) identifying a terminal lysine;
(2) identifying a terminal histidine and other histidines at residue positions immediately adjacent to the terminal histidine;
(3) identifying at least one lysine in about 6 to about 10 amino acid residues from at least one other lysine; and (4) identifying at least about 6% lysine;
A method comprising identifying a series of peptides comprising about 17 to about 30 amino acids.

  In a non-limiting aspect related to the present invention, a method for identifying a Replikin scaffold may include identifying a single or multiple individual members of a series of Replikin scaffolds.

  In a preferred non-limiting aspect with respect to the present invention, the method for identifying a Replikin backbone further comprises the identification of a second lysine immediately adjacent to the terminal lysine. Software in accordance with aspects of the present invention can include logic for obtaining and analyzing protein sequences to identify sequences using steps 1, 2, 3 and 4 above.

Tcl Programming Language Tcl (“Tool Command Language”, referred to as “Tickle”) has roots in the Unix® command shell, but has additional capabilities that are well suited for network communications, Internet functions, and graphical user interfaces Is a simple interpreted scripting language. Tcl was created in 1988 by John k. Ousterhout of the University of California, Berkeley. Although initially considered as a reusable and embeddable language core for a variety of software tools, it now includes applications including web scripting, test automation, network and systems management, and a variety of other fields. Widely used in

  In terms of the present invention, Genome Explorer and Dr. Peptide can be coded in Tcl / Tk, a script programming language that includes powerful capabilities for Internet access, user interface design, and string processing. Tcl / Tk is a program written in Tcl / Tk that can be ported to almost any available computer architecture and operated on most operating systems known to those skilled in the art. The source code for the specific execution of Genome Explorer and Dr. Peptide is provided in Appendix A and D. Specific implementations are provided by way of illustration and example only, and the invention is not in any way limiting the specific implementations illustrated.

Other uses of the three-point recognition method “Three-point recognition” is a proteomic method of identifying a particular protein type, so it is possible to use more than two different recognition points for other peptides. It should provide useful information about the type. Furthermore, the “three-point recognition” method is applicable to TOLL “innate” recognition of other recognins such as living lipopolysaccharides. Modified the three-point recognition method to identify other useful covalently linked organic molecule compounds that contain other covalently linked amino acids, nucleotides, carbohydrates, lipids, or combinations thereof It is also possible to do. In this aspect of the invention, sequences are screened for subsequences that contain three or more desired structural properties. When screening compounds consisting of covalently linked amino acids, lipids or carbohydrates, a subsequence of about 50 covalently linked units is (1) of the first amino acid, carbohydrate or lipid residue. At least one first amino acid, carbohydrate or lipid residue at 7-10 residues from the second; (2) at least one second amino acid, lipid or carbohydrate residue encoding; and (3 ) It should contain at least 6% of the first amino acid, carbohydrate or lipid residue. When screening a nucleotide sequence, a subsequence of about 21 to about 150 nucleotides is (1) a first amino acid that is within 18-30 nucleotides from a second codon encoding the first amino acid residue. (2) should contain at least one second amino acid residue and (3) encode at least 6% of said first amino acid residue.

  Several aspects of the invention are specifically illustrated and described herein. However, it will be appreciated that modifications and variations of the invention are encompassed by the above teachings and are within the scope of the appended claims without departing from the spirit and intended scope of the invention.

Example 1
Process for extraction, isolation and identification of Replikin and use of Replikin to target, label or destroy Replikin-containing organisms

a) Algae The following algae were collected from Bermuda water sites and extracted the same day or frozen at -20 ° C and extracted the next day. 1 gram of algae in a cold buffer (0-5 ° C.) in a Waring blender for 15 minutes in neutral buffer, eg 100 cc of a 0.005M phosphate buffer solution (“phosphate buffer”) at pH 7. Homogenized in an aliquot, centrifuged at 3000 rpm, the supernatant was condensed by osmosis and dialyzed against phosphate buffer at low temperature to produce a volume of about 15 ml. The volume of this extract solution was noted, an aliquot was taken for protein analysis, and the remainder was fractionated to obtain a protein fraction having a pK range of 1-4.

A preferred fractionation method is chromatography as follows. That is, the extract solution is fractionated in a cold room (4 ° C.) on a DEAE cellulose (Cellex-D) column equilibrated with 0.005 M phosphate buffer. Perform stepwise elution solvent changes using the following solutions:
Solution 1-1 to 4.04 g NaH2PO4 and 0.5 g NaH2PO4 are dissolved in 15 liters of purified water (0.005 mol, pH 7);
Solution 2—8.57 g NaH 2 PO 4 is dissolved in 2480 ml of purified water;
Solution 3-17.1 g of NaH2PO4 is dissolved in 2480 ml of purified water (0.05 mol, pH 4.7);
Solution 4-59.65 g NaH2PO4 is dissolved in 2470 ml purified water (0.175 mol);
Solution 5-101.6 g NaH2PO4 is dissolved in 2455 ml purified water (pH 4.3;
Solution 6-340.2 g NaH2PO4 is dissolved in 2465 ml purified water (1.0 mol, pX-i4.1;
Solution 7-283.63 g of 80% phosphoric acid (H3PO4) is made up in 2460 ml of purified water (1.0 mol, pH 10)

  The extract solution in a volume of 6-10 ml was transferred onto the column, the solution 1 was layered on top, a 300 ml tank of solution 1 was installed and dropped onto the column by gravity. A 3 ml aliquot of the eluate was collected and analyzed for protein content at OD280 until all of the protein to be removed with solution 1 had been removed from the column. Solution 2 was then applied to the column, and solutions 3, 4, 5, 6 and 7 continued in succession until all of the protein that could be removed with each solution was removed from the column. The eluates from solution 7 were combined and dialyzed against phosphate buffer to determine the protein content of both Dialysand and dialysate and both were analyzed by gel electrophoresis. In solution 7, one or two bands of peptides or proteins with a molecular weight between 3000 and 25,000 daltons are obtained. For example, after extraction and treatment as described above, the algae Caulerpa mexicana, Laurencia obtura, Cladophexa prolifera, Sargassum natans, Caulerpa verticillata, Halimeda tuna, and Penicillos capitatus all have this molecular weight free of any contaminants in solution 7 eluate. A sharp peptide band within the region was demonstrated. These proteins of protein 7 or their eluted bands are hydrolyzed and the amino acid composition is determined. Peptides thus obtained with a lysine composition of 6% or more are Replikin precursors. These Replikin peptide precursors are then determined for amino acid sequence and the Replikin determined by hydrolysis and mass spectrometry as detailed in US Pat. No. 6,242,578B1. Those meeting the criteria defined by the “3-point recognition” method are identified as Replikins. This procedure can also be used to obtain enzymes, bacteria, and any plant Replikin.

b) Viruses Isolate and identify Replikins in virus-infected cells using the same extraction and column chromatography separation methods described above in a) for algae.

c) Tumor cell in vivo and in vitro tissue culture Replikins in virus-infected cells are isolated and identified using the same extraction and column chromatographic separation methods described above in a) for algae. For example, Replikin precursor of astrocytin isolated from malignant brain tumors each treated as described above in a), mariignin (Aglyco10B) isolated from glioblastoma tumor cells in tissue culture, MCF7 suckling in tissue culture Animal cancer cells and P3J lymphoma cells in tissue culture produced Replikin precursors with lysine contents of 9.1%, 6.7%, 6.7%, and 6.5%, respectively. Hydrolysis and mass spectrometry of Aglyco10B as described in US Pat. No. 6,242,578B1 Example 10 yielded the amino acid sequence, ykagvaflhknndide, 16-mer Replikin.

Example 2:
As an example of diagnostic use of Replikin, as shown in FIGS. 2, 3, 4 and 7 of US Pat. No. 6,242,578B1, the corresponding antibody present in serum for diagnostic purposes is captured and the amount Aglyco10B or 16-mer Replikin can be used as an antigen for quantifying the amount.

  As an example of production of an agent to be attached to Replikin for labeling, nutrition or destruction purposes, injection of 16-mer Replikin into the body of a rabbit to produce specific antibodies against 16-mer Replikin is described in US Pat. 242, 578B1, Example 6 and in FIGS. 9A and 9B.

  As an example of the use of an agent to label Replikin, the use of an antibody against 16-mer Replikin to label specific cells containing this Replikin is described in US Pat. No. 6,242,578B1, FIG. This is shown in Example 6.

  As an example of the use of an agent to destroy Replikin, the use of an antibody against 16-mer Replikin to inhibit or destroy specific cells containing this Replikin is illustrated in US Pat. No. 6,242,578 B1. 6.

Example 3
Analysis of sequence data of influenza virus hemagglutinin protein or neuraminidase protein isolates for the presence and concentration of Replikin through the use of a computer program based on the three-point recognition system described herein or by visual scanning of the sequence carry out. An influenza virus isolate is obtained and the amino acid sequence of the influenza hemagglutinin and / or neuraminidase protein is obtained by any known method such as sequencing the hemagglutinin or neuramidase gene and deriving the protein sequence therefrom. . The sequences are scanned for the presence of new Replikins within each isolate, Replikin aging and Replikin concentrations. Comparison of Replikin sequences and concentrations to amino acid sequences obtained from isolates at an earlier time, such as about 6 months to about 3 years ago, is the seasonal influenza peptide vaccine with the highest probability of causing influenza in the upcoming influenza epidemic Or provide data used to predict the emergence of strains that form the basis for nucleic acid-based vaccines. Increasing concentrations of Replikin in a given strain of influenza virus over a period of about 6 months to about 3 years or more, especially the gradual increase, is a probable cause of future regional or pandemic influenza outbreaks As an emerging predictor of stocks.

  Replikin based peptide vaccines or nucleic acid based vaccines observed in emerging strains are generated. Emerging strains are identified as influenza virus strains that show the highest increase in the concentration of Replikin sequences within the hemagglutinin and / or neuraminidase sequences during the period. Preferably, the peptide or nucleic acid vaccine is based on or includes any Replikin sequence that has been observed to be conserved in emerging strains. The conserved Replikin is preferably a Replikin sequence that is present in the hemagglutinin or neuraminidase protein sequence for about 2 years and preferably longer. A vaccine can include any combination of Replikin sequences identified in an emerging strain.

  For vaccine production, the Replikin peptide (s) identified as useful for an effective vaccine can be any molecular biology technique and chemical synthesis including cloning, expression in host cells and purification therefrom. Synthesized by the method. The peptide is preferably admixed with a pharmaceutically acceptable carrier in an amount determined to elicit a therapeutic antibody response thereto. Generally, the dosage is from about 0.1 mg to about 10 mg.

  Influenza vaccines are preferably administered to patients in need thereof prior to the start of the “epidemic period”. The influenza season generally occurs in the second half of October and continues until the second half of April. However, the vaccine can be administered at any time during the year. Preferably, the influenza vaccine is administered once a year and is based on a Replikin sequence that is observed and preferably conserved within an emerging strain of influenza virus. Another preferred Replikin for inclusion in an influenza vaccine is a Replikin that has been demonstrated to have revived in an influenza strain after having been absent for one or more years.

Example 4
Analysis of sequence data of coronavirus nucleocapsids or spikes or coats or other protein isolates for the presence and concentration of Replikin through the use of a computer program based on the three-point recognition system described herein or through visual Implement by scanning. A coronavirus isolate is obtained and the amino acid sequence of the coronavirus protein is obtained by methods known in the art such as sequencing the protein gene and deriving the protein sequence therefrom. The sequences are scanned for the presence of new Replikins within each isolate, Replikin aging and Replikin concentrations. Comparison of Replikin sequences and concentrations to amino acid sequences obtained from isolates at earlier time points, such as about 6 months to about 3 years ago, is most likely to cause an outbreak of a pandemic, coronavirus peptide vaccine or nucleic acid based Provides data used to predict the emergence of strains that form the basis for vaccines. An increase in the concentration of Replikin in a given type or strain of coronavirus over a period of about 6 months to about 3 years or more, particularly its gradual increase is the probability of a regional or global epidemic such as SARS in the future It is a predictive judgment material of the emerging stocks as a high cause of.

  A peptide vaccine or nucleic acid type vaccine based on Replikin observed in an emerging strain of coronavirus is produced. Emerging strains are identified as coronavirus strains with the highest increase in Replikin sequences in the nucleocapsid sequence at that time. Preferably, the peptide or nucleic acid vaccine is based on or comprises any Replikin sequence that is observed to be conserved in the strain. A conserved Replikin is preferably a Replikin sequence that is present in a nucleocapsid protein sequence in about 2 years and preferably longer. The vaccine may comprise any combination of Replikin sequences identified in the emerging strain.

  For the production of vaccines, Replikin peptides or groups of peptides recognized as useful for effective vaccines are chemically synthesized and molecular biological techniques such as cloning, expression in host cells, and purification therefrom. It is synthesized by any method including The peptide is preferably mixed with a pharmaceutically acceptable carrier in an amount determined to elicit a therapeutic antibody response. Generally, the dosage is from about 0.1 mg to about 10 mg.

  The coronavirus vaccine can be administered to the patient at any point in the year. Preferably, the coronavirus vaccine is administered once, is observed to be present in the type of coronavirus, and is preferably based on the Replikin sequence stored therein.

Example 5
Analysis of the sequence data of isolates of tropical Plasmodium antigens for the presence and concentration of Replikin is performed through the use of a computer program based on the three-point recognition method described herein or by visual scanning of the sequences. A tropical malaria parasite isolate is obtained, and the amino acid sequence of the protein is obtained by any known method, such as sequencing the gene and deriving the protein sequence therefrom. The sequences are scanned for the presence of Replikin in each isolate, Replikin over time storage, and Replikin concentration. This information provides data used to form the base material for antimalarial or nucleic acid based vaccines.

  Peptide vaccines or nucleic acid type vaccines are produced based on Replikins observed in organisms that cause malaria. Preferably, the peptide or nucleic acid vaccine is based on or includes any Replikin sequence that has been observed to be present on the surface antigens of an organism. The vaccine can include any combination of Replikin sequences identified in a strain that causes malaria.

  For vaccine production, the Replikin peptide (s) identified as useful for an effective vaccine can be any molecular biology technique and chemical synthesis including cloning, expression in host cells and purification therefrom. Synthesized by the method. The peptide is preferably admixed with a pharmaceutically acceptable carrier in an amount determined to elicit a therapeutic antibody response thereto. Generally, the dosage is from about 0.1 mg to about 10 mg.

  The malaria vaccine is then administered to patients in need of it at any time during the year, especially before traveling to the tropical environment.

  Other aspects are for in vivo Replikins, including (but not limited to) viruses, trypanosomes, bacteria, fungi, algae, amoeba and plants that are complementary to the nucleotide sequence of Replikin-containing organisms. It contains an antisense nucleic acid molecule that is complementary to the mRNA or gene coding strand that encodes the organism.

Example 6
The amino acid sequences of five short SARS Replikins found in SARS coronavirus nucleocapsid, spike, and envelope proteins were synthesized and tested in rabbits to test the immune response to the Replikin sequences in SARS coronavirus. The following Replikin sequences were tested: (1) 2003 human SARS nucleocapsid (SEQ ID NO: 712); (2) 2003 human SARS spike protein (SEQ ID NO: 717); (3) 2003 human SARS spike protein (SEQ ID NO: 718) ); 2003 human SARS spike protein (SEQ ID NO: 719); (4) 2003 human SARS envelope protein (SEQ ID NO: 720); and (5) 2003 human SARS nucleocapsid protein (SEQ ID NO: 743). Each synthetic peptide was injected subcutaneously into rabbits. Test rabbits produced measurable specific antibodies against each of the five sequences that bind at a dilution of 1 or more in 10,000. The 21 amino acid SARS nucleocapsid Replikin antibody (SEQ ID NO: 712) demonstrated binding at one or more dilutions in 204,800. For previous unsuccessful tests to achieve a strong immune response without unnecessary side effects, obtained with all proteins or thousands of proteins or nucleic acids as a smallpox vaccine with a variety of small peptides, The ability of small molecule synthetic Replikin antigens to achieve a strong immune response has been shown to be significant for the effectiveness of SARS vaccines.

Example 7
43 amino acid Replikin sequence KNSTYPTIKRRSYNNTNQEDLLVLWWGIHHKKKKHKKKKKKHK-KLH designing vaccine V120304U2 is an influenza replikin of influenza "H5N1, labeled as" 2004 G5N1 Vietnam, highly pathogenic "" in Table 8 by the inventors Designed from a 29 amino acid Replikin backbone and two additional UTPE units (KKKKHK) on the C-terminus of the H5N1 backbone and an additional adjuvant covalently linked to the C-terminus of the two UTOPE units (major limpet hemocyanin (sequence KLH)) Rabbits and chickens were injected subcutaneously with 100 μg of vaccine vaccine V120304U2 antibodies before vaccination and 1 week after injection to 8 weeks after injection. The response was measured: the antibody response was prominent at 1 week and peaked at 3 to 4 weeks of vaccination, peak antibody response from 1: 12,000 dilution to over 1: 240,000 Antibody in enzyme-linked immunosorbent assay (ELISA) with peptide-GGG (goat gamma globulin) binding in solid phase (0.1 μg / 100 μl / well) in a high binding 96-well plate The serum was first diluted 50-fold and then further diluted in 2-fold serial dilutions, and the ELISA titer results were derived from an optical density at 405 nm of 0.2, and a non-linear regression of the serial dilution curve Measured from the estimated dilution factor derived from the analysis, detection was done with horseradish peroxidase-conjugated secondary antibody and ABTS (Boehringer Mannheim. The results from the test on two chickens and two rabbits are provided in Table 11. The individual well results from the test on rabbit D4500 are provided in Table 12. In combination with the results reported in Example 6, all six sequences of Replikin sequences for antibody responses in rabbits or chickens provided measurable antibody responses and were antigenic Proved.

  Appendix A-“Genome Explore” Tcl Application with Replikin Recognition Device

  Appendix B-Instructions for operating the Genome Explorer application

  (1) Move the application Genome (which looks like a PubMed double helix) to the desired location (or start the CD).

  (2) Start the application (double-click the icon).

(3) A dialog box with database (NCBI or LANL) and query [...] will be shown at the top. Select the database and enter the same string in [......] as the search [Protein] [NC] database for the PubMed query page “[......]”, influenza sequence database Search for [......] in the query page (LANL database).
NCBI
fetching ncbi.gif "
LANL
fetching lanl.gif "
(4) Press the search button.

  The application contacts the database via an internet connection and the query results are fetched. A protein page is then downloaded to access the amino acid sequence and description.

After fetching all pages, they are parsed from the database form into a more convenient local form.
Parsing.gif "

Finally, the fetched and parsed page is searched for matching peptide subsequences. Passed sequences are listed in the desktop folder “(passed) pass” as separate htm pages for each protein; all failed proteins are listed in a single desktop htm page “(failed) fail”.
searching.gif "

  Other entries in the dialog box allow several matched match criteria. The value given is the one originally requested.

  Analyze and search offline: Internet connection is not required at these stages.

  When the protein is checked, these identifiers are shown at the bottom of the dialog box and a red progress bar crosses the screen. “Total” count, total number of proteins checked so far in the query; “Pass” and “Fail” counts, number of passes or fail criteria. The progress bar proceeds separately from the fetch, analysis, and search phases.

  (5) Press the stop button to stop the active search. The progress text changes to “stopping” and then “stopping”.

  (6) Press the end button to end.

(7) The pass result can be viewed by opening the “pass” folder on the desktop and opening the htm page in the browser.
NCBI
report.ncbi.gif "
LANL
report.lanl.gif "

Each Replikin sequence in the Replikin Analysis page links to all history where a particular sequence is found in the current query. Entries in the Replikin history link back to the entry under Replikin analysis.
sequence-history.gif "

(8) The failure result can be viewed by opening the desktop “fail.htm” page in the browser. If there are no additional criteria, the search is a full text search of the database.
completed.gif "

Additional criteria can be added to the search with a + icon behind. Exclusion criteria include criteria that include, but exclude, pages with a given string. Exclusions and inclusions can be mixed to refine the search results.
include exclude.gif "

The “must have” criterion intends a string that must occur in the field.
must have.gif "

  The search can be restarted automatically after editing the additional criteria, or the search can be restarted by pressing the search button. If additional criteria are edited, the search is in progress, but the search is restarted with the new criteria. The search uses cached results; for databases and queries only: the string does not change, only the additional criteria change, and the fetch and parse steps are skipped. An internet connection is not required to refine previously fetched searches.

A little priority can be set with application priority.
preferences.gif "

  The working directory is where all fields and caches are listed. The default is the desktop.

  The save fetch page is intended for debugging purposes. This saves an intermediate query page for the working directory.

  The purge query cache removes all pages from the hidden cache. This happens automatically when the database or query string changes.

  The open result in the browser will open a Replikin report in the default browser when this is complete.

  Appendix C-Screen shot of "Genome Explorer"

Appendix D-"Dr. Peptide" Tcl application with complex amino acid pattern recognition device

It is a bar graph which shows the occurrence frequency of Replikin in various living organisms.

FIG. 5 is a graph showing the percentage of malignin per milligram of total membrane protein during anaerobic replication of glioblastoma cells.

It is a bar graph which shows the quantity of the anti-malignin antibody produced | generated in response to the exposure with respect to a lycognin 16mer.

FIG. 4A is a photograph of a blood smear taken with ordinary light and fluorescent light. FIG. 4B is a photograph of a blood smear taken with ordinary light and fluorescent light illustrating the presence of two leukemia cells. FIG. 4C is a photograph of the dense layer of glioma cells in the presence of anti-malignin antibody. 4D and 4E are photographs of the cell layer of FIG. 4C taken at 30 and 45 minutes after addition of anti-malignin antibody.

FIG. 4F is a bar graph showing the inhibition of growth of small cell lung carcinoma cells in vitro by anti-malignin antibodies.

2 is a plot of the amount of anti-malignin antibody present in the serum of a patient suffering from benign or malignant breast disease before and after surgery.

FIG. 4 is a block diagram illustrating aspects of the present invention in which a computer is used to implement a three-point recognition method for identifying Replikin sequences.

It is a graph which shows the concentration of Replikin observed in the hemagglutinin of influenza B and influenza A strain, and H1N1 at an annual pace from 1940 to 2001.

1 is a graph of Replikin concentrations observed in hemagglutinin of emerging strains defined by influenza A strains, H2N2 and H3N2, and its constituent Replikins at an annual pace from 1950 to 2001.

1 is a graph showing yearly Replikin counts for several virus strains, including coronavirus nucleocapsid Replikin, from 1917 to 2002.

FIG. 3 is a chart showing the average Replikin counts per year for nucleocapsid coronavirus isolates. FIG. 3 is a chart showing the average Replikin counts per year for nucleocapsid coronavirus isolates. 2 is a chart depicting average Replikin counts per year for nucleocapsid coronavirus isolates.

FIG. 6 is a chart showing Replikin counts per year for H5N1 hemagglutinin.

FIG. 5 is a conversion table showing amino acids encoded as one alphabetic character according to standards provided by the International Pure and Applied Chemistry Union (IUPAC).

A printout of human cancer proteins obtained by searching a protein database maintained by the National Center for Biotechnology Information (NCBI).

It is a conversion table showing the correspondence between nucleobase triplets and amino acids.

FIG. 6 is a graph showing a rapid increase in the concentration of Replikin patterns in the H5N1 lineage hemagglutinin protein of influenza before the increase of the three “bird flu” epidemics. FIG. 15 shows that increasing Replikin concentration (“Replikin Count”) of H5N1 hemagglutinin protein preceded three “avian influenza” epidemics. In H5N1 influenza, an increasing lineage-specific Replikin concentration (“Replikin Count, mean +/− SD) from 1995 to 1997 precedes the 1997 Hong Kong H5N1 epidemic (E1); the increase from 1999 to 2001 was 2001 And the increase in 2002-2004 was preceded by the epidemic (E3) in 2004. The decline in 1999 was caused by the mass disposal of poultry against the E1 epidemic in Hong Kong.

FIG. 6 is a table showing selected examples of Replikin patterns found in various organisms.

1 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 aspects of the present invention. FIG.

FIG. 5 is a simple flowchart illustrating a general method for locating a Replikin pattern in an amino acid sequence in accordance with an aspect of the invention.

4 is a flowchart illustrating a generalized method for locating multiple Replikin-like patterns in an amino acid sequence in accordance with an aspect of the present invention.

FIG. 4 is a source code listing that includes procedures for finding a Replikin pattern in an amino acid sequence in accordance with an aspect of the present invention.

FIG. 5 is a table showing Replikin scaffolds that occur at substantially fixed amino acid positions in different proteins.

FIG. 2 is a simplified block diagram of a computer system platform useful in the present invention.

Claims (31)

A method for identifying a Replikin skeleton in a virus or organism, comprising:
(1) terminal lysine and lysine directly adjacent to the terminal lysine;
(2) terminal histidine and histidine directly adjacent to the terminal histidine,
(3) lysine at about 6 to about 10 amino acid residues from other lysines; and (4) at least 6% lysine;
Identifying a series of Replikin backbone peptides comprising about 16 to about 30 amino acids comprising:   2. The method of claim 1, further comprising identifying an individual member or members of the series of Replikin backbone peptides.   And further comprising identifying an Exoskeleton Scaffold, wherein said series of Replikin scaffold peptides are identified in a first series of viruses, virus strains or organisms, and said first series of viruses The exoskeleton is identified in a late virus, virus strain or organism as compared with a virus strain or organism, wherein the exoskeleton skeleton contains about the same number of amino acids as the Replikin backbone And (1) terminal lysine and lysine immediately adjacent to the terminal lysine, (2) terminal histidine and histidine immediately adjacent to the terminal histidine, and (3) about from other lysines 2. The method of claim 1 wherein there is no lysine in 6 to about 10 amino acid residues.   Further, the step of identifying the second Replikin backbone peptide according to claim 1, the step of comparing the Replikin backbone peptide and the second Replikin backbone peptide, and the second Replikin backbone peptide not changing from the second Replikin backbone peptide The method for identifying a Replikin backbone peptide according to claim 1, comprising the step of selecting any Replikin backbone peptide as a vaccine.   (1) at least one lysine residue located 6 to 10 residues from the second lysine residue, wherein at least one lysine residue is a terminal lysine residue; (2) at least one histidine residue; and (3) An isolated or synthesized influenza virus peptide containing from 7 to about 50 amino acids comprising at least 6% lysine residues.   6. The isolated or synthesized influenza virus peptide of claim 5, wherein the isolated or synthesized influenza virus peptide is present in an emerging strain of influenza virus.   (1) comprising at least one lysine residue located 6 to 10 residues from the second lysine residue; (2) at least one histidine residue; (3) comprising at least 6% lysine residues. An isolated or synthesized influenza virus peptide containing 7 to about 50 amino acids, wherein the isolated or synthesized influenza virus peptide is present in the H5N1 strain of influenza virus .   8. The isolated or synthesized influenza virus peptide of claim 7, further comprising a terminal lysine.   8. The isolated or synthesized influenza virus peptide of claim 7, comprising the amino acid sequence, KKNSTYPTIKRSYNNTNNQEDLLLVLVGIHH. About 16 to about 30 amino acids, and (1) a terminal lysine and a lysine directly adjacent to the terminal lysine;
(2) terminal histidine and histidine directly adjacent to the terminal histidine,
(3) lysine at about 6 to about 10 amino acid residues from other lysines; and (4) at least 6% lysine;
An isolated or synthesized influenza virus peptide according to claim 5, wherein a homologue of the peptide is present in a plurality of heterologous specimens, virus strains or organisms of the virus, wherein Wherein the homologue is about 16 to about 30 amino acids, and (5) a terminal lysine and a lysine immediately adjacent to the terminal lysine;
(6) terminal histidine and histidine directly adjacent to the terminal histidine,
(7) lysine at about 6 to about 10 amino acid residues from other lysines; and (8) at least 6% lysine;
An influenza virus peptide comprising:   (1) at least one lysine residue located about 6 to about 10 residues from the second lysine residue; (2) at least one histidine residue; (3) at least 6% lysine residues A prophylactic or therapeutic viral vaccine comprising at least one isolated or synthesized peptide of influenza virus, comprising:   12. A prophylactic or therapeutic viral vaccine according to claim 11, wherein the isolated or synthesized peptide is present in an emerging strain of influenza virus.   12. A prophylactic or therapeutic viral vaccine according to claim 11, wherein the isolated or synthesized peptide is present in the H5N1 strain of influenza virus.   12. A prophylactic or therapeutic viral vaccine according to claim 11, comprising a peptide comprising an amino acid sequence comprising KKNSTYPTIKRSYNNTNQEDLLVLWGIHH.   It further includes any of SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736, SEQ ID NO: 737, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742. 12. A prophylactic or therapeutic viral vaccine according to claim 11 comprising.   15. The prophylactic or therapeutic viral vaccine of claim 14, further comprising SEQ ID NO: 732.   The prophylactic or therapeutic viral vaccine according to claim 11, further comprising a pharmaceutically acceptable carrier and / or adjuvant.   The prophylactic or therapeutic viral vaccine of claim 14, further comprising vaccine V120304U2.   A method of stimulating a subject's immune system to produce antibodies against influenza viruses, comprising: (1) at least one lysine residue located within 6 to 10 residues from the second lysine residue; Effectiveness of at least one isolated or synthesized influenza virus Replikin peptide comprising:) at least one histidine residue, and (3) 7 to about 50 amino acids comprising at least 6% lysine residues A method comprising the step of administering an amount.   20. The method of claim 19, further comprising a pharmaceutically acceptable carrier and / or adjuvant and further comprising preventing or treating influenza infection.   20. The method of claim 19, wherein the isolated or synthesized influenza virus peptide is present in an emerging virus.   20. The method of claim 19, wherein the isolated or synthesized influenza virus peptide is present in the H5N1 strain of influenza virus.   20. The method of claim 19, wherein the isolated or synthesized influenza virus peptide comprises an amino acid sequence comprising KKNSTYPTIKRRSYNNTNQEDLLLVLVGIHH. A method for producing a prophylactic or therapeutic viral vaccine, comprising identifying a Replikin scaffold comprising a plurality of Replikin scaffold peptides comprising about 16 to about 30 amino acids, and a preventive or therapeutic viral vaccine Isolating or synthesizing at least one Replikin scaffold peptide, wherein the Replikin scaffold peptide is:
(1) terminal lysine and lysine directly adjacent to the terminal lysine;
(2) terminal histidine and histidine directly adjacent to the terminal histidine,
(3) lysine at about 6 to about 10 amino acid residues from other lysines; and (4) at least 6% lysine;
Comprising a method. 25. The Replikin backbone peptide is present in an influenza virus.
The method described in 1. Applying a plurality of criteria to the data representing the protein sequence;
Identifying any subsequence in the protein sequence based on the criteria; and outputting the identified subsequence to a data file comprising:
The criteria are:
A set of amino acids {a} contained in the subsequence;
A set of amino acids excluded from the subsequence {b}; and a minimum and maximum allowable gap between members of the sets {a} and {b};
Comprising a method.   27. The method of claim 26, wherein the protein sequence is obtained via a network.   27. A machine-readable medium storing computer-executable instructions for performing the method of claim 26. Applying a plurality of criteria to the data representing the protein sequence;
Based on the criteria, identifying a subsequence in the protein sequence, wherein the identified subsequence is a predetermined tolerance of the distance between the lysine amino acids and between the histidine amino acid and the furthest lysine Having a predetermined tolerance of distance; and outputting the identified subsequence to a data file;
Comprising a method.   30. The method of claim 29, wherein the protein sequence is obtained via a network.   30. A machine-readable medium storing computer-executable instructions for performing the method of claim 29.

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