Patent Application
20090017052
This invention relates generally to identifying virulent and lethal strains of pathogenic viruses, pathogenic organisms and malignancies through identifying concentrations of the class of small peptides known as Replikins, and to diagnosis, prevention and treatment of disease from such virulent and lethal pathogens and malignancies.
Rapid replication is characteristic of virulence in, among other things, certain bacteria, viruses and malignancies. The inventors have described a quantitative chemistry common to rapid replication in different organisms, viruses and malignancies. The chemistry of rapid replication described by the inventors is present in a family of conserved small protein sequences related to rapid replication called Replikins. A correlation between increased concentrations of Replikin sequences and increased replication and virulence has been observed in a range of viruses and organisms. Replikin sequences, therefore, offer new targets for developing effective methods of predicting and treating viral outbreaks.
A Replikin sequence is an amino acid sequence of 7 to about 50 amino acids comprising a Replikin motif. A Replikin motif comprises (1) at least one lysine residue located at a first terminus of the motif and at least one lysine residue or at least one histidine residue located at a second terminus of the motif; (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues. A Replikin sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine. A Replikin peptide or Replikin protein is a peptide or protein consisting of a Replikin sequence.
The inventors have identified Replikin sequences in oncogenic cells and in viral and organismal proteins associated with rapid replication and virulence. Additionally, higher concentrations of Replikin sequences in the genomic code have now been associated with a variety of infectious and pathogenic agents including human cancer, HIV, plant viruses, and a range of pathogenic animal and human viruses. Further, the correlation between the concentration of Replikin sequences in viral or organismal proteins and major outbreaks of disease and the correlation between the concentration of Replikin sequences in malignancies and poor prognoses are both significant.
Replikin sequences have been observed to be conserved in human cancers generally and in many pathogenic organisms and viruses, including conservation in both intrastrain and interstrain influenza viruses, for as long as 90 years based on data going back to the 1917-18 flu pandemic. Concentration of Replikin sequences in viral genomes has been shown to increase prior to strain-specific outbreaks and increased mortality in SARS, in influenza, in H5N1 bird flu and now in many other viral and non-viral pathogens. An increase in concentration of production of proteins containing Replikin sequences also has been shown in cancer as replication increases.
Within the last century there have been three influenza pandemics, each strain specific: H1N1 in 1918; H2N2 in 1957; and H3N2 in 1968. The inventors have established that prior to each pandemic there was a strain-specific increase in the concentration of Replikin sequences within the strain. The strain-specific increase in Replikin concentration was followed by a decrease in Replikin concentration and several years later a rebound increase in Replikin concentration associated with a strain-specific rebound epidemic. The Replikin algorithm provided the first chemistry that correlated with influenza epidemics and pandemics.
A similar correlation between the outbreaks of H5NI (Bird Flu) between 1997 and 2007 and the concentration of Replikin sequences in the viral proteins has been demonstrated. Likewise, a correlation has been established between the global outbreak of SARS coronavirus in 2003 and an increase in the concentration of Replikin sequences in the proteins of coronavirus. In another study, Replikins in two strains of human HIV-1 virus demonstrated that the Replikin concentration in the rapidly replicating strain was six fold greater than that of the slowly replicating strain. No instances of rapid replication have been observed in all the viruses and organisms examined wherein the Replikin concentration did not significantly increase as compared to the Replikin concentration in the dormant state.
The Replikin algorithm was initially discovered in Glycoprotein 10B, a membrane glycoprotein isolated from brain glioblastoma multiforme, lymphoma and breast cancer cells (U.S. Pat. No. 6,242,578 B1). A constituent peptide of Aglyco 10B, malignin, was observed to be enriched in cell membranes tenfold during anaerobic replication while cell number was observed to increase only five-fold. This increase in membrane concentration of the malignin protein in rapid replication of glioma cells suggested an integral relationship of the Replikins in malignin to replication of the glioblastoma multiforme.
Hydrolysis and mass spectrometry of malignin yielded a 16-mer peptide that included the Replikin sequence kagvaflhkk (SEQ ID NO: 3658). This peptide, which is absent from the normal human genome, was assumed to be acquired. Homologues of the Replikin sequence were found in all tumor viruses (that is viruses that cause cancer), and in replicating proteins of algae, plants, fungi, viruses and bacteria.
When the glioma Replikin was synthesized in vitro and administered as a synthetic vaccine to rabbits, abundant antimalignin antibody was produced. This production of abundant antimalignin antibody established that the peptide alone is an epitope, that is, it is a sufficient basis for an immune response observed in cancer patients wherein antimalignin antibodies are naturally produced. A 16-mer peptide containing the glioma Replikin produced both IgM and IgG forms of the antibody.
A study of 8,090 serum specimens from cancer patients and controls demonstrated that the concentration of antimalignin antibody increases with age in healthy individuals, as the incidence of cancer in the population increases, and increases further two to three-fold in early malignancy, regardless of cell type. In vitro this antibody was observed to be cytotoxic to cancer cells at picograms (femtomoles) per cancer cell, and in vivo the concentration of antimalignin antibody related quantitatively to the survival of cancer patients. As shown in glioma cells, the stage in cancer at which cells have only been transformed to the immortal malignant state but remain quiescent or dormant, now can be distinguished from the more active life-threatening replicating state which is characterized by the increased concentration of Replikins.
Using the sequence of the glioma Replikin peptide (kagvaflhkk) (SEQ ID NO: 3658) as a template, and constructing a “3-point-recognition” method to visually scan protein sequences of several different organisms, a new class of peptides, the Replikins, was revealed in organisms as diverse as algae, yeast and viruses. Surprisingly, these peptides were found to be concentrated in larger “replicating” and “transforming” proteins.
An infrequent occurrence of homologues was observed in “virus peptides” as a whole (1.5%), and in other peptides not designated as associated with malignant transformation or replication such as “brain peptides” and “neuropeptides” (together 8.5%). A surprisingly high frequency of occurrence of homologues was identified in tumor viruses, transforming proteins and cancer cell proteins. For example, 100% of identified tumor viruses contain Replikin sequences. 85% of transforming proteins contained Replikin sequences and 97% of cancer proteins contained Replikin sequences.
Further, Replikins were identified in such proteins as Saccharomyces cerevisiae replication binding protein; the replication associated protein A of maize streak virus; the replication-associated protein of Staphylococcus aureus; the DNA replication protein of bovine herpes virus 4; and the mealigrid herpes virus 1 replication binding protein. Replikin-containing proteins also are associated frequently with redox functions, and protein synthesis or elongation, as well as with cell replication.
The highest concentration of Replikin sequences in an organism or virus that had been analyzed and reported was 111 Replikin sequences per 100 amino acids in the extraordinarily-rapidly-replicating parasitic protozoa Plasmodium falciparum (reportedly responsible for 90% of malarial deaths in humans) (herein sometimes referred to as malaria). P. falciparum has been observed to replicate 11,000 times in 48 hours during passage of the parasite from liver to blood in the host.
A significant feature of Replikin sequences observed in P. falciparum was a marked overlapping of Replikin structures throughout malarial proteins. For example, there are nine overlapping Replikins in the 39 amino acid sequence of SEQ ID NO. 3667 (Replikin concentration=23.1/100 amino acids); and 15 overlapping Replikins in the 41 amino acids of SEQ ID NO. 3668 (Replikin concentration=36.6/100 amino acids). Both of these overlapping Replikin structures occur in blood stage trophozoites and schizonts. This mechanism of lysine multiples was also seen in the Replikins of cancer proteins such as in gastric cancer transforming protein, ktkkgnrvsptmkvth (SEQ ID NO: 3669), and in transforming protein P21B (K-RAS 2B) of lung, khkekmskdgkkkkkks (SEQ ID NO: 3670).
In monitoring Replikin sequences in influenza virus, the inventors have additionally identified a sub-family of conserved Replikin sequences known as Replikin Scaffolds or Replikin Scaffold sequences. Replikin Scaffolds were initially identified in conserved structures in particularly virulent influenza viruses. Included among these strains were the viruses causing the pandemics of 1918, 1957, 1968 and virulent strains of the H5N1 “bird flu” strain of influenza virus. Analogues of Replikin Scaffold sequences have since been identified in the virulent and rapidly replicating SARS coronavirus. See U.S. Published Application No. 2007/0026009.
Scaffolding of Replikin sequences homologous but not identical to the algorithm of the identified Replikin Scaffold has also been identified in P. falciparum. Replikin scaffolding in general has been related to an increase in Replikin concentrations in pathogenic genomes where it has been identified. In P. falciparum, scaffolding contributes significantly to the very high Replikin concentration noted in the proteins of the protozoa.
Virulent and lethal outbreaks of influenza are a continuing challenge to world health and the medical practitioner is increasingly aware of the continued threat of virulent and lethal influenza pandemics that require new methods of predicting virulence and lethality and will require new methods and compounds for treatment. Influenza is an acute respiratory illness of global importance. Despite international attempts to control influenza virus outbreaks through vaccination, influenza infections remain an important cause of morbidity and mortality. Worldwide influenza pandemics have occurred at irregular and previously unpredictable intervals throughout history and it is expected that influenza pandemics will continue to occur in the future. The impact of pandemic influenza is substantial in terms of morbidity, mortality and economic cost.
Influenza vaccines remain the most effective defense against influenza virus, but because of the ability of the virus to mutate, and the availability of non-human host reservoirs, it is expected that influenza will remain an emergent or re-emergent infection. Global influenza surveillance indicates that influenza viruses may vary within a country and between countries and continents during an influenza season. Virologic surveillance is of importance in monitoring antigenic shift and drift. Disease surveillance is also important in assessing the impact of epidemics. Both types of information have provided the basis of vaccine composition and use of antivirals. However, traditionally there has been only annual post hoc hematological classification of the increasing number of emerging influenza virus strains, and no specific chemical structure of the viruses was identified as an indicator of approaching influenza epidemic or pandemic. Until recently, the only basis for annual classification of influenza virus as active, inactive or prevalent in a given year was the activities of the virus hemagglutinin and neuraminidase proteins.
There is a need in the art for methods of predicting increases in virulence and lethality of influenza prior to outbreaks. There is likewise a need in the art for methods of preventing and treating outbreaks caused by virulent strains of influenza. Because of the annual administration of influenza vaccines and the short period of time when a vaccine can be administered, strategies directed at improving vaccine coverage are of critical importance.
Equine influenza is a common upper respiratory disease of the horse currently caused by the H3N8 strain of equine influenza virus (EIV). Typical symptoms of equine influenza include a dry hacking cough, nasal discharge, and fever. The viral disease is considered enzootic in Europe, the United States and parts of Asia. Significant outbreaks have also been observed in South America, China, and India.
The first outbreak of equine influenza in Japan since 1972 was recently reported and 2007 saw the first ever report of equine influenza in Australia. So far, no fatalities have been reported. Equine influenza is, however, sometimes fatal in young foals.
Quarantine has been thought to be the best prevention against the spread of equine influenza. South Africa, Australia and Japan have used quarantine of imported horses to stop the spread of, among other diseases, equine influenza. The quarantine practice has apparently not been fully successful suggesting possible incidental transfer of the disease through human handlers of the horses.
The influenza virus is highly mutable and, as a result, development of long-term therapies has been difficult. Vaccines generally have needed to be updated as virulent mutants of the virus have arisen. Annual review of worldwide outbreaks of the virus provides data for recommended production of vaccines against the most relevant strains of virus. Significant time elapses between identification of the most relevant strains and commercialization of vaccines.
There is a need in the art for methods of identifying emerging equine influenza viruses prior to outbreaks so that preventive measures may be taken against such emerging viruses. There is likewise a need in the art for methods of preventing and treating outbreaks caused by virulent strains of EIV including vaccines.
Foot and Mouth Disease is a highly contagious and sometimes fatal viral disease of cattle, pigs and other animals including bovids with cloven hooves cause by foot and mouth disease virus (FMDV). FMDV is a single-stranded RNA aphthovirus of the Picornaviridae virus family. There are said to be seven different FMDV serotypes: 0, A, C, SAT-1, SAT-2, SAT-3, and Asia-1.
There is a need in the art for methods of predicting increases in virulence of FMDV prior to outbreaks of Foot and Mouth Disease. There is likewise a need in the art for methods of preventing and treating outbreaks of Food and Mouth Disease caused by virulent strains of FMDV.
West nile virus (WNV), in a small percentage of infected humans, causes encephalitis and other serious neuroinvasive diseases. In about four percent of reported cases of WNV infection, the resulting neuroinvasive disease results in death. WNV is flaviviridae virus that was first observed in North America in 1999 and is now considered endemic in the United States. The virus is spread to humans through mosquito (and related insect) bites. Infection with WNV causes diseases such as encephalitis, meningitis and meningoencephalitis in less than about one percent of infected humans. In about 20 percent of infected humans, less severe illness, characterized by fever, headache, tiredness, aches and sometimes rashes, may occur. Of the total number of U.S. cases of WNV infection reported, about four percent have resulted in death.
WNV is a single-stranded sense RNA virus and is a member of the Japanese encephalitis virus antigenic complex, which includes several medically important viruses associated with human encephalitis: Japanese encephalitis, St. Louis encephalitis, Murray Valley encephalitis, and Kunjin encephalitis, an Australian subtype of WNV.
Since introduction of the disease to the United States in 1999, there have been more than 16,000 reported cases of WNV in humans and more than 650 reported deaths. In addition, more than 21,000 cases have been reported in horses. Currently, the only available approved strategies to combat WNV in humans are nationwide active surveillance in conjunction with mosquito control efforts and individual protection with insect repellents. There is a need in the art, therefore, for methods of predicting increases in virulence of WNV prior to epidemics. There is likewise a need in the art for methods of preventing and treating outbreaks caused by virulent strains of WNV.
Two severe viral diseases now endemic in swine in many countries and presently causing great economic losses worldwide are Porcine Reproductive and Respiratory Syndrome (PRRS) and Porcine Circovirus Associated Diseases (PCVAD), caused by porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV), respectively. Each disease has a significant impact on the hog industry and, in both diseases, current control measures are proving inadequate.
PRRS is a relatively recently recognized disease in pigs. The infectious virus is classified in the family Arteriviridae and order Nidovirales and did not have a standardized name in the past but is now known as porcine reproductive and respiratory syndrome virus (PRRSV). The disease is characterized by reproductive failure, death in young pigs and mild respiratory disease.
The pig is the only known host for PRRSV but evidence suggests that another host or hosts may have existed prior to identification of PRRS in the United States in 1987 and Europe in 1990. PRRS is now endemic in the United States and many European countries. Evidence of infection (whether serological or virological or both) has been found in Japan, Korea, the Philippines, Vietnam, South America and the Caribbean.
The disease has been associated with reproductive failure in sows and respiratory disease in all stages of pig development. Clinical signs of the disease include: fever, anorexia, depression, reduced conception rates, abortion, week piglets, respiratory distress and increased rates of other endemic diseases.
PRRSV is a positive-sense single-stranded small envelope RNA virus with at least nine open reading frames (ORFs) in its genome encoding about 20 putative proteins: ORF 1a and 1b encode replication proteins; ORF 2a and 2b encode unknown structure proteins; ORF 3, 4 and 5 encode envelope proteins; ORF 6 encodes membrane proteins and ORF 7 encodes nucleocapsid proteins.
Two types of PRRSV have been identified: European (Type I) and North American (Type II). The two types share about 60% sequence identity. PRRSV strains are known to differ markedly in pathogenicity. In 2006, highly pathogenic outbreaks of PRRSV occurred in China and Mexico. The cost of PRRSV infection to the U.S. pork industry has been estimated at between $560 million and $761 million annually. PRRSV infection has been associated with a reduction in the number of pigs weaned per litter, a reduction in birthing rate, increased mortality, reduced feed conversion and reduced average daily weight gain.
Porcine Circovirus Associated Diseases (PCVAD) have also only recently been recognized in pigs (1996). PCVAD is a term used to define the entire range of disease associated with porcine circovirus (PCV) infection. The range of disease in pigs includes: Postweaning Multisystemic Wasting Syndrome (PMWS); respiratory illness; pneumonia; diarrhea; reproductive disorders and high mortality. PCVAD symptoms may include detection of PCV within lesions that form on growing pigs, inflammation in, for example, the spleen, thymus, intestines, lymph nodes, lung, kidney, liver, and tonsils, and depletion of lymphoid cells. PCV infection is thought to pose no apparent risk to human health. PCVAD is presently severely affecting the Canadian swine industry.
Two antigenically distinct types of PCV have been identified. Porcine Circovirus 1 (PCV1), which may be non-pathogenic, and Porcine Circovirus 2 (PCV2), which appears to be the strain that causes PCVAD. PCV1 and PCV2 share about 65% amino acid identity in open reading frame 2 of the virus genome.
The incidence of PCV infection associated disease has increased by 4% between 2000 and 2006 in Canada and new outbreaks have been observed in Western Canada. In some studies, more than 80% of Canadian pigs have been found to be infected with PCV2 at slaughter. In infected herds, an increase in mortality rates has also been observed. As incidence of PCV infection has increased, pork production has decreased due to pig death and decreased productivity. Production in Canada in 2006 is expected to decrease 1.5 percent below 2005 production due to PCV-influenced disease.
There is a need in the art for methods of predicting increases in virulence of PRRSV and PCV prior to outbreaks. There is likewise a need in the art for methods of preventing and treating outbreaks caused by virulent strains of PRRSV and PCV.
White spot syndrome virus (WSSV) (also known as white spot baculoform virus) and taura syndrome virus (TSV) are global lethal pathogens in shrimp.
Taura syndrome is a viral disease in shrimp that significantly impacts the shrimp farming industry worldwide. Taura Syndrome is caused by the taura syndrome virus (TSV), which is a member of the Discistroviridae family in the genus Cripavirus that has a single positive stranded genome of about 10,000 nucleotides. The genome contains two open reading frames (ORF). ORF 1 reportedly contains coding for a helicase, a protease and an RNA-dependent RNA polymerase. ORF2 reportedly contains coding for three capsid proteins.
Taura syndrome is now considered endemic in the Americas and outbreaks have been observed in Asia. Infected shrimp generally have a red tail, are anorexic and erratic in their behavior, tail muscles may become opaque and the cuticle may become soft. Mortality rates between 5% and 95% have been observed during the acute phase of the disease. Shrimp that survive outbreaks of TSV seem to be refractory to reinfection while remaining infectious.
White spot syndrome (WSS) is a highly contagious and lethal viral infection of shrimp often destroying entire farm populations within several days of observation of the first symptoms. The first reported epidemic of the disease was in Taiwan in 1992 and the disease is now known to be present in all shrimp growing regions globally except Australia. The virus has a wide host range including most cultured penaeid shrimp including Fenneropenaeus indicus, Penaeus monodon, Litopenaeus vannamei, and Marsupenaeus japonicas, other non-penaeid shrimp, crabs, spiny lobsters and others.
WSSV is a rod-shaped double-stranded DNA virus. The complete DNA sequence of WSSV genome has reportedly been assembled into a circular sequence of 292,967 base pairs. Clinical signs of WSSV infection include white spots on the carapace, often reddish discoloration, and reduction in food consumption and loss of energy. There is a need in the art for methods of preventing and treating viral infections of shrimp such as TSV and WSSV by manipulating the replicating function of Replikin sequences and for identifying molecular targets related to the replicating function of Replikin sequences for treatment of virulent viral.
The present invention provides a method of identifying a first virus, first organism or first malignancy with a higher lethality than at least one second virus of the same species as the first virus, second organism of the same species as the first organism or second malignancy of the same species as the first malignancy which comprises comparing the Replikin Count of the Replikin Peak Gene of the first virus, first organism or first malignancy to the Replikin Count of the Replikin Peak Gene of at least one second virus, second organism, or second malignancy to determine that the virus, organism or malignancy with the higher Replikin Count is the more lethal.
In one embodiment, the first malignancy is a lung malignancy, a brain malignancy, a breast malignancy, an ovarian malignancy or a lymph malignancy. In a specific embodiment, the first malignancy is a non-small cell lung carcinoma.
In another embodiment, the first organism is a Mycobacterium tuberculosis, Mycobaterium mucogenicum, Staphylococcus aureus, or Plasmodium falciparum.
In a further embodiment, the virus is influenza virus, foot and mouth disease virus, west nile virus, porcine respiratory and reproductive syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, coronavirus, ebola virus, gemini leaf curl virus, hemorrhagic septicemia virus, or tobacco mosaic virus.
In a specific embodiment, the first virus is a strain of Influenza A virus of H1N1, H2N2, H3N2, H5N1, or H3N8.
In a further embodiment, said at least one Replikin sequence within the protein or protein fragment of the identified Replikin Peak Gene is isolated from influenza A strain H5N1 and is selected from the group consisting of SEQ ID NOS: 1685-1691, SEQ ID NOS: 1702-1717. In a further embodiment, said at least one Replikin sequence within the protein or protein fragment of the identified Replikin Peak Gene is isolated from equine influenza virus (H3N8) and is selected from the group consisting of SEQ ID NOS: 547-562.
The present invention further provides a isolated or synthesized Replikin Peak Gene of a virus, organism or malignancy wherein said Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus, a cell of the organism or a malignant cell of the malignancy consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein or protein fragment of the virion of the virus, the cell of the organism or the malignant cell of the malignancy.
In one embodiment, the isolated or synthesized Replikin Peak Gene is the portion of a protein or protein fragment consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to all other proteins or protein fragments in the virion of the virus, in the cell of the organism or in the malignant cell of the malignancy.
In a specific embodiment, the isolated or synthesized Replikin Peak Gene is isolated from a lung malignancy, a brain malignancy, a breast malignancy, an ovarian malignancy, or a lymph malignancy. In another specific embodiment, the isolated or synthesized Replikin Peak Gene is isolated from a non-small cell lung carcinoma or glioblastoma multiforme.
In yet another embodiment, the isolated or synthesized Replikin Peak Gene of is isolated from Mycobacterium tuberculosis, Mycobacterium mucogenicum, Staphylococcus aureus, or Plasmodium falciparum.
According to a further embodiment, the isolated or synthesized Replikin Peak Gene is isolated from influenza virus, foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, coronavirus, ebola virus, gemini leaf curl virus, hemorrhagic septicemia virus, or tobacco mosaic virus.
In one embodiment, the isolated or synthesized Replikin Peak Gene is from influenza virus, particularly an Influenza A virus. In a specific embodiment, the Influenza A virus is a strain H1N1, H2N2, H3N2, H5N1 or H3N8. In another specific embodiment, the Replikin Peak Gene is isolated from the pB1 gene area of an influenza virus.
According to another embodiment, the isolated or synthesized Replikin Peak Gene is from foot and mouth disease virus. In a specific embodiment, the isolated or synthesized Replikin Peak Gene is identified within the VP1 gene of a foot and mouth disease virus.
In yet another embodiment, the isolated or synthesized Replikin Peak Gene is from a west nile virus. In a specific embodiment, the isolated or synthesized Replikin Peak Gene is isolated from the envelope protein of west nile virus.
In a further embodiment, the isolated or synthesized Replikin Peak Gene is from a porcine respiratory and reproductive syndrome virus. In a specific embodiment, the isolated or synthesized Replikin Peak Gene is isolated from a nucleocapsid protein of a porcine respiratory and reproductive syndrome virus.
In yet another embodiment, the isolated or synthesized Replikin Peak Gene is from a porcine circovirus. In a specific embodiment, the isolated or synthesized Replikin Peak Gene is isolated from a replicase protein of a porcine circovirus.
In still a further embodiment, the isolated or synthesized Replikin Peak Gene is from a white spot syndrome virus. In a specific embodiment, the isolated or synthesized Replikin Peak Gene is isolated from a ribonucleotide reductase protein of a white spot syndrome virus.
In yet another embodiment, the isolated or synthesized Replikin Peak Gene is from a tobacco mosaic virus.
In a further embodiment, the isolated or synthesized Replikin Peak Gene is from a hemorrhagic septicemia virus in fish. In a specific embodiment, the isolated or synthesized Replikin Peak Gene is isolated from a glycoprotein in a hemorrhagic septicemia virus.
In another specific embodiment, the isolated or synthesized Replikin Peak Gene comprises a sequence of SEQ ID NO: 1741, SEQ ID NO: 3664, SEQ ID NO: 3660, SEQ ID NO: 3665, SEQ ID NO: 1996, SEQ ID NO: 1665, SEQ ID NO: 1684, SEQ ID NO: 1701, SEQ ID NO: 546, SEQ ID NO: 124, SEQ ID NO: 130, SEQ ID NO: 311, SEQ ID NOS: 341-344, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NOS: 233-238, SEQ ID NO: 415, SEQ ID NO: 421, SEQ ID NO: 438, SEQ ID NO: 451, SEQ ID NO: 462, SEQ ID NO: 498, SEQ ID NO: 669, SEQ ID NO: 1168, SEQ ID NO: 1531, SEQ ID NO: 1548, or SEQ ID NO: 1939.
The present invention further provides an immunogenic composition comprising the isolated or synthesized Replikin Peak Gene. In a specific embodiment, the immunogenic composition comprises a Replikin sequence of SEQ ID NOS: 2902-2925, SEQ ID NOS: 2312-2544, SEQ ID NOS: 2701-2711, SEQ ID NOS: 2713-2718, SEQ ID NOS: 3282-3285, 3287-3291, 3293, 3295, 3297, 3299, 3300, 3302, 3304, 3306, and 3308, SEQ ID NOS: 1685-1691, SEQ ID NOS: 1702-1717, SEQ ID NO: 106, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NOS: 125-129, SEQ ID NOS: 131-156, SEQ ID NOS: 233-244, SEQ ID NOS: 286-290, SEQ ID NOS: 312-323, SEQ ID NOS: 354-366, SEQ ID NOS: 368-380, SEQ ID NOS: 383-393, SEQ ID NOS: 395-401, SEQ ID NOS: 403-414, SEQ ID NOS: 291-307, SEQ ID NOS: 308-310, SEQ ID NOS: 324-327, SEQ ID NOS: 328-340, SEQ ID NOS: 416-419, SEQ ID NOS: 422-437, SEQ ID NOS: 440-445, SEQ ID NOS: 452-457, SEQ ID NOS: 464-476, SEQ ID NOS: 482-484 and SEQ ID NOS: 487-492, SEQ ID NOS: 547-562. SEQ ID NOS: 663-667, SEQ ID NOS: 670-1166, SEQ ID NOS: 1169-1529, SEQ ID NOS: 1532-1542, SEQ ID NO: 1548, SEQ ID NOS: 3788-3823), or SEQ ID NOS 1637-1663.
A non-limiting embodiment of the present invention provides computer readable medium having stored thereon instructions which, when executed, cause the processor to perform a method for identifying a Replikin Peak Gene of a virus, organism or malignancy comprising identifying, within amino acid sequences or nucleic acid sequences that encode amino acid sequences of said virus, organism or malignancy, the portion of the genome, or protein or protein fragment of said virus, said organism or said malignancy consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, or protein or protein fragment of the malignancy, organism or virus.
In one embodiment, the computer readable medium comprises instructions which, when executed, cause the processor to perform a method for predicting an increase in lethality or virulence of said virus, organism or malignancy that comprises said identified Replikin Peak Gene or an outbreak of said virus or organism that comprises said identified Replikin Peak Gene by: (1) determining that the Replikin Count of said Replikin Peak Gene or that the Replikin Count of a protein or gene area comprising said Replikin Peak Gene is higher than another Replikin Peak Gene or a protein or gene area comprising said other Replikin Peak Gene identified within the genome or within a protein or protein fragment of at least one other virus of the same species as said virus, at least one other organism of the same species as said organism or at least one other malignancy of the same type as said malignancy wherein said other virus, said other organism or said other malignancy is isolated at an earlier time point than said virus, said organism or said malignancy, and (2) predicting an increase in lethality or virulence of said virus, organism or malignancy or predicting an outbreak of said virus or organsism.
The invention also provides a method of predicting the strain, the host or the geographic region of an outbreak or increase in lethality or virulence of a virus or organism by (1) identifying a Replikin Peak Gene or a protein or gene area comprising a Replikin Peak Gene within the genome of a first virus or organism of a first strain, from a first host, or isolated from a first geographic region or within a protein or protein fragment of the first virus or organism that has a higher Replikin Count than a Replikin Peak Gene or protein or gene area comprising a Replikin Peak Gene identified within the genome or within a protein or protein fragment of at least one second virus of the same species as the first virus or at least one second organism of the same species as the first organism wherein said first virus or said first organism is isolated at a later time point than said first virus or said first organism and is the same strain, from the same or another host or isolated from the same or another geographic region as the first virus or first organism, and (2) predicting an outbreak or an increase in lethality or virulence of said first strain, in said first host or within said first geographic region of said first virus or organism.
In one embodiment, the protein or gene area comprising said Replikin Peak Gene within the genome of a first virus or organism is identified as having a higher Replikin Count than said protein or gene area comprising a Replikin Peak Gene identified within the genome or within a protein or protein fragment of said at least one second virus or organism.
In another embodiment, the first virus or first organism is isolated at least six months to three years later than the second organism or said second virus. In a specific embodiment, the first organism or first virus is Mycobacterium tuberculosis, Mycobaterium mucogenicum, Staphylococcus aureus, and Plasmodium falciparum, influenza virus, foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, coronavirus, ebola virus, gemini leaf curl virus in tomato plants, hemorrhagic septicemia virus, or tobacco mosaic virus. In another embodiment, the Staphylococcus aureus is methicillin-resistant.
In a further embodiment, the influenza virus a strain of Influenza A virus. In a specific embodiment, the first virus is an influenza virus of the strain H1N1, H2N2, H3N2, H5N1 or H3N8.
In a further embodiment of the invention, the protein or gene area comprising the Replikin Peak Gene is the pB1 gene area of the influenza virus.
In yet another embodiment, the protein or gene area is a nucleocapsid protein of porcine respiratory and reproductive syndrome virus.
In a further embodiment, the protein or gene area is an envelope protein of west nile virus.
In a further embodiment, the protein or gene area is a VP1 protein of foot and mouth disease virus.
In still another embodiment, the protein or gene area is an ATP-ase of Plasmodium falciparum.
In yet a further embodiment, the protein or gene area is a replicase protein of porcine circovirus.
In another embodiment, the protein or gene area is a ribonucleotidease of said white spot syndrome virus.
The present invention further provides a method of identifying a first virus, organism or malignancy associated with higher lethality, higher virulence or more rapid replication than a second virus of the same species as the first virus, a second organism of the same species as the first organism or a second malignancy of the same type as the first malignancy comprising identifying a Replikin Peak Gene encoded within the genome of at least one virion of the first virus, or at least one cell of the first organism, or at least one malignant cell of the first malignancy, or within a protein or protein fragment of at least one virion of the first virus, or at least one cell of the first organism, or at least one malignant cell of the first malignancy that has a higher Replikin Count than a Replikin Peak Gene identified encoded within the genome of at least one virion of the second virus, or at least one cell of the second organism, or at least one malignant cell of the second malignancy or within a protein or protein fragment of at least one virion of the second virus, or at least one cell of the second organism, or at least one malignant cell of the second malignancy wherein said first virus, first organism or first malignancy has higher lethality, higher virulence or more rapid replication than said second virus, second organism or second malignancy, and wherein the Replikin Peak Gene is defined as a protein or protein fragment having the highest concentration of continuous Replikin sequences per 100 amino acids as compared to the remaining proteins or protein fragments in the same virion of the virus, the same cell of the organism, or the same malignant cell, or the portion of the genome encoding the protein or protein fragment.
Further provided is a method of identifying a first virus, first organism or first malignancy with a higher lethality than at least one second virus of the same species as the first virus, second organism of the same species as the first organism or second malignancy of the same species as the first malignancy comprising comparing the Replikin Count of the whole genome of a virus, organism or malignancy to the Replikin Count of the whole genome of at least one second virus, second organism, or second malignancy to determine that the virus, organism or malignancy with the higher Replikin Count is the more lethal.
According to a specific embodiment, the first virus is a coronavirus, a foot and mouth disease virus, a white spot syndrome virus, a taura syndrome virus, a porcine circovirus, or an influenza virus.
In one specific embodiment, the first virus is an H5N1 strain of influenza virus.
In another specific embodiment, the influenza virus is an Influenza A virus. In a further specific embodiment, the Influenza A virus is H1N1, H2N2, H3N2, H5N1 or H3N8.
According to another embodiment, the Replikin Peak Gene is isolated from the pB1 gene area of an influenza virus.
The present invention also provides method for obtaining an isolated or synthesized Replikin Peak Gene of a virus, organism or malignancy for diagnosis, prevention or treatment of an infection of said virus or said organism or for diagnosis, prevention or treatment of said malignancy comprising: (1) obtaining a plurality of isolates of virus of the same species, a plurality of organisms of the same species, or a plurality of malignancies of the same type; (2) analyzing the protein sequences or protein sequence fragments of each individual isolate of the plurality of isolates of virus, a cell of each individual organism of the plurality of organisms, or a malignant cell of each individual malignancy of the plurality of malignancies for the presence and concentration of Replikin sequences; (3) identifying the protein sequence or the protein sequence fragment having the highest concentration of continuous Replikin sequences in the malignant cell of each individual malignancy, the cell of each individual organism or each individual virus isolate; (4) selecting the protein sequence or protein sequence fragment having the highest concentration of continuous Replikin sequences among the plurality of isolates of virus, the plurality of organisms, or the plurality of malignancies; (5) identifying the amino acid sequence of the selected protein sequence or protein sequence fragment as the Replikin Peak Gene of the plurality of virus isolates, organisms or malignancies; and (6) isolating or synthesizing the identified Replikin Peak Gene of at least one of the plurality of virus isolates, organisms or malignancies wherein the isolated or synthesized identified Replikin Peak Gene is useful for diagnosis, prevention or treatment of said infection of said virus or said organism or said malignancy.
Further provided is an immunogenic composition comprising at least one isolated or synthesized Replikin Peak Gene isolated according to the above method. In a specific embodiment, the immunogenic composition is isolated from an emerging strain of a virus or organism, and optionally further comprises a pharmaceutically acceptable carrier.
The present invention also provides a vaccine comprising at least one isolated or synthesized Replikin Peak Gene. In a specific embodiment, the vaccine comprises a Replikin Peak Gene isolated from an emerging strain of virus or organism. In another specific embodiment, the vaccine comprises SEQ ID NO: 1741, SEQ ID NO: 3664, SEQ ID NO: 3660, SEQ ID NO: 3665, SEQ ID NO: 1996, SEQ ID NO: 1665, SEQ ID NO: 1684, SEQ ID NO: 1701, SEQ ID NO: 546, SEQ ID NO: 124, SEQ ID NO: 130, SEQ ID NO: 311, SEQ ID NOS: 341-344, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NOS: 233-238, SEQ ID NO: 415, SEQ ID NO: 421, SEQ ID NO: 438, SEQ ID NO: 451, SEQ ID NO: 462, SEQ ID NO: 498, SEQ ID NO: 669, SEQ ID NO: 1168, SEQ ID NO: 1531, SEQ ID NO: 1548, positions 81-204 of SEQ ID NO: 3787, or SEQ ID NO: 1939.
In yet a further embodiment, the vaccine comprises a Replikin Peak Gene isolated from a virus.
In a specific embodiment, the virus is influenza virus, foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, coronavirus, ebola virus, gemini leaf curl virus, hemorrhagic septicemia virus, or tobacco mosaic virus.
In one embodiment, the Replikin Peak Gene in the vaccine is isolated from Influenza A, or specifically strains H1N1, H2N2, H3N2, H5N1 or H3N8.
In another embodiment, the vaccine comprises a Replikin Peak Gene isolated from an organism.
In a further embodiment, Replikin Peak Gene is isolated from Mycobacterium tuberculosis, Mycobaterium mucogenicum, Staphylococcus aureus, or Plasmodium falciparum. In a specific embodiment, the Staphylococcus aureus is methicillin-resistant.
In still another embodiment, the Replikin Peak Gene is isolated from a malignancy.
In a specific embodiment, the Replikin Peak Gene is isolated from a lung malignancy, a brain malignancy, a breast malignancy or a lymph malignancy. In another embodiment, the Replikin Peak Gene is isolated from a non-small cell lung carcinoma. In a further embodiment, the Replikin Peak Gene is isolated from glioblastoma multiforme.
The present invention further provides an immunogenic composition comprising a Replikin Peak Gene, optionally in combination with a pharmaceutically acceptable carrier. In one embodiment, the immunogenic composition comprises SEQ ID NO: 1741, SEQ ID NO: 3664, SEQ ID NO: 3660, SEQ ID NO: 3665, SEQ ID NO: 1996, SEQ ID NO: 1665, SEQ ID NO: 1684, SEQ ID NO: 1701, SEQ ID NO: 546, SEQ ID NO: 124, SEQ ID NO: 130, SEQ ID NO: 311, SEQ ID NOS: 341-344, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NOS: 233-238, SEQ ID NO: 415, SEQ ID NO: 421, SEQ ID NO: 438, SEQ ID NO: 451, SEQ ID NO: 462, SEQ ID NO: 498, SEQ ID NO: 669, SEQ ID NO: 1168, SEQ ID NO: 1531, SEQ ID NO: 1548, or SEQ ID NO: 1939.
The present invention further provides an isolated or synthesized Replikin sequence isolated from a protein or protein fragment a Replikin Peak Gene or isolated from a protein comprising a Replikin Peak Gene.
In one embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from Mycobacterium tuberculosis, Mycobacterium mucogenicum, Staphylococcus aureus, or a Plasmodium falciparum. In a specific embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from Mycobacterium mucogenicum. In a further embodiment, the Replikin Peak Gene is SEQ ID NOS: 2902-2925. In another specific embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from Plasmodium falciparum. In a further embodiment, the Replikin Peak Gene is one of SEQ ID NOS: 2312-2544, SEQ ID NOS: 2701-2711, SEQ ID NOS: 2713-2718, SEQ ID NOS: 3282-3285, 3287-3291, 3293, 3295, 3297, 3299, 3300, 3302, 3304, 3306, or SEQ ID NO: 3308.
In another embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from influenza virus, foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, coronavirus, ebola virus, gemini leaf curl virus, hemorrhagic septicemia virus, or tobacco mosaic virus.
In a specific embodiment, the influenza virus is Influenza A virus. In another specific embodiment, the Influenza A virus is H1N1, H2N2, H3N2, H5N1 or H3N8. In a further specific embodiment, the Influenza A virus is H5N1 and the Replikin sequence is one of SEQ ID NOS: 1685-1691, SEQ ID NOS: 1702-1716 or SEQ ID NO: 1717. In a further specific embodiment, the Influenza A virus is H3N8 and the Replikin sequence is one of SEQ ID NOS: 547-561 or SEQ ID NO: 562.
In another embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from foot and mouth disease virus. In a specific embodiment, the Replikin sequence from the foot and mouth disease virus is one of SEQ ID NO: 106, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NOS: 125-129, SEQ ID NOS: 131-155 or SEQ ID NO: 156.
In still another embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from west nile virus. In a specific embodiment, the Replikin sequence from the west nile virus is one of SEQ ID NOS: 233-243 or SEQ ID NO: 244.
In a further embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from porcine reproductive and respiratory virus. In a specific embodiment, the Replikin sequence from porcine reproductive and respiratory virus is one of SEQ ID NOS: 286-290, SEQ ID NOS: 312-323, SEQ ID NOS: 354-366, SEQ ID NOS: 368-380, SEQ ID NOS: 383-393, SEQ ID NOS: 395-401, SEQ ID NOS: 403-413 or SEQ ID NO: 414.
In another embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from porcine circovirus. In a specific embodiment, the Replikin sequence from porcine circovirus is one of SEQ ID NOS: 291-307, SEQ ID NOS: 308-310, SEQ ID NOS: 324-327, SEQ ID NOS: 328-340, SEQ ID NOS: 416-419, SEQ ID NOS: 422-437, SEQ ID NOS: 440-445, SEQ ID NOS: 452-457, SEQ ID NOS: 464-476, SEQ ID NOS: 482-484, SEQ ID NOS: 487-491 or SEQ ID NO: 492.
In still a further embodiment, the Replikin sequence is from a Replikin Peak Gene isolated from white spot syndrome virus. In a specific embodiment, the Replikin sequence from white spot syndrome virus is one of SEQ ID NOS: 663-667, SEQ ID NOS: 670-1166, SEQ ID NOS: 1169-1529, SEQ ID NOS: 1532-1542 and SEQ ID NO: 1548.
According to the present invention provided is a vaccine for prevention and/or treatment of an viral or organismal infection or a malignancy wherein the vaccine comprises at least one isolated or synthesized Replikin sequence within a protein or protein fragment of a Replikin Peak Gene or a protein comprising a Replikin Peak Gene identified in said virus, organism, or malignancy.
In a further embodiment, the at least one isolate or synthesized Replikin sequence in the vaccine is one of SEQ ID NOS: 2902-2925, SEQ ID NOS: 2312-2544, SEQ ID NOS: 2701-2711, 2713-2718, SEQ ID NOS: 3282-3285, 3287-3291, 3293, 3295, 3297, 3299, 3300, 3302, 3304, 3306, 3308, SEQ ID NOS: 1685-1691, SEQ ID NOS: 1702-1717, SEQ ID NOS: 547-562, SEQ ID NO: 106, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NOS: 125-129, and SEQ ID NOS: 131-156, SEQ ID NOS: 233-244, SEQ ID NOS: 286-290, SEQ ID NOS: 312-323, SEQ ID NOS: 354-366, SEQ ID NOS: 368-380, SEQ ID NOS: 383-393, SEQ ID NOS: 395-401, SEQ ID NOS: 403-414, SEQ ID NOS: 291-307, SEQ ID NOS: 308-310, SEQ ID NOS: 324-327, SEQ ID NOS: 328-340, SEQ ID NOS: 416-419, SEQ ID NOS: 422-437, SEQ ID NOS: 440-445, SEQ ID NOS: 452-457, SEQ ID NOS: 464-476, SEQ ID NOS: 482-484 SEQ ID NOS: 487-492, SEQ ID NOS: 663-667, SEQ ID NOS: 670-1166, SEQ ID NOS: 1169-1529, SEQ ID NOS: 1532-1542, SEQ ID NO: 1548, SEQ ID NOS: 1637-1662, or SEQ ID NO: 1663.
In one embodiment, the vaccine is for prevention and/or treatment of a viral infection. In a specific embodiment, the vaccine is for a viral infection is caused by influenza virus, foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, coronavirus, ebola virus, gemini leaf curl virus, hemorrhagic septicemia virus, or tobacco mosaic virus.
In another specific embodiment, the influenza virus is Influenza A virus. In a further specific embodiment, the Influenza A virus is H1N1, H2N2, H3N2, H5N1 or H3N8 Influenza A virus.
In another specific embodiment, the virus is hemorrhagic septicemia virus.
In another embodiment, the vaccine is for prevention and/or treatment of an organismal infection.
In one specific embodiment, the organismal infection is caused by Mycobaterium mucogenicum, Mycobacterium tuberculosis, Staphylococcus aureus, or Plasmodium falciparum. In a further specific embodiment, the Staphylococcus aureus is methicillin-resistant.
In another embodiment, the vaccine is for prevention of a malignancy.
In one specific embodiment, the malignancy is a lung malignancy, a brain malignancy, a breast malignancy, an ovarian malignancy, or a lymph malignancy. In a further specific embodiment, the malignancy is non-small cell lung carcinoma or glioblastoma multiforme.
The invention also provides an immunogenic compound comprising at least one isolated or synthesized Replikin sequence within the protein or protein fragment of a Replikin Peak Gene or within a protein comprising a Replikin Peak Gene wherein said Replikin Peak Gene is identified in a virus, an organism or a malignancy, optionally further comprising a pharmaceutically acceptable carrier.
In another aspect, the present invention provides a method of stimulating the immune system, comprising administering in an animal at least one isolated or synthesized Replikin sequence identified within a protein or protein fragment of a Replikin Peak Gene or within a protein or gene area comprising a Replikin Peak Gene identified in a virus, organism, or malignancy. In a specific embodiment, the animal is a human.
The invention further provides an antibody to at least one isolated or synthesized Replikin sequence within a protein or protein fragment of Replikin Peak Gene or within protein or gene area comprising a Replikin Peak Gene.
Also provided by the present invention is a method of identifying a lethal strain of malignancy, organism or virus comprising: (1) obtaining a plurality of isolates of said malignancy, organism or virus; (2) identifying the Replikin Peak Gene in each isolate of the plurality of isolates of said malignancy, organism or virus; (3) analyzing the amino acid sequence of a protein or protein fragment of the Replikin Peak Gene of each isolate of the plurality of isolates for the presence and concentration of Replikin sequences; (4) comparing the concentrations of Replikin sequences in each of the proteins or protein fragments of the Replikin Peak Gene of each isolate of the plurality of isolates to the concentration of Replikin sequences in each of the proteins or protein fragments of the Replikin Peak Gene of each of the other isolates of the plurality of isolates; and (5) identifying the isolate having the highest concentration of continuous Replikin sequences in the protein or protein fragment of the Replikin Peak Gene as a virulent or lethal strain of said malignancy, organism or virus.
Further provided is a method of selecting a peptide from a malignancy, organism or virus for inclusion in a preventive or therapeutic vaccine or immunogenic compound for a malignancy, organism or virus comprising identifying at least one difference in the amino acid sequence of an otherwise conserved Replikin sequence or Replikin Peak Gene between at least two isolates of said malignancy, organism or virus and correlating the identified at least one difference in the amino acid sequence with the highest virulence, morbidity or host mortality among the at least two isolates and selecting an otherwise conserved Replikin sequence, Replikin Peak Gene or Replikin sequence within a Replikin Peak Gene having the identified at least one amino acid sequence difference as the peptide for inclusion in a preventive or therapeutic vaccine or immunogenic compound.
In one embodiment, the method further comprises predicting the isolate comprising the selected conserved Replikin sequence or Replikin Peak Gene having the at least one difference in the amino acid sequence to be lethal isolate of said malignancy, organism or virus.
In a specific embodiment, the malignancy, organism or virus is a malignancy.
In another specific embodiment, the malignancy is a lung malignancy, a brain malignancy, a breast malignancy or a lymph malignancy. In a further specific embodiment, the malignancy is a non-small cell lung carcinoma or a glioblastoma multiforme.
In another aspect, the malignancy, organism or virus is an organism.
In a first specific embodiment, the organism is Mycobacterium tuberculosis, Mycobaterium mucogenicum, Staphylococcus aureus, or Plasmodium falciparum. In another specific embodiment, the Staphylococcus aureus is methicillin-resistant.
In another aspect, the malignancy, organism or virus is a virus.
In one specific embodiment, the virus is influenza virus, foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, coronavirus, ebola virus, gemini leaf curl virus, hemorrhagic septicemia virus or tobacco mosaic virus.
The invention further provides a method of determining a source of a case of lung malignancy comprising identifying at least one peptide in a Replikin Peak Gene of a lung cancer cell that is also present in a Replikin Peak Gene of an isolate of tobacco mosaic virus, wherein the peptide is involved with the source of the lung malignancy.
In one embodiment, a plurality of peptides is identified in the Replikin Peak Gene of the lung cancer cell wherein each one of the plurality of peptides is also identified in the Replikin Peak Gene of an isolate of tobacco mosaic virus.
In another embodiment, the at least one peptide in the Replikin Peak Gene of the lung cancer cell and the at least one peptide in Replikin Peak Gene of the isolate of tobacco mosaic virus is a peptide of about 10 amino acids or less comprising at least two lysines and at least one histidine.
In a further embodiment, the at least one peptide in the Replikin Peak Gene of the lung cancer cell and the at least one peptide in Replikin Peak Gene of the isolate of tobacco mosaic virus is a peptide of about 10 amino acids or less comprising at least three lysines and at least one histidine.
In yet another embodiment, the at least one peptide in the Replikin Peak Gene of the lung cancer cell and the at least one peptide in the Replikin Peak Gene of the isolate of tobacco mosaic virus is about 7 amino acids or less comprising at least three lysines and at least one histidine.
In a further embodiment, the at least one peptide in the Replikin Peak Gene of the lung cancer cell and the at least one peptide in the Replikin Peak Gene of the isolate of tobacco mosaic virus is about 4 amino acids comprising three lysines and one histidine.
In a specific embodiment, the at least one peptide in the Replikin Peak Gene of the lung cancer cell and in the Replikin Peak Gene of the isolate of tobacco mosaic virus is KHKK (SEQ ID NO: 1584).
In another embodiment, the more than one KRKK (SEQ ID NO: 1584) peptide is identified in the Replikin Peak Gene of the lung cancer cell and in the Replikin Peak Gene of the isolate of tobacco mosaic virus.
In one specific embodiment, at least 10 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the lung cancer cell and at least 10 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the isolate of tobacco mosaic virus.
In another specific embodiment, at least 20 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the lung cancer cell and at least 20 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the isolate of tobacco mosaic virus.
In a third specific embodiment, at least 30 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the lung cancer cell and at least 30 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the isolate of tobacco mosaic virus.
In a fourth specific embodiment, at least 50 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the lung cancer cell and at least 50 KHKK (SEQ ID NO: 1584) peptides are identified in the Replikin Peak Gene of the isolate of tobacco mosaic virus.
The present invention further provides a method of identifying a first case of malignancy of the lung having a higher rate of replication, aggressive growth pattern or lethality as compared with a second case of malignancy of the lung comprising identifying a Replikin Peak Gene in a malignant cell from a first case of malignancy of the lung that has a higher Replikin Count in the Replikin Peak Gene than a Replikin Peak Gene identified in a malignant cell from a second case of malignancy of the lung.
In one embodiment, first and second cases of malignancy of the lung are non-small cell lung malignancies.
Further provided is an isolated or synthesized Replikin Peak Gene in a lung malignancy for diagnosis, prevention or treatment of lung cancer by the method comprising: (1) obtaining at least one malignant cell from a lung malignancy; (2) analyzing the protein sequences or protein sequence fragments of the at least one malignant cell for the presence and concentration of Replikin sequences; (3) identifying the protein sequence or the protein sequence fragment having the highest concentration of continuous Replikin sequences in the at least one malignant cell; (4) selecting the protein sequence or protein sequence fragment having the highest concentration of continuous Replikin sequences; (5) identifying the amino acid sequence of the selected protein sequence or protein sequence fragment as the Replikin Peak Gene; and (6) isolating or synthesizing the identified Replikin Peak Gene of the at least one malignant cell, wherein the isolated or synthesized identified Replikin Peak Gene is useful for diagnosis, prevention or treatment of lung cancer.
In one aspect, the lung malignancy is a non-small cell lung malignancy.
In another aspect, at least one isolated or synthesized Replikin sequence within the protein or protein fragment of the identified Replikin Peak Gene for diagnosis, prevention or treatment of lung cancer.
In a specific embodiment, the at least one isolated or synthesized Replikin sequence within the protein or protein fragment of the identified Replikin Peak Gene is one of SEQ ID NOS: 1585-1635 of SEQ ID NO: 1636.
The invention also provides an immunogenic composition for prevention and treatment of lung cancer, wherein the immunogenic composition comprises at least one isolated or synthesized Replikin sequence within the protein or protein fragment of the identified Replikin Peak Gene.
Also provided is method of stimulating the immune system, comprising administering in an animal the at least one isolated or synthesized Replikin sequence identified within the Replikin Peak Gene of the lung malignancy for prevention, treatment or diagnosis of lung cancer in an animal. In a specific embodiment, the animal is a human.
In another embodiment, the present invention provides a method of identification of a lethal form of lung cancer comprising: (1) obtaining at least one malignant cell from a plurality of lung tumors; (2) identifying the Replikin Peak Gene in the at least one malignant cell of each of the plurality of lung tumors; (3) analyzing the amino acid sequence of a protein or protein fragment of the Replikin Peak Gene in the at least one malignant cell of each of the plurality of lung tumors for the presence and concentration of Replikin sequences; (4) comparing the concentrations of Replikin sequences in each of the proteins or protein fragments of the Replikin Peak Gene in the at least one malignant cell of each of the plurality of lung tumors; and (5) identifying the lung tumor having the highest concentration of continuous Replikin sequences in the protein or protein fragment of the Replikin Peak Gene as a lethal form of lung cancer.
In a further embodiment, the present invention provides a method of identification of a more lethal form of lung cancer among at least two lung cancers, comprising: (1) obtaining at least one malignant cell from each of at least two lung cancers; (2) identifying the Replikin Peak Gene in the at least one malignant cell of each of the at least two lung cancers; (3) analyzing the amino acid sequence of a protein or protein fragment of the Replikin Peak Gene in the at least one malignant cell of each of the at least two lung cancers for the presence and concentration of Replikin sequences; (4) comparing the concentrations of Replikin sequences in each of the proteins or protein fragments of the Replikin Peak Gene in the at least one malignant cell of each of the at least two lung cancers; and (5) identifying the lung cancer having the highest concentration of continuous Replikin sequences in the protein or protein fragment of the Replikin Peak Gene as the more lethal form of lung cancer.
The invention further provides a method of determining an expected increase in lethality or virulence of a virus or organism which method comprises: (1) obtaining a plurality of isolates of said virus or organism wherein each isolate is isolated within a known time period and wherein at least two of said isolates is isolated about six months to about 5 years later than at least two other of said isolates; (2) identifying a Replikin Peak Gene in each isolate of said plurality of isolates; (3) analyzing the identified Replikin Peak Gene of each isolate of the plurality of isolates to determine the Replikin Count of each Replikin Peak Gene of each isolate of the plurality of isolates, or analyzing a protein, protein fragment, or gene area comprising the identified Replikin Peak Gene of each isolate of the plurality of isolates to determine the Replikin Count of the protein, protein fragment, or gene area of the plurality of isolates; (4) determining a mean Replikin Count within the Replikin Peak Gene or within the protein, protein fragment, or gene area comprising said identified Replikin Peak Gene for each known time period; (5) comparing the mean Replikin Count within the Replikin Peak Gene or within the protein, protein fragment, or gene area for each known time period one to another; (6) identifying an increase in the mean Replikin Count between at least two known time periods; and (7) identifying an expected increase in lethality or virulence of said virus, or organism within about six months to about three years following said identified increase in the mean Replikin Count.
In one specific embodiment, the known time period is about 1 year. In another specific embodiment, the increase in mean Replikin Count occurs over one year. In a further specific embodiment, the increase in mean Replikin Count occurs over three years. In another embodiment, the increase in mean Replikin Count is significant between at least two known time periods. In a further embodiment, the increase in mean Replikin Count has a significance of p=<0.001.
Replikin analysis was performed separately for H5N1 Replikin Peak Genes of each host group, namely, goose, duck, chicken and human. Low levels of Replikin count, below 4, were observed in each host group until 2005-2006. In 2005-2006 epidemics began to increase in Asian countries. While duck H5N1 counts decreased in 2006, they continued to increase in chicken H5N1 in 2006. Human RPG activity was upregulated in 2005-2006 and overtook RPG activity in chickens. This transition of Replikin Count increase from duck to chicken to human is in agreement with epidemiological evidence of the order of transfer of the virus between hosts. Changes in Replikin Count in the Replikin Peak Gene of the H5N1 isolates as in
Replikin analysis was performed separately for human H5N1 RPGs of each country. The results are shown for the Replikin Count for all data available on PubMed each year from 2003-2006. Low levels of Replikin count, below 4, were observed in each host group until 2005-2006, when human H5N1 increased in Asian countries. Human RPG activity was upregulated in 2005-2006 most prominently in Indonesia. The country most likely to first experience the increased human mortality was predicted in 2006 to be Indonesia. This prediction was proven correct in 2007 where incidence of human morbidity and mortality in the Indonesian outbreak were exceptionally high and evidence of possible human to human transmission was observed. Changes in Replikin Count in the Replikin Peak Gene of the H5N1 isolates such as in
Although SARS was first identified in 2003, Applicants wondered whether the emergence of the SARS strain of coronavirus might have been presaged in the activity of the whole group of coronaviruses. The pre-pandemic increase in both nucleocapsid and spike coronavirus proteins is in accord with, and might have served as a warning of, the finding that a coronavirus would be responsible for the 2003 first SARS emergence. It may be seen that the Replikin Count rose between 1995 and 2002, consistent with the SARS coronavirus outbreak, which emerged at the end of 2002 and persisted into 2003. The decline in Replikin Count correctly signaled the end of the SARS outbreak and had already begun its return to pre-outbreak levels when the outbreak emerged. A similar decline occurred on termination of Influenza A epidemics and pandemics (
As used herein, a Replikin Peak Gene (RPG) (or sometimes a Replikin Peak Gene Area-RPGA) is to mean a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has a highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) when compared to other segments or named genes of the genome. Generally, a whole protein or gene or gene segment that contains the amino acid portion having the highest concentration of continuous Replikin sequences is also referred to as the Replikin Peak Gene. More than one RPG may be identified within a gene, gene segment, protein, or protein fragment. An RPG may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine. For diagnostic, therapeutic and preventive purposes, an RPG may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine or may likewise have neither a terminal lysine nor a terminal histidine so long as the terminal portion of the RPG contains a Replikin sequence or Replikin sequences defined by the definition of a Replikin sequence, namely, an amino acid sequence having about 7 to about 50 amino acids comprising:
As used herein, a Replikin sequence is an amino acid sequence having about 7 to about 50 amino acids comprising:
A Replikin sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine. A Replikin peptide or Replikin protein is a peptide or protein consisting of a Replikin sequence. A Replikin sequence may also be described as a Replikin sequence of about 7 to about 50 amino acids comprising or consisting of a Replikin motif wherein the Replikin motif comprises:
The term “Replikin sequence” can also refer to a nucleic acid sequence encoding an amino acid sequence having about 7 to about 50 amino acids comprising:
As used herein, “animal” includes mammals, such as humans.
As used herein, the term “peptide” or “protein” refers to a compound of two or more amino acids in which the carboxyl group of one amino acid is attached to an amino group of another amino acid via a peptide bond. As used herein, “isolated” or “synthesized” peptide or biologically active portion thereof refers to a peptide that is, after purification, substantially free of cellular material or other contaminating proteins or peptides from the cell or tissue source from which the peptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized by any method, or substantially free from contaminating peptides when synthesized by recombinant gene techniques or a protein or peptide that has been isolated in silico from nucleic acid or amino acid sequences that are available through public or private databases or sequence collections. An “encoded” or “expressed” protein, protein sequence, protein fragment sequence, or peptide sequence is a sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence with any codon known to one of ordinary skill in the art now or hereafter. It should be noted that it is well-known in the art that, due to redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon and still result in an identical amino acid sequence. As will be understood by one of skill in the art, a method of identifying a Replikin amino acid sequence also encompasses a method of identifying a nucleic acid sequence that encodes a Replikin amino acid sequence wherein the Replikin amino acid sequence is encoded by the identified nucleic acid sequence.
As used herein, “reservoir” is any source of Replikin sequences that may be shared with a virus, organism or malignancy including any host of a virus, organism or malignancy, any food source of a host of the virus, organism or malignancy, any vector of virus, organism or malignancy, or any substance wherein the genetic information of a virus, organism or malignancy that may be shared, mingled, mixed, exchanged, or come into the proximity of the Replikin sequences of the reservoir.
As used herein, “different time periods” or “different time points” is any two time periods or points that may be differentiated one from another. For example, an isolate of virus isolated during the year 2004 is isolated in a different time period than an isolate of the same virus isolated during the year 2005. Likewise, an isolate of virus isolated in May 2004 is isolated in a different time period than an isolate of the same virus isolated in June 2004. When comparing Replikin concentrations of different isolates, it is preferred to use comparable time periods for comparison. For example, an isolate from 2004 is preferably compared to at least one other isolate from some other year such as 2002 or 2005. Likewise, an isolate from May 2004 is preferably compared to at least one isolate from some other month of some year, for example, an isolate from December 2003 or from June 2004. An isolate is any virus isolated from a natural source wherein a natural source includes, but is not limited to, a reservoir of a virus, a vector of a virus or a host of a virus. “Obtaining” an isolate is any action by which an amino acid or nucleic acid sequence within an isolate is obtained including, but not limited to, isolating an isolate and sequencing any portion of the genome or protein sequences of the isolate, obtaining any nucleic acid sequence or amino acid sequence of an isolate from any medium, including from a database such as PubMed, wherein the nucleic acid sequence or amino acid sequence may be analyzed for Replikin concentration, or any other means of obtaining the Replikin concentration of a virus isolated from a natural source at a time point.
As used herein, an earlier-arising virus or organism or a virus or organism isolated at an earlier time period is a specimen of a virus or organism collected from a natural source of the virus or organism on a date prior to the date on which another specimen of the virus or organism was collected from a natural source. For viruses, a natural source includes, but is not limited to, a reservoir of a virus, a vector of a virus, or a host of the virus. A later-arising virus or organism or a virus or organism isolated at a later time period is a specimen of a virus or organism collected from a natural source of the virus (including, but not limited to, a reservoir, a vector, or a host) or a natural source of the organism on a date subsequent to the date on which another specimen of the virus or organism was collected from a natural source.
As used herein, “emerging strain” refers to a strain of a virus identified as having an increased or increasing concentration of Replikin sequences in one or more of its protein sequences relative to the concentration of Replikins in other strains of such organism. The increased or increasing concentration of Replikins occurs over a period of preferably at least about six months, at least about one year or at least about three years, but may be a much shorter period of time for highly mutable viruses. An emerging strain of virus indicates an increase in lethality, virulence or replication.
As used herein, “bird” is any avian species including migratory and domestic birds, wherein said migratory and domestic birds includes, for example, chickens, ducks of all kinds, geese, pigeons, gulls, seabirds etc.
As used herein, “outbreak” is an increase in virulence, morbidity or mortality in a viral disease as compared to a baseline of an earlier occurring epidemiological pattern of infection in the same viral disease. One of ordinary skill in the art will know how to determine an epidemiological baseline. As used herein, “morbidity,” is the number of cases of a disease caused by the virus, either in excess of zero cases in the past or in excess of a baseline of endemic cases in the past. Therefore the baseline of endemic cases, in epidemiological terms, may, for example, relate to whether no or some cases were present in a geographic region in the immediate past. The past, in epidemiological terms, may mean more than one year and can mean several years or more as understood by one of ordinary skill in the art. The past may also mean less than one year as determined by one of ordinary skill in the art. In the case of annually-recurrent common influenza, for example, the baseline reflects an annual recurrence of common influenza.
As used herein, “mutation” refers to a change in the structure and properties of a virus or organism caused by substitution of amino acids. In contrast, the term “conservation” as used herein, refers to conservation of particular amino acids due to lack of substitution. A “point mutation” may refer to a change in a single amino acid residue or may refer to a change in a small number of amino acid residues.
As used herein, “segment” or “portion” of a genome, protein or protein fragment refers to any nucleic acid sequence of any size within a genome or any amino acid sequence of any size within a protein or protein fragment wherein the termini of the nucleic acid sequence may be any two nucleic acid residues within the genome and the termini of the amino acid sequence may be any two amino acid residues within the protein or protein fragment.
As used herein, “Replikin Count” or “Replikin Concentration” refers to the number of Replikins per 100 amino acids in a protein, protein fragment, virus, or organism. A higher Replikin concentration in a first strain of a virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier-arising or later-arising strain of the virus or organism having a lower Replikin concentration.
As used in this patent application, the term “continuous Replikin sequences” means a series of two or more Replikin sequences that are overlapped or are directly covalently linked.
As used herein a “Replikin Scaffold” refers to a series of conserved Replikin peptides wherein each of said Replikin peptide sequences comprises about 16 to about 34 amino acids, and preferably about 27 to about 33 amino acids and further comprises: (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine; (3) a lysine within 6 to 10 amino acid residues from another lysine; and (4) about 6% lysine. “Replikin Scaffold” also refers to an individual member or a plurality of members of a series of Replikin Scaffolds.
In an influenza virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 28 to about 30 amino acid residues. In a white spot syndrome virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 31 amino acid residues. In a taura syndrome virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 33 amino acid residues.
I. Replikin Count in Replikin Peak Gene is Predictive of and Related to Virulence and Lethality in Malignancies, Influenza and Other Pathogens and Replikin Peak Genes and Associated Replikin Sequences are Useful for Diagnostic, Therapeutic and Predictive Purposes
A virus Replikin gene related to lethality and virulence was first identified by Applicants in human H5N1 Influenza And was labeled a Replikin Peak Gene. Replikin Peak Genes were subsequently isolated in silico in numerous other viruses, bacteria, and protozoa. Replikin Peak Genes have now been associated with lethality in plant, fish, crustacea and vertebrate hosts. Because of their association with lethality, virulence and rapid replication, Replikin Peak Genes are now available as excellent targets for therapeutic and preventive treatments for a wide range of malignancies and pathogens.
Replikins, a class of peptides related to rapid replication, are 7 to 50 amino acids long, containing at least 2 lysine groups 6 to 10 amino acids apart, at least 1 histidine group, and at least 6% lysine. The phenomenon of the association of Replikins with rapid replication and virulence has been fully described in U.S. Pat. No. 7,189,800, U.S. Pat. No. 7,176,275, U.S. application Ser. No. 11/355,120, U.S. application Ser. No. 10/860,050 and U.S. application Ser. No. 10/105,232. Both Replikin concentration (number of Replikins per 100 amino acids) and Replikin composition have been correlated with the functional phenomenon of rapid replication.
Using an algorithm constructed to identify, count, and track Replikin sequences historically, Replikins were analyzed in 130,488 protein and genome sequences, representing all the accession numbers for common strains of influenza and some other lethal virus isolations published between 1917 and 2007 and reported on PubMed. Genomic areas with the highest concentration of continuous Replikins were isolated and named Replikin Peak Genes (RPGs).
Analysis of all publicly available protein and genome sequences for lethal Influenza A strains, including HSN 1, revealed 10,182 RPGs. RPGs were found to be present in isolates from all outbreaks of lethal influenza between 1917 and 2007 and the number of Replikin sequences per 100 amino acids (Replikin Count) in the identified RPGs was consistently observed to be above four and increased to as high as 29. In a significant control in Influenza B virus, which is non-lethal in humans, the Replikin Count in all 371 RPGs in Influenza B between 1940 and 2006 were found never to exceed four. Replikin Counts below four in the RPG of Influenza B virus contrasts with lethal Influenza A strains (with Replikin Counts as high as 29). RPG Replikin Counts during quiescent (or non-lethal) periods of Influenza A, were regularly observed to be four or below.
Replikin Counts below four for non-lethal isolates of influenza may be compared to highly lethal or virulent viruses such as ebola virus, which has been observed to have a Replikin Count of 32, Porcine Reproductive and Respiratory Virus (PRRSV) in pigs, which has been observed to have a Replikin Count of 43, gemini yellow leaf curl virus in tomato plants observed to have a Replikin Count of 56, hemorrhagic septicemia virus in fish observed to have a Replikin Count of 59, and white spot syndrome virus in shrimp, which has been observed to have a Replikin Count of 106. All of these viruses were observed to return to low counts during quiescent periods. Increased Replikin Counts in RPGs also were found in Mycobacterium tuberculosis (28), in methicillin-resistant Staphylococcus aureus (81), in Plasmodium falciparum (malaria) (153), and in lung cancer (261).
Analysis of Replikin Counts in genomic and proteonomic sequences alone prospectively correctly predicted: 1) the order of lethality in shrimp of four strains of taura syndrome virus (prediction was made blind in a laboratory study); 2) a 2007 increase in H5N1 percent mortality in humans; and 3) the country in which the increased percent mortality would occur most significantly, namely, Indonesia.
In addition to high Replikin Counts, analysis of rapidly replicating, virulent and lethal virus has revealed a series of conserved Replikin peptides associated with rapid replication, virulence and lethality known as Replikin Scaffolds. Replikin Scaffolds were observed in influenza virus strains where, for example, a 29-amino acid Replikin Scaffold has been conserved for 90 years in the genome of successive influenza virus strains. The scaffold has been present in each of the lethal influenza pandemics of 1918, 1957 and 1968 and in each lethal H5N1 outbreaks. Repeating signatures such as a KHKK (SEQ ID NO: 1584) signature has been observed in Replikin sequences within RPGs of lethal malignancies, viruses and organisms. The KHKK (SEQ ID NO: 1584) signature has been observed eleven times within the RPG of the protozoa that causes most malaria, P. falciparum. The KHKK (SEQ ID NO: 1584) signature has been observed 20 times within the RPG of a tobacco mocaic virus that induced exacerbated cell death in a pepper plant. The KHKK (SEQ ID NO: 1584) signature has been observed 57 times in non-small cell lung carcinoma within 52 Replikins observed within the 18 amino acid RPG identified in chromosome 9 of a non-small cell lung carcinoma. The presence of such a high number of KHKK (SEQ ID NO: 1584) signatures within the 18 amino acid RPG of the non-small cell lung carcinoma is explained by overlapping of the signatures. Overlapping of Replikin sequences and repeated signatures such as KHKK (SEQ ID NO: 1584) has now been associated with lethality, virulence and rapid replication. Together, these data indicate that a Replikin gene is quantitatively associated with lethal functions, and may be a mobile agent of lethality transferring between strains and species.
Whether Replikins can arise by synthesis de novo or are transferred from one organism or virus to another (or both) is yet to be determined. There is some beginning evidence for both. In one experiment, Replikin synthesis and/or transfer was facilitated in the laboratory in glioblastoma multiforme cells growing in tissue culture. The event, which facilitated the synthesis and/or transfer, was induced anoxia. Whether the anoxia stimulates increased rate of Replikin synthesis or membrane impairment facilitates Replikin transfer, or both, is yet to be determined.
Counting of Replikin sequences within a malignancy, a virus, a protozoon, a plant or an animal is aided by computer review of databases of gene and protein sequences. Bacteria were accepted as real when the light microscope permitted them to be seen as discrete entities, sufficiently discrete that they could be counted. Similarly, viruses were accepted as real when the electron microscope permitted them to be seen as discrete entities, sufficiently discrete that they could be counted. Likewise, Replikins can now be accepted as real since the “computer microscope” permits them to be seen as discrete entities, sufficiently discrete that they can be counted. Hence, the Replikin Count, or determination of number of Replikin Sequences in 100 amino acids in any given genomic or proteomic sequence, is facilitated on a large scale by computer analysis and comparison of Replikin Counts has provided the necessary evidence to associate increased Replikin Counts (in both whole genomes and Replikin Peak Genes) with lethality.
Visualization and counting of Replikin sequences in a wide range of genomes has now revealed that Replikin sequences are not scattered throughout the genome of lethal, virulent and rapidly replicating entities but, instead, are concentrated in particular areas of the genome. The concentration of Replikin sequences in a particular area of the genome has now been identified as a Replikin Peak Gene (RPG). Concentration of Replikin sequences in a RPG provides a magnification of the Replikin Count and a magnification of the developmental, growth and disease associations with the presence of Replikin Sequences. See, e.g.,
The magnification effect of analyzing the Replikin Count of a Replikin Peak Gene as compared to Replikin Counts from other parts of a genome or the whole genome is demonstrated in
By means of visual and software inspection, Applicants have analyzed 130,488 protein and genome sequences from common strains of influenza and other lethal viruses, isolated from 1917 to 2007 and accessible in PubMed. Replikin sequences in these 130, 488 sequences have been identified, counted and annually tracked. This extensive analysis revealed the Replikin Peak Gene that has not been found to be quantitatively related to lethality in several hosts, including plants, fish, crustacea and vertebrates, such as humans.
II. Prediction of Pathogenic Outbreaks and Lethal Malignancies
Prediction of epidemics and future outbreaks of viruses such as Influenza A (including H1N1, H2N2, H3N2, H3N8 and H5N1), foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, white spot syndrome virus, taura syndrome virus, tobacco mosaic virus, coronavirus, and SARS virus, may be made, for example, by reviewing the Replikin concentration of isolates of a virus strain and comparing the Replikin concentration for a particular time period with Replikin concentrations from another time period. Prediction of outbreaks or increases in virulence or lethality of organism may also be made, for example, by reviewing the Replikin concentration of isolates of an organism and comparing the Replikin concentration for a particular time period with Replikin concentrations from another time period. Organisms for which outbreaks or increases in virulence and lethality may be predicted include, for example, P. falciparum, M mucogenicum and S. aureus.
The difference in time period may be, for example, one month, six months, one year, three years or more. Preferably, the difference in time period is six months to three years. Also preferably, the difference in time period is one year. A significant increase in Replikin concentration from one year to the next and preferably over one, two, three or five years provides predictive value of an emerging strain of virus or organism that may begin an outbreak. A viral or other pathogenic outbreak may be predicted within about six months to about one to about three-years from the observation of a significant increase in Replikin concentration. The outbreak is preferably predicted within about one to about two years. An outbreak of virus or other pathogen, therefore, may be predicted within 1 to about 2 years as demonstrated in
Significant increases may be observed over a time period of more than one year, such as three, four, five or more years. An outbreak may likewise be predicted within about six months to about one year or more from the initial observation of an observable decrease in Replikin concentration following a significant increase.
The correlation between Replikin concentration and viral outbreaks noted throughout this application provide a method of predicting outbreaks of virus and other pathogens by monitoring increases or decreases in Replikin concentration in the RPG of isolates of the virus or other pathogen. Likewise, the lethality of an organism may be predicted by comparing the Replikin Count of the identified RPG of a malignancy with the Replikin Count of the identified RPG of another malignancy of the same type.
III. Replikin Peak Gene Sequences in Diagnostics and Therapies
High Replikin concentrations and RPGs have been shown to be related to rapid replication, viral outbreaks, epidemics, morbidity and host mortality in, for example, influenza virus strains, including H5N1, in SARS coronavirus, in shrimp in taura syndrome virus and in white spot syndrome virus, in foot and mouth disease virus, porcine reproductive and respiratory syndrome virus and in porcine circovirus, and in malignancies such as non-small cell lung carcinoma, among others. Because Replikin sequences in general (and particularly RPGs) are chemically defined, the sequences may be synthesized by organic chemistry rather than biological techniques, and thus are more specific, more reproducible and more reliable than other targets for diagnostics and therapeutics. The chemically defined Replikin sequences identified by the applicants are likewise potentially freer of adverse reactions that are characteristic of biologically derived vaccines and antibodies.
In addition to the diagnostic power of Replikin technology shown in these examples, it is clear that recognition for the first time of this class of virus peptides, and the discovery that they are related to rapid replication, virus outbreaks and high morbidity and mortality, makes the Replikins, and particularly the Replikin Peak Gene structures illustrated herein, new conserved prime targets for treatment and vaccines against these and other viruses.
Presence of the Replikin Peak Gene correlates with an increase in virulence in various species and an increase in mortality rate in humans in influenza virus, malaria and lung cancer and in pigs in PRRSV and porcine circovirus. Because an increase in virulence and mortality rate can be correlated with the Replikin Peak Gene (RPG), portions or fragments of the RPG are available as preferred targets for treatment with vaccines, antibodies or other blocking agents. Replikins in the gene are further preferred targets for identification of virulent strains of virus and other pathogens and for prediction of outbreaks of virus and other pathogens.
IV. Immunogenic Compounds, Vaccines, Antibodies and Blocking Agents
The observations of specific Replikins and their concentration in proteins of viral and organismal pathogens and malignancies provides the first specific quantitative early chemical correlates of outbreaks and provides for production and timely administration of vaccines tailored specifically to treat the prevalent emerging or re-emerging strain virus in a particular region of the world. By analyzing the protein sequences of isolates of a virus for the presence, concentration and/or conservation of Replikins, virus outbreaks and epidemics can be predicted and treatments developed. Furthermore, the severity of such outbreaks can be significantly lessened by administering a peptide immunogenic compound or vaccine based on the Replikin sequences identified herein or using the methods provided herein or Replikin sequences found to be most abundant or shown to be on the rise in virus isolates over a given time period, such as about one to about three years.
Vaccine products against SARS Replikin sequences and H5N1 influenza virus Replikin Scaffolds have been demonstrated by Applicants. See, e.g., U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 (Examples 6 and 7), incorporated herein by reference. Replikin sequences added to the feed source of shrimp have likewise imparted measurable resistance to challenges with taura syndrome virus. See Example 19. To date, all Replikin sequences tested in rabbit or chicken have induced an immune response and the glioma Replikin sequence (SEQ ID NO: 3658) has been identified and synthesized in peptides that induce and immune response and react with natural antibody responses in humans. See U.S. Pat. No. 6,638,505.
An immunogenic compound or peptide vaccine of the invention may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in particular virus strains. Preferably, the peptide vaccine is a Replikin Peak Gene or a Replikin sequence isolated within a Replikin Peak Gene. Further, the peptide vaccine may be based on Replikin sequence(s) shown to be increasing in concentration over a given time period and conserved for at least that period of time. A vaccine may also include a conserved Replikin peptide(s) in combination with a new Replikin(s) peptide or may be based on new Replikin peptide sequences. The Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences, although vaccines based on peptides containing only Replikin sequences, Replikin Peak Genes or Replikin sequences identified within a Replikin Peak Gene are preferred. Preferably, vaccine compositions of the invention also contain a pharmaceutically acceptable carrier and/or adjuvant.
The immunogenic compounds and vaccines of the present invention can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
The vaccine of the present invention may be administered to any animal capable of producing antibodies in an immune response. For example, the vaccine of the present invention may be administered to a rabbit, a chicken, a pig or a human. Because of the universal nature of Replikin sequences, a vaccine of the invention may be directed at a range of strains of virus or a particular strain of virus.
V. Increased Replikin Counts in Replikin Peak Gene of pB1 Area of Influenza A Strains Correlates with Pandemics and Lethal Outbreaks
Applicants have identified Replikin Peak Genes as a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has the highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) as compared to other segments or named genes of a genome. The inventors have likewise identified gene areas or proteins or protein fragments containing the highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) as Replikin Peak Genes.
Increased Replikin Counts in the Replikin Peak Gene identified in the pB1 gene area of influenza A strains has now been correlated by Applicants with pandemics and lethal outbreaks of influenza. These findings correspond to the Applicants' discovery that quantitative measurement of the concentration of Replikin peptides in proteins allows for correlation of Replikin peptide concentration per 100 amino acids with virulence, morbidity, mortality, epidemics and pandemics in malignancies, and organismal and viral infections. A correlation between increased Replikin Counts in the RPG of malignancies and pathogens has been established by Applicants in, for example, human pandemic influenza viruses, H5N1 (“Bird Flu”) influenza virus, white spot syndrome virus, foot and mouth disease virus, west nile virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, equine influenza virus, tobacco mosaic virus, malaria and non-small cell lung malignancies, among others. An increase in Replikin Count in these pathogens and malignancies allows for prediction of increased lethality or virulence and prediction of forthcoming outbreaks of infections.
A. Replikin Peak Gene in H5N1 Associated with Lethal Outbreak
Applicants initially identified a Replikin Peak Gene in the pB1 gene area of the genome of the H5N1 strain of influenza virus (e.g., SEQ ID NO: 1684) and observed that outbreaks of the H5N1 virus and lethality in infections from the virus correlated with increases in Replikin Count in the identified Replikin Peak Gene.
Table 1 provides mean Replikin Count and standard deviation from mean for publicly available sequences at PubMed for each of the eight gene areas in isolates of H5N1 between 2003 and 2006. Where no data is available for a given year, the year is not included in the table.
Analysis of the number of Replikin sequences present in the areas in the genome adjacent to the pB1 area revealed no more than a two-fold increase in Replikin Count in the seven other areas of the genome as compared to an eight-fold increase (p<0.001) in the Replikin Count in the pB1 area between years 2003 to 2006. The specificity of the localization of the upregulated RPG in the pB1 area is underlined by the fact that other parts of the polymerase gene area of which pB1 is a part, namely, the pB2 and pA gene areas do not have the same amount of increase in Replikin Count even though the gene areas are immediately adjacent to pB1.
In the illustration, the standard deviation of the means is shown in light gray columns on top of the means, rather than in the usual ‘T’ symbols, to emphasize the diverse expanding virus population with regard to the Replikin Count. As Replikin Count increases in a population, a diversity of Replikin Counts may be observed as the lethality and virulence of the virus increases. An increasing standard deviation within a virus population is, therefore, itself an index of viral outbreaks. Here in
Examples 1-3 are provided below as examples of analysis of Replikin Peak Genes in sequences publicly available in accession numbers at PubMed. Examples 2 and 3 illustrate how identification of a Replikin Peak Gene allows for a magnification of the effect of increases in Replikin Count in an isolate where the increase may be correlated with and predict increases in virulence and lethality. For example, Example 2 provides a 2003 isolate of H5N1 from Hong Kong with a whole pB1 gene area (SEQ ID NO: 1683) Replikin Count of 2.0 and an RPG Replikin Count of 14.6. Example 3 provides a 2006 isolate from Indonesia with a whole pB1 gene area Replikin Count of 17.8 and an RPG Replikin Count of 22.5. Indonesia experienced a highly lethal outbreak of H5N1 with evidence of human to human transmission in 2007. The high Replikin Counts in isolates from Indonesia in 2006 allowed the inventors to prospectively predict the lethal Indonesian outbreak.
The Replikin count of the whole genome from the 2006 Indonesian isolate demonstrates a significant increase as compared to the 2003 Hong Kong isolate. The isolation of the Replikin peak gene (RPG) area, that is the area of the genome which shows the highest concentration (count) of continuous Replikins per 100 amino acids, magnifies the effect. For this reason, whole genome counts are used for first approximations of Replikin count increases, and where more detailed specific gene areas or open reading frame data is not available. However, when available, the RPG is used for more definitive “higher power” examinations. This is illustrated in
B. Increasing Replikin Count in RPG of Influenza A Associated with Pandemics and Lethality
The inventors have now associated an increase in Replikin Count in the RPG of Influenza A virus with pandemics, epidemics and lethal outbreaks of influenza.
Over a ninety year period, the graph in
Over the same ninety year period, constant low Replikin Counts of less than four may be observed during quiescent non-lethal periods between epidemic outbreaks in all 3 pandemic strains of Influenza A including H1N1, H2N2, H3N2 and low Replikin Counts of less than four may also be observed in human H5N1 and H5N1 in chicken in relatively quiescent periods. Low Replikin Counts may likewise be observed in non-lethal Influenza B through the entire period of observation. In particular, the absence of increase in Replikin Count above five in Influenza B corresponds to the absence of any observed lethal outbreak. As such, during the observation period, Influenza B is always non-lethal. This absence of Replikin Counts of five or more in non-lethal Influenza B provides an important control for the study of Replikin Count as a correlate of lethality. In Influenza A, an increase in Replikin Count indicates an increase in lethality and a decrease in Replikin Count indicates a decrease in lethality.
Analysis of publicly available sequences for isolates of Influenza B virus between 1940 and 2007 is provided below in Table 2. Years in which not data was available are not included in the table.
While the Replikin Count in non-lethal Influenza B remains remarkable constant, the Replikin Count in Influenza A shows significant variation that correlates with outbreaks, epidemics and pandemics. For example, mean Replikin Count of the RPG in
The dominance of H5N1 over other Influenza A strains between 1990 and 2007 is also evident in
Dramatic increases in Replikin Count may be observed just before outbreak in the rebound epidemic of H1N1 beginning in the 1930's, in the pandemics of H2N2 and H2N3, which occurred in 1957 and 1968, respectively, and the outbreaks of H5N1 between 1997 and 2007. The largest increase in Replikin Count may be observed in the pB1-F2 area of the genome, which is contained within the pB1 area of the genome and contains an identified RPG (e.g., SEQ ID NO: 1723). The next largest increase in Replikin Count may be observed in the pB1 area of the genome, which is contained in the polymerase area of the genome. The smallest increase in Replikin Count may be observed in the polymerase area of the genome. It may be observed, therefore, that the Replikin Count becomes magnified as measured within the pB1 area as compared to the polymerase area and within the pB1-F2 area as compared to the pB1 area.
As in
C. Replikin Count in RPG of H5N1 Directly Correlates with Human Mortality
The inventors have now demonstrated that increased Replikin Counts in the RPG of H5N1 influenza virus (e.g., SEQ ID NO: 1684) may be directly correlated with human mortality.
Magnification of Replikin Count may be observed in
In
Table 3 provides mortality data for H5N1 infections from 2005 through 2007 and does not include earlier mortality data. Mortality data prior to 2005 has not been included in Table 3 because data prior to 2005 is inconsistent and understood by those of skill in the art to contain errors including errors caused by underreporting. The first generally agreed occasion when there were human deaths caused by proven H5N1 infection was in Hong Kong in 1997-1998. (This is probably incorrect, however, since there probably was mortality between 1959, when H5N1 was first reported, and 1997). The usual figures cited for 1997 are: 30 human cases, 8 deaths with mortality rate of about 27%. The number of cases (morbidity) and the number who died (mortality) that were not reported is unknown, but suspected to be significant. These errors are usually high in geographic areas where the medical care is less structured and scientific and the reporting is incomplete. Press reports between 1998 and 2002 were few, scattered, and not in agreement. Mortality data between 2005 and 2007 appear to be more consistent and have a higher level of reliability. Table 3, therefore, contains data from these years.
D. Replikin Count in RPG Correctly Identifies Host Lethality and Geographic Location of Outbreaks
The inventors have now demonstrated that Replikin Count in a Replikin Peak Gene provides a method for predicting and identifying outbreaks of pathogens such as H5N1 influenza by host and by geographic area.
Increased Replikin Counts in pathogens in particular hosts is predictive of an increase in probability of an outbreak of the pathogen. For example,
Increased Replikin Counts in pathogens in particular geographic areas is predictive of an increase in lethality of the pathogen in the identified geographic area. For example,
E. Replikin Peak Genes as Predictors of Outbreaks
Identification of the pB1 Replikin Peak Gene as a more significant gene area for changes in Replikin concentration effecting virulence reflects the same phenomenon in equine influenza as demonstrated in the pB1 gene area of H5N1. See
F. Replikin Concentration in Replikin Peak Gene of pB1 Area Correlates with Equine Influenza Epidemics
As with other influenza strains, an increase in Replikin concentration in equine influenza virus (EIV) has likewise been shown to be predictive of an increase in virulence of the virus and allows for prediction of forthcoming outbreaks or increases in morbidity and, in extreme cases, mortality. A review of publicly available amino acid sequences of isolates of EIV that demonstrates an increase in Replikin Count in the genome or a genome segment, or in a protein or protein fragment of the virus over time or between isolates is used as a predictor of an increase in outbreaks and morbidity in horses, donkeys, mules and other effected animals. Publicly available sequences for isolates of EIV from PubMed or other public or private sources may be analyzed by hand or using the FluForecast® search tool. (REPLIKINS LLC, Boston, Mass.).
Applicants have established a correlation between Replikin Count in the pB1 gene area (RPG) in EIV and an increase in virulence of the virus resulting in epidemics. The Applicants have reviewed publicly available amino acid sequences of isolates of EIV having accession numbers at www.pubmed.com and have identified increases in Replikin concentration in the Replikin Peak Gene of the pB1 gene area of the genome of the virus that relate to and predict an increase in outbreaks.
Applicants' initial analysis determined the Replikin Peak Genes within publicly available sequences of the pB1, pB2 and pA proteins of the H3N8 strain of influenza virus by analyzing publicly available sequences for the gene areas of the pB1, pB2 and pA proteins and identifying the protein segment having the highest concentration of continuous Replikin sequences within each gene area.
Applicants then compared the mean Replikin concentration in the identified Replikin Peak Gene for each of the three gene areas for isolates in each year having publicly available sequence information between 1977 and 2007. Applicants further analyzed all publicly available whole genome sequences for H3N8 between 1977 and 2007.
Replikin Counts of the RPGs of the pA and pB2 genomic areas, which are immediately adjacent to the pB1 area in the H3N8 genome, remain below 5 and do not increase to the extent of the Replikin Count of the RPG of the pB1 area. These observed increases in the pB1 area and absence of increases in the pB2 and pA areas are in direct agreement with the data on H5N1 influenza reflected in
The range of Replikin Counts in the RPGs of H3N8 may be observed to be similar to the range of Replikin Counts in other Influenza A species. See, e.g.,
The data for
In
Specifically, Series 3 in
Specifically, Series 4 in
VII. Methods of Predicting and Treating Outbreaks of Foot and Mouth Disease Virus (FMDV) Using RPGs and Related Replikin Sequences
An increase in Replikin concentration in the VP1 protein (containing an RPG of the virus genome) of foot and mouth disease virus (FMDV) is predictive of an increase in virulence and lethality of the virus and allows for prediction of forthcoming outbreaks or increases in virulence or lethality. Applicants have reviewed all publicly available amino acid sequences of isolates of FMDV having accession numbers at www.pubmed.com between 1969 and 2006 and have identified increases in Replikin concentration in the VP1 protein of FMDV (e.g., SEQ ID NO: 157) that relate to and predict certain known outbreaks of Foot and Mouth Disease.
Applicants reviewed Accession No. ABM63320 (SEQ ID NO: 157), which provides the amino acid sequence of the entire serotype-O FMDV VP1 polyprotein, and identified two RPGs. The first RPG begins at amino acid residue 925 and continues through amino acid residue 1018 and was isolated in silico as SEQ ID NO: 124. Five Replikin sequences were isolated (SEQ ID NOS: 125-129) in the first RPG, which gave the first RPG a Replikin Count of 6.3. The first RPG represents the Replikin Peak Gene of a fragment of the VP1 polyprotein.
The second Replikin Peak Gene begins at amino acid residue 1300 and continues through amino acid residue 1481 and was isolated in silico as SEQ ID NO: 130. Twenty-six Replikin were isolated in the second RPG (SEQ ID NOS: 131-156). The second Replikin Peak Gene Area has a Replikin Count of 14.3 and represents the Replikin Peak Gene of the entire reported VP1 polyprotein. Conserved Replikins within the RPG at SEQ ID NO: 130 are also contained, for example, in sequence fragments reported at Accession Nos. ABA46641, AAG43385, AAP81678 and ABG77564. Likewise, parts of the RPG of SEQ ID NO: 124 are contained in these accession numbers.
In the amino-terminal of SEQ ID NO: 157 (Accession No. ABM63320) SEQ ID NOS: 158-160 were isolated as Replikins. In the mid-molecule, SEQ ID NOS: 161-194 were isolated as Replikins. In the carboxy-terminal, SEQ ID NOS: 195-213 were isolated as Replikins. Each of these Replikin sequences is a preferred sequence for immunogenic compositions and vaccines and for other diagnostic, therapeutic and predictive purposes as described herein.
Prediction of the listed epidemics as well as future outbreaks may be made, for example, by reviewing the Replikin Counts of isolates of FMDV and comparing the Replikin Counts of the VP1 protein or the RPG within the VP1 protein for a particular year with Replikin Counts from other years. A significant increase in Replikin Count from one year to the next and preferably over one, two or three years provides predictive value of an emerging strain of FMDV that may begin an outbreak of Foot and Mouth Disease. A Foot and Mouth Disease outbreak may be predicted within about six months to about one year or more from the observation of a significant increase in Replikin Count.
More preferably, an outbreak of Foot and Mouth Disease may be predicted within about six months to about one year from the observation of a significant increase in Replikin count over two or three years. An outbreak may likewise be predicted within about six months to about one year from the initial observation of a decrease in Replikin Count following a significant increase. Using this method, Applicants predicted the Aug. 3, 2007 outbreak of FMDV in the United Kingdom months prior to the outbreak.
The data for
A. Prediction using VP1 Protein of All Serotypes
In addition to FMDV VP1 proteins of serotype-O, Applicants also analyzed publicly available sequences for isolates of all reported serotypes of FMDV VP1 protein from PubMed. The data is provided in Table 6 below. Note the increase in Replikin Count correlated with two epidemics in the United Kingdom (and other European countries) in 2001 and in the United Kingdom in 2007. Also note the low Replikin Counts during quiescence. Replikin Count increases from 1.6 in 1998, to 2.5 in 1999, to 2.7 in the year of the epidemic, 2001. Then post-epidemic, three lower Replikin Count years are noted, 1.5 in 2002, 1.5 in 2003, and 1.1 in 2005 (there were no publicly available sequences from 2004). The Replikin Count then rose to 2.8 in 2006 just prior to the outbreak in 2007. Note that the p values are less than 0.001 with respect to previous Replikin Counts.
B. Prediction Using VP1 Protein of Serotype C
Table 7 provides Replikin Count data for isolates of serotype-C FMDV for some years between 1955 and 2006. Note the significant increases over the low value in Replikin Count in 1998 and 1999 (prior to the 2001 epidemic in the UK) and the significant increase over the low value in 2006 (prior to the 2007 outbreak in the UK). Years having no available data are not reflected in the table.
The correlation between Replikin concentration and viral outbreaks noted above and illustrated in
The epidemiology and virology FMDV is different from the epidemiology and virology of some other viruses discussed herein such as Influenza virus. Nevertheless, a correlation between increases in Replikin Count in the FMDV VP1 protein and outbreaks of the virus provides compounding data establishing a shared phenomenon of rapid replication and virulence with an overwhelming number of other tested viruses and organisms.
C. Replikins Conserved in Serotype O FMDV RPGs
In serotype-O of FMDV, two conserved Replikin sequences contained within the Replikin Peak Gene are hkqkivapvk (SEQ ID NO: 91) and hpsearhkqkivapvk (SEQ ID NO: 92). A point mutant of the hpsearhkqkivapvk sequence to hptearhkqkivapvk (SEQ ID NO: 93) (mutation underlined) reportedly occurred in isolates from 1967 and 2007. The Replikin sequence hkqkivapvk (SEQ ID NO: 91) has been conserved from 1962 to 2006. The Replikin sequence hpsearhkqkivapvk (SEQ ID NO: 92) has been conserved from 1962 to 2006 except for the point mutation hptearhkqkivapvk (SEQ ID NO: 93), which is present in isolates reportedly having caused the 1967 outbreak (isolate O1BFS) and now the 2007 outbreak in the United Kingdom. These isolated conserved Replikin sequence are embodiments of the invention of particular preference for predictive, diagnostic and therapeutic capacity.
Table 8 provides the accession numbers of isolates between 1962 and 2006 containing the conserved sequence hkqkivapvk (SEQ ID NO: 91) and the amino acid position within the VP1 protein sequence where the conserved Replikin sequence begins.
Table 9 provides the accession numbers of FMDV isolates between 1962 and 2006 containing the conserved sequence hpsearhkqkivapvk (SEQ ID NO: 92) or the point mutation hptearhkqkivapvk (SEQ ID NO: 93) and the amino acid position within the VP1 protein sequence where the conserved Replikin sequence begins.
Accession No. AAG43385 (SEQ ID NO: 107) reports an FMDV serotype O isolate from 1999 that partly contains the RPG of SEQ ID NO: 124 and contains the conserved sequence SEQ ID NO: 91. In SEQ ID NO: 107, no Replikin sequences were identified in the amino-terminal. Replikin sequence SEQ ID NO: 108 was identified in the mid-molecule. Replikin sequence SEQ ID NO: 91 was identified in the carboxy-terminus.
Accession No. AAP81678 (SEQ ID NO: 111), reports an FMDV serotype 0 isolate from 1962 that partly contains the RPG of SEQ ID NO: 124 and contains the conserved sequence SEQ ID NO: 91. Accession No. ABA46641 (SEQ ID NO: 114) likewise reports an FMDV serotype O isolate from 1962 that partly contains the RPG of SEQ ID NO: 124 and contains the conserved sequence of SEQ ID NO: 91 and the conserved sequence of SEQ ID NO: 92 but for a single unknown residue at position 199 (SEQ ID NO: 115). In SEQ ID NO: 114, no Replikin sequences were identified in the amino-terminus or mid-molecule portion of the sequence. SEQ ID NOS: 115 and 116 were isolated in the carboxy-terminus.
Accession No. ABG77564 (SEQ ID NO: 118) reports an FMDV serotype O isolated from 2006 that partly contains the RPG of SEQ ID NO: 124 and contains the conserved sequence SEQ ID NO: 91. In SEQ ID NO: 118, no Replikins were identified in the amino terminus of the sequence. SEQ ID NOS: 119-121 and 91 were identified as Replikins in the mid-molecule. And no Replikins were identified in the carboxy terminus.
In addition to the diagnostic power of Replikin technology shown in these examples, it is clear that recognition for the first time of this class of virus peptides, and the discovery that they are related to rapid replication, virus outbreaks and high morbidity and mortality, makes the Replikins, and particularly the Replikin Peak Gene structures illustrated here, new conserved prime targets for treatment and vaccines in FMDV and other viruses. For example, the Replikin sequences (SEQ ID NOS: 91-93) provide invariant targets for such a vaccine. Likewise, the RPGs of SEQ ID NOS: 124 and 130 and the Replikin sequence identified in the accession number sequences (SEQ ID NOS: 108, 115-116 and 119-121) are preferred sequences for immunogenic compositions and vaccines. An embodiment of the invention, therefore, is a vaccine comprising at least one of the sequences SEQ ID NOS: 91-93 or SEQ ID NOS: 108, 115-116 and 119-121 or any combination thereof.
VIII. Methods of Predicting and Treating Outbreaks of West Nile Virus Using RPGs and Related Replikin Sequences
Applicants have now demonstrated a correlation between an increase in Replikin Count in a Replikin Peak Gene of the west nile virus (WNV) (e.g., SEQ ID NO: 245) and outbreaks, morbidity and mortality in the viral disease. See
Review of publicly available sequences of isolates of WNV from 1982-2007 revealed a Replikin Peak Gene in the envelope protein of west nile virus that has now been associated with virulence and lethality. In comparison with morbidity and mortality data in the United States between 1999 and 2006, an association between Replikin Count in the envelope protein of west nile virus and morbidity and mortality data is clear. See
The data for
Upon analysis of Replikin Counts of publicly available sequences from the entire genome of WNV and comparison with WNV morbidity and mortality data from the United States Center for Disease Control, the applicants observed that the mean Replikin Count of WNV increased significantly between years 2000, 2004, 2005 and 2006, respectively. As seen in Table 11, the mean Replikin Count of 2.8±0 observed in 2000 was found to be significantly different (p<0.001) from the mean Replikin Count of 3.8±1.7 observed in 2004, the mean Replikin Count observed in 3.8±1.7 in 2004 was found to be significantly different (p<0.01) from the mean Replikin Count observed in 4.5±1.8 in 2005, and, finally, the mean Replikin Count observed in 4.5±1.8 in 2005 was found to be significantly different (p<0.001) from the mean Replikin Count observed in 6.0±1.1 in 2006.
In the summer of 2007, Applicants reviewed the data for the whole WNV genome in publicly available sequences as provided in Table 11 and expressly predicted that a virulent increase in infection of WNV would likely follow the significant increase observed between each of the analyzed years. Immediately after Applicants' prediction, the California Department of Public Health confirmed Applicants' prediction by reporting that infections of WNV in California through Aug. 2, 2007 had been three times greater than infections seen in the previous year and a health emergency for three California counties was declared.
The epidemiology and virology of WNV is different from the epidemiology and virology of some other viruses discussed herein such as influenza, FMDV, PRRSV and PCV. Nevertheless, a correlation between increases in Replikin Count in the WNV envelope protein and morbidity and mortality provides compounding data establishing a shared phenomenon of rapid replication and virulence with an overwhelming number of other tested viruses and organisms.
In WNV and the other viruses and pathogens described herein, prediction of epidemics and future outbreaks may be made, for example, by (1) reviewing the Replikin Counts of isolates of WNV and identifying a RPG, for example, and RPG in the envelope protein (e.g., SEQ ID NO: 245), (2) comparing the Replikin Counts in the RPG, in the protein or gene area containing the RPG, or in the whole virus genome for a particular year with Replikin Counts from other years. A significant increase in Replikin Count from one year to the next and preferably over one, two or three years provides predictive value of an emerging strain of WNV that may begin an outbreak of more highly virulent WNV. A WNV outbreak may be predicted within about six months to about one year or more from the observation of a significant increase in Replikin Count.
More preferably, an outbreak of WNV may be predicted within about six months to about one year from the observation of a significant increase in Replikin Count over two or three years or, as in inventors prediction in 2007, following the observation of strongly significant increases over several years such as wherein Replikin Counts between 2000, 2004 and 2006 had p values of less than at least 0.001 and frequently less than 0.001. As such, significant increases may be observed over a time period of more than one year, such as three, four, five or more years. An outbreak may likewise be predicted within about six months to about one year from the initial observation of an observable decrease in Replikin Count following a significant increase. Using this method, Applicants prospectively predicted the beginnings of a 2007 outbreak of WNV. The method may also employ isolates of individual strains or isolates of all strains of WNV.
An embodiment of the invention provides a segment of the genome or a protein or segment of a protein of the WNV in which the expressed gene or expressed gene segment has the highest concentration of Replikins, or Replikin Count (number of Replikins per 100 amino acids), when compared to other segments or named genes of the genome, namely the RPG. An RPG (SEQ ID NO: 245) in Accession No. ABA54585 is reported in Example 7 below. Twelve Replikin sequences (SEQ ID NOS: 246-257) are identified in the RPG diagnostic, preventive, therapeutic and predictive applications. These Replikin sequences are preferred embodiments of immunogenic compositions and vaccines. The invention further provides Replikin sequences within the identified RPG that are conserved in the genome over time and, as such, are available as relatively invariant preferred targets for diagnosis and manipulation of rapid replication and virulence in WNV through immunogenic responses and vaccines.
IX. Methods of Predicting and Treating Outbreaks of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Using Replikin Sequences
An increase in Replikin concentration in PRRSV is predictive of an increase in virulence of the virus and allows for prediction of forthcoming outbreaks or increases in mortality. A review of publicly available amino acid sequences of isolates of PRRSV that demonstrate an increase in Replikin concentration in the genome or a genome segment, or in a protein or protein fragment of the virus over time or between isolates is used as a predictor of an increase in outbreaks and morbidity and mortality of pigs infected with PRRSV. Publicly available sequences for isolates of PRRSV from PubMed or other public or private sources may be analyzed by hand or using proprietary search tool software (ReplikinForecast™ from REPLIKINS LLC, Boston, Mass.).
The inventors have now identified a Replikin Peak Gene in the nucleocapsid protein of the Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) and have demonstrated a correlation between increased Replikin Count in the nucleocapsid protein of PRRSV between 2004 and 2007 and major outbreaks of PRRSV in China. Example 8.
The invention provides RPGs and Replikin sequences within the identified RPGs for diagnostic, preventive and therapeutic applications. For example, each Replikin sequences identified within an identified RPG in PRRSV and other viruses, organisms and malignancies is available for diagnostic and therapeutic applications including vaccines, immunogenic compositions and antibody therapies. The entire Replikin Peak Gene sequence or fragments thereof are likewise available for diagnostic, preventive, therapeutic and predictive applications. Further, the presence of the Replikin Peak Gene in an isolate of the virus is indicative of rapid replication.
As discussed herein, applicants have identified RPGs of available PRRSV isolates within the nucleocapsid protein of PRRSV. Identification of these RPGS is different, for example, from the Replikin Peak Gene previously identified by applicants in H5N1 influenza in one polymerase area, namely the RNA-directed RNA polymerase or pB1 protein. Identification of Replikin Peak Genes in different structures of different viruses is made possible through the strict criteria for a Replikin sequence as defined by the applicants. The proprietary software ReplikinForecast™ (licensable from REPLIKINS LLC, Boston, Mass.) provides an efficient survey of publicly available Replikin sequences and identification and isolation in silico of the Replikin Peak Gene.
The size of a Replikin Peak Gene, both in terms of the number of amino acids and the Replikin Count, will depend upon the size of the sequence of the entire genome, protein or fragment thereof that has been isolated and reported. The invention further provides Replikin sequences within the identified Replikin Peak Gene or Area that are conserved in the genome over time and, as such, are available as relatively invariant targets for diagnosis and manipulation of rapid replication and virulence in PRRSV.
Further, the following RPGs have been identified in PRRSV isolates from China reported at Accession Nos. AAM18565, AAP81809 and ABL60920, respectively:
The identified RPG sequences are identical across the 2000, 2003 and 2006 isolates except for point mutations at positions 45 and 46 (underlined in bold). These sequences are, therefore, relatively invariant targets for diagnosis and manipulation of rapid replication and virulence in PRRSV and are available as vaccines against the disease.
Point mutations, such as in positions 45 and 46 in the above-listed Chinese isolates, provide excellent predictive capacity. In the highly virulent and fatal Chinese variant disclosed in 2006 at ABL60920 (SEQ ID NO: 343), the asparagine and arginine at positions 45 and 46 are the same residues in the same relative positions as asparagine and arginine at residues 21 and 22 in the RPG of the highly virulent PRRSV 2006 Mexican isolate publicly available at Accession No. ABF 19568 (comparable mutated residues underlined in bold):
These two RPG sequences are, therefore, especially predictive of virulence and are preferred sequences for immunogenic compositions and vaccines. Identification of these residues in other RPG sequences in PRRSV provides a high likelihood of virulence and an excellent target for attack of the virus through antibody therapies, vaccines and other treatments.
X. Methods of Predicting and Treating Outbreaks of Porcine Circovirus (PCV) Using Replikin Sequences
An increase in Replikin concentration in PCV is predictive of an increase in virulence of the virus and allows for prediction of forthcoming outbreaks or increases in mortality. A review of publicly available amino acid sequences of isolates of PCV that demonstrate an increase in Replikin concentration in the genome or a genome segment, or in a protein or protein fragment of the virus over time or between isolates is used as a predictor of an increase in outbreaks and morbidity and mortality of pigs infected with PCV. Publicly available sequences for isolates of PCV from PubMed or other public or private sources may be analyzed by hand or using software described herein.
Applicants have now established a correlation between Replikin Counts in PCV and an increase in virulence. Applicants reviewed publicly available amino acid sequences of isolates of PCV having accession numbers at www.pubmed.com and identified increases in Replikin Counts in the genome of the virus that predict an increase in outbreaks and mortality of pigs infected with PCV.
The data for
In particular, the Replikin Count of PCV is observed at 9.4 (±10.8) in 1997 and decreases rapidly to 2.9 (1.2) in 2000. Replikin Count then rises to 3.5 (±1.4) in 2002 and rises again to 3.9 (±1.2) through 2007. During this time period, the virulence and mortality observed in swine herds in Canada (with additional reported incidence in Central America) were increasing. The large standard deviation seen in 1997-1999 evidences a virus population that is undergoing rapid change in the concentration of Replikin sequences in the genome and points to forthcoming changes in virulence, morbidity and mortality.
Prediction of epidemics and future outbreaks may be made, for example, by reviewing the Replikin Counts of RPGs or other portions of isolates of PCV or PRRSV or other virus or pathogen and comparing the Replikin Counts for a particular year with Replikin Counts from other years. A significant increase in Replikin Count from one year to the next and preferably over one, two or three or more years provides predictive value of an emerging strain of PCV that may begin an outbreak of more highly virulent and/or more highly lethal PCV.
A PCV outbreak may be predicted within about six months to about one year or more from the observation of a significant increase in Replikin Count. More preferably, an outbreak of PCV may be predicted within about six months to about one year from the observation of a significant increase in Replikin Count over two or three years or following the observation of strongly significant increases over several years such as wherein Replikin Counts of PCV between 2000 and 2002 and between 2005 and 2007 increased with p values each year over lowest mean Replikin Count in the series of less than 0.001.
Significant increases may be observed over a time period of more than one year, such as three, four or five years or more. An outbreak may likewise be predicted within about six months to about one year or more from the initial observation of an observable decrease in Replikin Count following a notable increase. For example, the marked decrease from 1997 to 2000 in PCV Replikin Counts predicts the increase of incidence and mortality in viral infections beginning in 2000 and continuing through at least 2006 (morbidity and mortality data for 2007 have not been made available at this time). Using this method, Applicants, for example, prospectively predicted the beginnings of a 2007 outbreak of WNV. See
The inventors have identified a Replikin Peak Gene in the replicase protein of the Porcine Circovirus (PCV). Examples of the identification of a Replikin Peak Gene (RPG) in an isolate of PCV in Manitoba, Canada in 1997 and an RPG in an isolated of PCV in China in 2007 are provided in Example 9 (SEQ ID NOS: 520 and 525). Example 9 demonstrates comparably high Replikin Counts of the identified RPGs and provides prediction that the isolated strains of the virus have high virulence. Example 9 further provides RPGs and Replikin sequences within the identified RPGs as targets for production of immunogenic compositions and vaccines.
The invention provides Replikin sequences within the identified Replikin Peak Gene gene or gene segment for diagnostic, preventive and therapeutic applications. SEQ ID NOS: 324-328 are Replikin sequences provided in an RPG from Accession No. AAC59472. See Example 9. SEQ ID NOS: 329-340 are provided in an RPG from Accession No. ABP68657. See Example 9. For example, each of the above-listed sequences as Replikin sequences identified within an identified RPG are available for diagnostic and therapeutic applications including vaccines and antibody therapies. The entire Replikin Peak Gene sequence or fragments thereof are likewise available for diagnostic, preventive, therapeutic and predictive applications. Further, the presence of the Replikin Peak Gene in an isolate of the virus is indicative of rapid replication.
Replikin Peak Genes (RPG) have also been identified in PCV isolates in Accession Nos. AAC98885, AAL01075 and ABP68667 (SEQ ID NOS: 481, 438, and 451). See Example 9. For each identified RPG, continuous, non-interrupted and overlapping Replikin sequences have been identified for predictive and therapeutic applications.
Applicants have to date identified RPGs of available PCV isolates both within open reading frame 1 in a putative replicase protein and within open reading frame 11 in a predicted 1.8 kD protein. Identification of Replikin Peak Genes in different structures of different viruses is made possible through the strict criteria for a Replikin sequence as defined by the applicants. The size of a Replikin Peak Gene, both in terms of the number of amino acids and the Replikin Count, will depend upon the size of the sequence of the entire genome, protein or fragment thereof that has been isolated and reported. The invention further provides Replikin sequences within the identified Replikin Peak Gene that are conserved in the genome over time and, as such, are available as relatively invariant targets for diagnosis and manipulation of rapid replication and virulence in PCV.
XI. Conservation of Replikin Structure Relates to Virulence and Lethality
The conservation of any structure is critical to whether that structure provides a stable invariant target to attack and destroy or to stimulate. Replikin sequences have been shown to generally be conserved. When a structure is tied in some way to a basic survival mechanism of the organism, the structures tend to be conserved. A varying structure provides an inconstant target, which is a good strategy for avoiding attackers, such as antibodies that have been generated specifically against the prior structure and thus are ineffective against the modified form. This strategy is used by influenza virus, for example, so that a previous vaccine may be quite ineffective against the current virulent virus.
Certain structures too closely related to survival functions, however, apparently cannot change constantly. An essential component of the Replikin structure is histidine (h), which is known for its frequent binding to metal groups in redox enzymes and is a probable source of energy needed for replication. Since the histidine structure remains constant, Replikin sequence structures remain all the more attractive a target for destruction or stimulation.
A. Replikin Conservation in HIV
Conservation of Replikin sequences has been observed in trans-activator (Tat) proteins in isolates of HIV. Tat (trans-activator) proteins are early RNA binding proteins regulating lentiviral transcription. These proteins are necessary components in the life cycle of all known lentivirases, such as the human immunodeficiency viruses (HIV). Tat is a transcriptional regulator protein that acts by binding to the trans-activating response sequence (TAR) RNA element and activates transcription Initiation and/or elongation from the LTR promoter. HIV cannot replicate without tat, but the chemical basis of this has been unknown. In the HIV tat protein sequence from 89 to 102 residues, we have found a Replikin that is associated with rapid replication in other organisms. The amino acid sequence of this Replikin is hclvckqkkglgisygrkk (SEQ ID NO: 3666) In fact, Applicants found that this Replikin is present in every HIV tat protein. Some tat amino acids are substituted frequently, as shown in Table 12, by alternate amino acids (in small size fonts lined up below the most frequent amino acid, the percentage of conservation for the predominant Replikin (hclvcfqkkglgisygrkk) (SEQ ID NO: 3314). These substitutions have appeared for most of the individual amino acids. However, the key lysine and histidine amino acids within the Replikin sequence, which define the Replikin structure, are conserved 100% in the sequence; while substitutions are common elsewhere in other amino acids, both within and outside the Replikin, none occurs on these key histidine amino acids. The sequences listed in Table 12 are SEQ ID NO: 3314 and the denoted variations of formula peptide SEQ ID NO: 3315.
The substitutions cannot be considered to be at random because amino acids were substituted except for the lysines and histidines which define the Replikin structure. It is not just that lysine per se is “immune” to substitution, because the lysine not 6 to 10 amino acids from another lysine was freely substituted, while those lysines which do define the Replikin structure were not substituted.
B. Conservation in Replikin Peak Genes in H5N1 in Humans and Chickens
A series of conserved Replikin sequences (SEQ ID NOS: 1-11 and 14) were isolated in silico by Applicants in human and chicken isolates of H5N1 influenza virus. SEQ ID NO: 1 was identified in the following accession numbers in the following years at the following amino acid residue positions: (1997) AAK49342 beginning at position 134, AAK49340, 134, AAF74320, 134, AAF74319, 134, AAF74318, 134, AAF74317, 134, AAK49344, 134, AAK49343, 134, AAK49341, 134, AAK49339, 134, AAK49338, 134; (1998) AAK49345, 134; (2003) BAE07200, 134; (2004) AAW59551, 131, AAW59549, 129, ABE97897, 123, ABE97896, 123, ABE97895, 123, ABE97892, 123, ABE97891, 123, AAV32651, 134, AAV32643, 134; (2005) ABG78563, 109, ABG78562, 109, ABF56657, 127, ABF56656, 127, ABF56655, 127; (2006) ABK34973, 134, ABL31779, 134, ABL31765, 134, ABL31754, 134, ABL07029, 134, ABL07018, 119, ABL07007, 134, ABI49406, 134, ABI36481, 134, ABI36470, 134, ABI36451, 134, and ABI36440, 134.
SEQ ID NO: 11 was identified in the following accession numbers in the following years at the following amino acid residue positions: (2003) BAE07200, beginning at position 19; (2004) AAW59551, 16, AAW59549, 14, ABE97897, 8, ABE97896, 8, ABE97895, 8, ABE97894, 8, ABE97893, 8, ABE97892, 8, ABE97891, 8, ABE97890, 8, ABE97889, 8, ABE97888, 8, AAV35115, 19, AAV32651, 19, AAV32643, 19; (2005) ABC72649, 19, ABF56657, 12, ABF56656, 12, ABF56655, 12; (2006) ABK34973, 19, ABL31779, 19, ABL31765, 19, ABL31754, 19, ABL31743, 19, ABI49414, 19, ABL07029, 19, ABL07018, 4, ABL07007, 19, ABI49406, 19, ABI36481, 19, ABI36470, 19, ABI36451, 19, ABI36440, 19, ABI36429, 19.
SEQ ID NO: 14 was identified in the following accession numbers in 2006 at the following amino acid residue positions: ABL31777, beginning at position 41, ABI49393, 41, ABL07016, 41, ABL07005, 41, ABI49404, 41, ABI36472, 41, ABI36461, 41, ABI36452, 41, ABI36441, 41, and ABI36430, 41.
SEQ ID NO: 14 was isolated in silico from the pB1 gene area sequence disclosed at Accession No. ABI36441 (SEQ ID NO: 15). Replikin sequences (SEQ ID NOS: 16-17) were identified in the amino-terminus. Replikin sequences (SEQ ID NOS: 18-32) were identified in the mid-molecule. No Replikin sequences were identified in the carboxy-terminus. Sixteen Replikin sequences in 90 amino acid residues gave a Replikin Count of 17.8.
SEQ ID NO: 14 was also isolated in silico from Accession No. ABI36430 (SEQ ID NO: 33). Replikin sequences (SEQ ID NOS: 34-35) were identified in the amino-terminus. Replikin sequences (SEQ ID NOS: 36-49) were identified in the mid-molecule. No Replikin sequences were identified in the carboxy-terminus.
SEQ ID NO: 14 was also isolated in silico from Accession No. ABL07027 (SEQ ID NO: 50). Replikin sequences (SEQ ID NOS: 51-52) were identified in the amino-terminus. Replikin sequences (SEQ ID NOS: 53-68) were identified in the mid-molecule. Replikin sequences (SEQ ID NO: 69-71) were identified in the carboxy-terminus.
SEQ ID NO: 2 was identified in the following accession numbers in the following years at the following amino acid residue positions: (1997) Q9WLS3, 184, O89749, 184, AAK49358, 184, AAF74316, 184, AAK49362, 184, AAK49357, 184, AAK49356, 184, CAB95863, 184; (2003) BAE07199, 184; and (2004) ABL97546, 184, ABE97545, 184, ABE97544, 184, ABE97543, 184, ABE97542, 184, ABE97540, 184, ABE97564, 179, ABC72648, 184, ABK34974, 184.
SEQ ID NO: 3 was identified in the following accession numbers in the following years at the following amino acid residue positions: (1997) Q9WLS3, 184, O89749, 184, AAK49358, 184, AAF74316, 184, AAF74315, 184, AAF74314, 184, AAK49362, 184, AAK493761, 184, AAK49359, 184, AAK49357, 184, AAK49356, 184, CAB95863, 184; (1998) AAK49363, 184; (2003) BAE07199, 184; (2004) ABE97546, 184, ABE97545, 184, AGE97544, 184, ABE97543, 184, ABE97542, 184, ABE97541, 184, ABE97540, 184, ABE97539, 184, ABE97538, 184, ABE97537, 184, ABE97536, 184, AAV35116, 184, AAV32644, 184; (2005) ABG78564, 184, ABC72648, 184; and (2006) ABK34974, 184.
SEQ ID NO: 7 was identified in the following accession numbers in the following years at the following amino acid residue positions: (2003) BAE07200, 128; (2004) AAW59551, 125, AAW59549, 123, ABE97897, 117, ABE97896, 117, ABE97895, 117, ABE97894, 117, ABE97893, 117, ABE97892, 117, ABE97891, 117, ABE97890, 117, ABE97889, 117, ABE97888, 117, AAV32651m 128, AAV32643, 128; (2005) ABG78563, 103, ABG78562, 103, ABF56657, 121, ABF56656, 121, ABF56655, 121; and (2006) ABL31779, 128, AB31765, 128, ABL31754, 128, ABL31743, 128, ABI49414, 128, ABI49395, 128, ABL07029, 128, ABI36470, 128, ABI36451, 128, ABI36440, 128, ABI36429, 128.
SEQ ID NO: 8 was identified in the following accession numbers in the following years at the following amino acid residue positions: (1997) Q9WLS3, 184, O89749, 184, AAK49360, 168, AAK49356, 168, AAF74316, 168, AAK49362, 168, AAK49359, 168, AAK49357, 168, AAK49356, 168, CAB5863, 168; (2003) BAE07199, 168; (2004) ABE97546, 168, ABE97545, 168, ABE97544, 168, ABE97543, 168, ABE97542, 168, ABE97541, 168, ABE97539, 168, ABE97538, 168, ABE97537, 168, ABE97536, 168, AAV35116, 168, AAV32644, 168; (2005) ABG78564, 163, ABC72648, 168; and (2006) ABK34974, 168.
The series of conserved Replikin sequences discussed above are preferred embodiments of the invention and are particularly useful as immunogenic compounds and vaccines and the presence of these sequences has particular predictive value for timing, geographic position and lethality of H5N1 outbreaks.
C. Conservation in Replikin Scaffolds in Influenza A strains
Table 13, below, provides support for the role of Replikin Scaffolds as Replikin Peak Genes in lethal outbreaks of influenza in humans and birds. In Table 13, the history of the Goose Replikin and its homologues are tracked from 1917 to the present outbreak of avian H5N1 virus. Table 13 demonstrates conservation of the “scaffold” homology of the Goose Replikin in virulent strains of influenza.
Table 13 illustrates the history, by year or smaller time period, of the existence in the protein structure of the Goose Replikin and its homologues in other influenza Replikins. Table 13 further illustrates the history of amino acid substitutions in those homologues and the conservation of certain amino acids of the Replikin structure which are essential to the definition of a Replikin and the function of rapid replication supplied by Replikins.
Table 13 illustrates a Fixed Replikin Peak Gene Scaffold with ordered non-random substitution in the 90 year conservation of influenza virus Replikin peptides, from a 1917 goose flu and 1918 human pandemic to a 2007H5N1 ‘Bird Flu’ homologue.
The Goose Replikin is a 29 amino acid peptide RPG in the hemagglutinin protein of influenza virus beginning with kk and ending with hh (SEQ ID NO: 3672). Replikins may contain overlapping Replikins. This Replikin Scaffold appears in the virus genome only when the Replikin count rises above 3, and disappears again when the clinical outbreak is over and the Replikin count declines to less than 3.
D. Replikin Scaffold in 2007 Isolate of H1N1
A Replikin Scaffold hemagglutinin Replikin Peak Gene has now been identified in one human case of H1N1 isolated in 2007 in Thailand. This evidence suggests H1N1 is making a comeback. The H1N1 Replikin Scaffold that has been identified is knglypnlsksyannkekevlvlwgvhh (SEQ ID NO: 2011), which is associated with a whole hemagglutinin Replikin Count of 8.1, and Replikin Count in the RPG of 28. The Replikin Count in the RPG of the 2007 Thailand isolate is higher than the Replikin Count in the RPG of an H1N1 isolate from the 1918 pandemic, Accession No: IRUZL, which has a Replikin Count in its RPG of 19. Example 5 provides the inventors analysis of the 2007 Thailand isolate.
E. Homologous Replikin Scaffold Sequences in Influenza, WSSV, and TSV
The inventors have further established a relationship between virulent influenza virus and shrimp viruses WSSV and TSV in the Replikin Scaffold portions of the viruses as may be seen in Table 14 below. Although there is extensive substitution, several short Replikins of the Shrimp white spot syndrome virus demonstrates significant homologies to the influenza virus Replikin sequences, especially with regard to length and key lysine (k) and histidine (h) residues. Similar, but less extensive, homologies are seen in taura syndrome virus. These homologies suggest that the sequences are derived from a shared reservoir and/or that similar mechanisms of Replikin production are used in both virus groups.
TSV is less virulent than WSSV and the structure of the TSV Replikin Scaffold is less closely related to influenza virus than are the structures of WSSV Replikin Scaffolds. In year 2000, TSV had a Replikin concentration of 2.7. Between 2001 and 2004, TSV had a lower mean Replikin concentration, as low as 0.7, and its Replikin Scaffold disappeared. In 2005 the Replikin Scaffold reappeared, with an increase in lysines and histidines, and a commensurate increase in Replikin concentration to 1.8, followed by an increase in TSV outbreaks in 2006-2007. See Table 19.
F. Replikin Peak Genes Provide Increased Predictive and Therapeutic Capacity
Since the identification of the Replikin structure, correlation between increased concentrations of Replikin sequences and increased replication and virulence has been observed in a range of viruses and organisms. These observations are made more accurate by the present isolation in silico of Replikin Peak Genes. While increased concentration of Replikin sequences in the genome of a virus offers both advance warning and new targets for developing effective methods of predicting and treating viral outbreaks, identification of an increase in concentration of Replikin sequences in a Replikin Peak Gene of a genome or protein heightens the predictive capacity of the change in Replikin concentration and the efficacy of new targets.
For example, more precise predictions of increased virulence are now available through identification of a Replikin Peak Gene in, among other viruses, the H5N1 strain of influenza (
By monitoring changes in concentrations of Replikin sequences in viral genomes generally, emerging viral diseases can be identified in virus reservoirs and vectors in advance of their appearance in animal or human hosts. Identification of the emerging viruses and the Replikin sequences within the virus genome allows for appropriate, advance control efforts, including isolation and quarantine, and provide sufficient time for the synthesis and testing of vaccines specific to the sequences of the emerging virus.
As discussed above, the inventors have identified the pB1 gene area of the H3N8 strains of influenza virus (SEQ ID NO 545) as the region of the genome of the virus having the highest concentration of Replikin sequences. A Replikin Peak Gene has also been identified in H5N1 influenza virus and has been correlated with epidemics, increased virulence, morbidity and human mortality. (
The invention provides Replikin sequences within the identified Replikin Peak Gene gene or gene segment (gene area) for diagnostic, preventive and therapeutic applications. For example, each Replikin sequence identified within an identified RPG is available for diagnostic and therapeutic applications including vaccines and antibody therapies. The entire Replikin Peak Gene sequence or fragments thereof are likewise available for diagnostic, preventive, therapeutic and predictive applications. Further, the presence of the Replikin Peak Gene in an isolate of the virus is indicative of rapid replication. For each identified RPG, continuous, non-interrupted and overlapping Replikin sequences have been identified for predictive and therapeutic applications. The size of a Replikin Peak Gene or Replikin Peak Gene Area, both in terms of the number of amino acids and the Replikin Count, will depend upon the size of the sequence of the entire genome, protein or fragment thereof that has been isolated and reported.
The invention further provides Replikin sequences within the identified Replikin Peak Gene or Replikin Peak Gene Area that are conserved in the genome over time and, as such, are available as relatively invariant targets for diagnosis and manipulation of rapid replication and virulence in EIV.
Point mutations within an RPG provide excellent predictive capacity when the point mutation is correlated with high virulence and provide an excellent target for attack of the virus through antibody therapies, vaccines and other treatments, as well as excellent predictive capacity when such point mutations are identified in emerging strains of the virus.
A further aspect of the invention provides utilizing software that searches for Replikin Peak Genes and enables the discovery of the point or points in the genome that have the highest concentration of Replikins, the years in which they have occurred, the strain or strains in which they occur, the host or hosts in which they occur, the geographic locations in which they occur, their increase or decrease in the above years, strains, hosts and geographic locations and point or small mutations that are correlatable with virulence.
The in silico detection of the Replikin Peak Gene by software methodology now permits both host and geographic localization of upregulated Replikin gene activity both in viruses and in other organisms. As seen in this study, the annual RPG Replikin analysis, by its correlation with a function such as epidemic outbreak or increase in lethality, can for the first time actually provide evidence for the correlation with the function.
The Replikin count in the whole genome or RPG make possible the prediction in advance of epidemic outbreaks of high mortality infections, such as those caused by influenza viruses, as seen for H5N1 in
It may be concluded that Replikins represent a specific class of peptides that are widely distributed, conserved, quantitative markers of lethality. While not wishing to be bound by theory, evidence from the apparent transfer of conserved Replikin structures between strains suggest they may be mobile agents of lethality, transferring horizontally between carrier viruses to reach multicellular hosts, where they may replicate rapidly with lethal consequences. As newly recognized targets for prevention and therapy, Replikins offer a platform from which specifically to control rapid replication and lethality of organisms and cells, without necessarily destroying them.
G. Conserved Replikins in PCV for Diagnostics and Therapies
In review of the publicly available sequences for Porcine Circovirus, the applicants have identified three Replikin sequences from Accession No. ABQ 10608 that are conserved across many isolates from 1997 or 1998 through 2007: kngrsgpqphk (SEQ ID NO: 345); hlqgfanfvkkqtfnk (SEQ ID NO: 346) and kkqtfnkvkwylgarch (SEQ ID NO: 347). Because these sequences are conserved, they have predictive value and provide novel and preferred targets for diagnostic and therapeutic applications such as, for example, vaccines. Furthermore, two of these sequences, hlqgfanfvkkqtfnk (SEQ ID NO: 346) and kkqtfnkvkwylgarch (SEQ ID NO: 347) are contained within the identified RPG of Accession No. ABQ 10608. These sequences, therefore, are of preferred value in predicting virulent strains when such strains contain the sequences. Also, the sequences provide preferred targets for diagnostic and therapeutic applications such as, for example, vaccines. Table 15 provides the accession numbers of isolates of PCV between 1997 and 2007 containing the conserved sequence kngrsgpqphk (SEQ ID NO: 345) and the amino acid position within the PCV protein sequence wherein the conserved Replikin sequence begins.
Table 16 provides the accession numbers of PCV isolates between 1997 and 2007 containing the conserved sequence hlqgfanfvkkqtfnk (SEQ ID NO: 346) and the amino acid position within the PCV protein sequence wherein the conserved Replikin sequence begins.
Table 17 provides the accession numbers of PCV isolates between 1998 and 2007 containing the conserved sequence kkqtfnkvkwylgarch (SEQ ID NO: 347) and the amino acid position within the PCV protein sequence wherein the conserved Replikin sequence begins.
XII. Relationship between Replikin Peak Gene and Lethality in Tobacco Mosaic Virus and Lung Malignancy
As established above, the Replikin Peak Gene correlates with activity of viruses such as pandemic influenza, Bird Flu, west nile virus and Bird Flu H5N1, among many others. It has surprisingly now been discovered that the highest activity to date of the Replikin Peak Gene was found in lung cancer (SEQ ID NO: 1741). Although viruses have been amply confirmed to be associated with the causation of several cancers since the work of Rous in sarcoma at the beginning of the last century, and viruses are the basis of current anti-cancer vaccines, how viruses are related to cancer is still not well understood. The antimalignin antibody in serum (AMAS) test is an FDA-permitted Medicare-approved early detection method for cancer that measures production of antibody against peptides containing a key Replikin sequence, namely, the glioma Replikin peptide, kagvaflhkk (SEQ ID NO: 3658), but how AMAS detects cancer regardless of cell type has not been fully understood. Results from separate studies in the areas of viruses and cancer now have converged with the isolation by the inventors of Replikins in both viruses and cancers that are concentrated in proteins where the concentration of Replikins has been related to rapid replication.
Higher Replikin Counts in RPGs have now been associated consistently with a higher percent lethality in the host; whether the host is a plant, fish, shrimp, or vertebrate, including human cases of H5N1 bird flu. The increase in count has frequently been detected one year or more before outbreaks have become clinically apparent. In addition to the correlation of high counts with virulence and lethality, structures specific to Replikins have been found by the inventors. For example, a 29-amino acid Replikin scaffold (beginning with SEQ ID NO: 3672) conserved for 90 years, appeared in the genome of successive influenza virus strains and each of the lethal pandemics and lethal H5N1 outbreaks. Additionally, repeating specific Replikin sequence signatures in RPGs of certain pathogens and malignancies have been identified and correlated with lethality. For example, an identical signature (SEQ ID NO: 1584) was found to repeat eleven times in the RPG of protozoan P. falciparum, 20 times in the RPG of tobacco mosaic virus which included exacerbated cell death in a pepper plant, with exacerbated cell death induced by Tobacco Mosaic virus, and 57 times (by overlapping) within 52 Replikins in the 18 amino acid RPG of non-small cell lung carcinoma.
While the inventors do not wish to be bound by theory, both of the above two studies support the impression that Replikins are mobile agents of lethality. Pathogenic viruses may just be the carrier of the lethal mobile agents. The highest Replikin Count in a Replikin Peak Gene that has to date been observed in highly lethal non-small cell lung cancer. The Replikin Count was observed to be 289 Replikin sequences per 100 amino acids. Other cancers, such as breast and ovarian have likewise been observed to have very high Replikin Counts in their Replikin Peak Genes with counts above 40 Replikin sequences per 100 amino acids. An RPG was identified and a Replikin Count of 129 was observed in Accession No. EAW84344 in lymphoblastic leukemia. An RPG was likewise identified and a Replikin Count of 23 was observed in Accession No. EAX09769 in myeloid leukemia.
Replikins chemically synthesized in the laboratory were found experimentally to be immuno-stimulants, producing strong antibody responses in chickens and rabbits. It appears that the antibodies measured in the AMAS test are against the Replikins' chemistry of rapid replication rather than the histological diagnosis of cancer or the cell type. Thus, for example, histologically proven prostate cancer that is “quiescent” (over 90% of such cancers) has low antibody levels in the AMAS test. But when these cells replicate rapidly, antibody levels measured by the AMAS test increase markedly. AMAS warning frequently precedes detection of the production of Prostate-Specific Antigen (PSA), an antigen that is frequently assayed because of its relationship to prostate cancer. AMAS probably precedes PSA because PSA measures protein fragments, the antigens that must be released by the cancer cells into the blood, while AMAS measures antibody to the peptide changes in the cancer cells, an earlier detectable event.
Since humans are host to and inhabited by thousands of viruses and bacteria that live symbiotically within the body, unless some event like rapid replication creates disease, no pathogenesis exists. Therefore, it may be important to learn how to control symbiosis between host humans and symbiotic viruses and bacteria without necessarily aiming to destroy the organism outright, especially when destruction proves difficult.
Peptides isolated from cancer cells grown in tissue culture have been found to contain Replikin sequences. When stimulated by anoxia, the cell number in these tissue cultures increased five-fold per week. Surprisingly, however, Replikin sequence concentration increased ten-fold per week (twice that of cell number), demonstrating a correlation of Replikin count with rapid replication in cancer tissue culture. When the structure of these Replikin-containing peptides was determined, separately synthesized chemically, and administered to rabbits, the peptides produced specific antimalignin antibodies in abundance. The production of antimalignin antibodies in response to the Replikin-containing peptides provided evidence to close the circle of confirmation that AMAS is measuring Replikins activity in malignancy.
In addition to early detection by the AMAS test of the activity of the group of Replikins that are unique to cancer, Replikins are widely distributed markers of, and probably agents of, lethality. As newly recognized targets for prevention and therapy, Replikins offer a platform from which to control rapid replication and lethality of organisms and cells, without necessarily destroying them.
XIII. Replikin Count Correlates with Virulence and Lethality in Shrimp Taura Syndrome Virus
Applicants have likewise demonstrated in a blind study using an independent laboratory testing taura syndrome virus (TSV) in shrimp that virulence and mortality in shrimp correlates with Replikin Count in TSV. The inventors analyzed the genome of the TSV of four main isolates from Hawaii, Belize, Thailand and Venezuela to provide predictions ranking the virulence and mortality rate of each isolate. An independent laboratory tested each isolate in shrimp and provided blind data on mortality. The data demonstrate a quantitative linear correlation between Replikin concentration and mortality. See Example 18. Despite differences in epidemiology, virology and host, all of these data lend further support for the value of Replikin concentration in predicting outbreaks of pathogens and lethality of pathogens and malignancies.
XIV. Replikin Concentration in Replikin Peak Gene of Ribonucleotide Reductase Gene Area Correlated with a WSSV Epidemic
An increase in Replikin concentration in white spot syndrome virus (WSSV) is predictive of an increase in virulence of the virus and allows for prediction of forthcoming outbreaks or increases in morbidity and, in extreme cases, mortality. A review of publicly available amino acid sequences of isolates of WSSV that demonstrate an increase in Replikin Count in the genome or a genome segment, or in a protein or protein fragment of the virus over time or between isolates is used as a predictor of an increase in outbreaks in shrimp. Publicly available sequences for isolates of WSSV from PubMed or other public or private sources may be analyzed by hand or using proprietary search tool software (ReplikinForecast™ available in the United States from REPLIKINS LLC, Boston, Mass.).
Applicants have established a correlation between Replikin concentrations in WSSV and an increase in virulence of the virus resulting in epidemics. Applicants reviewed publicly available amino acid sequences of isolates of WSSV having accession numbers at www.pubmed.com and have identified a remarkable increase in Replikin concentration in the Replikin Peak Gene of the ribonucleotide reductase gene area of the genome of the virus (e.g., SEQ ID NO: 669). The remarkable increase occurred just prior to a significant outbreak of WSSV in shrimp in 2001.
A. Analysis of Annual Replikin Count of WSSV
Applicants analyzed publicly available sequences for isolates of WSSV from PubMed. The data is contained in Table 18 and graphically described in
B. Prediction and Treatment of WSSV Outbreaks
Prediction of epidemics and future outbreaks may be made, for example, by reviewing the Replikin Counts of isolates of WSSV and comparing the Replikin Count for a particular year with Replikin Counts from other years. A significant increase in Replikin Count from one year to the next and preferably over one, two, three or five years or more provides predictive value of an emerging strain of WSSV that may begin an outbreak of more highly virulent WSSV. A WSSV outbreak may be predicted within about six months to about one year, to about three, to about five years or more from the observation of a significant increase in Replikin concentration. The outbreak is preferably predicted within about one to about three years and more preferably within about one to about two years. An outbreak of WSSV, therefore, may be predicted within 1 to about 2 years as demonstrated in
Significant increases may be observed over a time period of more than one year, such as three, four, five or more years. An outbreak may likewise be predicted within about six months to about one year or more from the initial observation of an observable decrease in Replikin concentration following a notable increase.
The correlation between Replikin concentration and viral outbreaks noted above provide a method of predicting outbreaks of WSSV by monitoring increases or decreases in Replikin Count in the RPG of isolates of WSSV. The method may employ isolates of individual strains or isolates of all strains of WSSV.
XV. Replikin Count in TSV Epidemic
An increase in Replikin concentration in taura syndrome virus (TSV) is predictive of an increase in virulence and lethality of the virus and allows for prediction of forthcoming outbreaks or increases in lethality.
The TSV is less virulent than WSSV and the structure of the TSV Replikin Scaffold is less closely related to influenza virus than are the structures of WSSV Replikin Scaffolds.
XVI. Software
A further aspect of the invention provides utilizing software that searches for Replikin Peak Genes and enables the discovery of the point or points in the genome that have the highest concentration of Replikins, the years in which they have occurred, the strain or strains in which they occur, the host or hosts in which they occur, the geographic locations in which they occur, their increase or decrease in the above years, strains, hosts and geographic location and point or small mutations that are correlatable with virulence.
XVII. SARS Replikin Concentration Correlates with Epidemics
An increase in Replikin concentration in coronaviruses also correlates with the SARS coronavirus epidemic. In particular, as may be seen in
XVIII. Passive Immunity
In another aspect of the invention, isolated Replikin peptides may be used to generate antibodies, which may be used, for example to provide passive immunity in an individual. Various procedures known in the art may be used for the production of antibodies to Replikin sequences. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that are linked to a cytotoxic agent may also be generated. Antibodies may also be administered in combination with an antiviral agent. Furthermore, combinations of antibodies to different Replikins may be administered as an antibody cocktail.
Monoclonal antibodies to Replikins may be prepared by using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) or other techniques may be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Replikin-specific single chain antibodies.
Antibodies to any peptides observed to be present in an emerging or re-emerging strain of virus and combinations of such antibodies are useful in the treatment and/or prevention of viral infection, especially RPG peptides and Replikin sequences isolated within RPG peptides.
Antibody fragments that contain binding sites for a Replikin may be generated by known techniques. For example, such fragments include but are not limited to F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries can be generated (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
In another aspect of the invention, immune serum containing antibodies to one or more Replikins obtained from an individual exposed to one or more Replikins may be used to induce passive immunity in another individual or animal. Immune serum may be administered via i.v. to a subject in need of treatment. Passive immunity also can be achieved by injecting a recipient with preformed antibodies to one or more Replikins. Passive immunization may be used to provide immediate protection to individuals who have been exposed to an infectious organism. Administration of immune serum or preformed antibodies is routine and the skilled practitioner can readily ascertain the amount of serum or antibodies needed to achieve the desired effect. One of the reasons that vaccines directed towards a particular protein antigen of a disease causing agent have not been fully effective in providing protection against the disease (such as foot and mouth vaccine which has been developed against the VP1 protein or large segments of the VP1 protein) is that the best antibodies have not been produced, that is—it is likely that the antibodies to the Replikins have not been produced.
For example, either epitopes other than Replikins present in the larger protein fragments may interfere according to the phenomenon of antigenic primacy and/or because the hydrolysis of larger protein sequences into smaller sequences for processing to produce antibodies results in loss of integrity of any Replikin structure that is present, e.g., the Replikin is cut in two and/or the histidine residue is lost in the hydrolytic processing. The present studies suggest that for a more effective vaccine to be produced, the Replikin sequences, and no other epitope, should be used as the vaccine. For example, a vaccine of the invention can be generated using any one of the Replikin peptides identified by the three-point recognition system. A more preferred vaccine comprises at least one Replikin sequence isolated in an RPG. Another preferred vaccine comprises an RPG peptide. Among the preferred Replikin peptides for use in a virus vaccine are those conserved Replikins observed to “re-emerge” after an absence from the amino acid sequence for one or more years.
The Replikin peptides of the invention, alone or in various combinations are administered to a subject, preferably by i.v. or intramuscular injection, in order to stimulate the immune system of the subject to produce antibodies to the peptide. Generally the dosage of peptides is in the range of from about 0.1 μg to about 10 mg. In another embodiment, the dosage of the peptides is in the range from about 10 μg to about 1 mg. In a preferred embodiment, the dosage of the peptides is in the range from about 50 μg to about 500 μg. The skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.
XIX. A Control Test of Reliability of Method of Predicting Outbreaks with Replikin Count
Table 3, which contains H5N1 data above, provides Replikin Count data across eight gene areas and an increased correlation is observed between mortality data and the whole virus, the polymerase gene and the pB1 gene area (Replikin Peak Gene). See also, e.g.,
In Table 3 the structures that are correlated have, to the knowledge of the Applicants, not been correlated before, that is, the inventors have examined the relationship of one internal virus structure to another internal virus structure or structures (e.g., three-way relationship between whole virus gene area, polymerase and Replikin Peak Gene area) and have examined the external relation of these two or more internal structures to a host result of the virus infection, that is, percent mortality.
Table 3 represents consistent reproducible data, on repeated trials, which is the essence of the reliability of any method. For example, Table 3 provides independent data on (1) whole virus concentration of Replikins, (2) just polymerase concentration of Replikins, and (3) just the Replikin Peak Gene concentration of Replikins. The data is then correlated with H5N1 mortality three times, namely in 2003, 2004 and 2005. The absence of significant changes in the pA and pB2 gene areas provides a control. In each case, the method measures Replikin concentration three ways, each of which correctly predict mortality, independently, thereby confirming the method, and further illustrating in the process, the magnifying function of the Replikin Peak Gene.
The inventors queried Accession No. ABB54523 at www.pubmed.com. Accession No. ABB54523 discloses the amino acid sequence of SEQ ID NO: 1664, deduced from the genomic information of an H3N2 strain of Influenza A virus isolated in 1968 in Memphis. Upon analysis of SEQ ID NO: 1664, the inventors observed a Replikin Peak Gene having continuous Replikin sequences beginning at residue 15 (histidine) and continuing through residue 85 (lysine) (SEQ ID NO: 1665).
The inventors isolated the RPG (SEQ ID NO: 1665) in silico. SEQ ID NO: 1665 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of influenza. Seventeen Replikin sequences (SEQ ID NOS: 1667-1682) were identified in the RPG of SEQ ID NO: 1665 for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 1667-1674 were identified in the amino-terminal of the sequence disclosed in Accession No. ABB54523 (SEQ ID NO: 1664), SEQ ID NOS: 1675-1682 were identified in the mid-molecule of the sequence.
The Replikin Count of the amino acid sequence (SEQ ID NO: 1664) disclosed at ABB54523 was seventeen Replikin sequences in 90 total amino acids for a Replikin Count of 18.9. The Replikin Count of the RPG (SEQ ID NO: 1665) was seventeen Replikin sequences in 71 total amino acids for a Replikin Count of 23.9.
The inventors queried Accession No. BAE07199 at www.pubmed.com. Accession No. BAE07199 discloses an amino acid sequence deduced from the genomic information of the RNA polymerase gene of an H5N1 strain of Influenza A virus isolated in 2003 in Hong Kong. The inventors analyzed the whole pB1 gene area (SEQ ID NO: 1683) of the polymerase sequence. Upon analysis of SEQ ID NO: 1683, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 168 (lysine) and continue through residue 215 (lysine).
The inventors isolated the RPG (SEQ ID NO: 1684) in silico. SEQ ID NO: 1684 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of influenza. Seven Replikin sequences (SEQ ID NOS: 1685-1691) were identified in the RPG of SEQ ID NO: 1684 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 1685-1691 were identified in the amino-terminal of the sequence disclosed in Accession No. BAE07199 (SEQ ID NO: 1683), Replikin sequences SEQ ID NOS: 1692-1694 were identified in the mid-molecule of the sequence, and Replikin sequence SEQ ID NOS: 1695-1699 were identified in the carboxy-terminal of the sequence.
The Replikin Count of the whole pB1 area sequence (SEQ ID NO: 1683) was 15 Replikin sequences in 757 total amino acids for a Replikin Count of 2.0. The Replikin Count of the RPG (SEQ ID NO: 1684) was seven Replikin sequences in 48 total amino acids for a Replikin Count 14.6.
The inventors queried Accession No. ABI36257 at www.pubmed.com. Accession No. ABI36257 discloses an amino acid sequence deduced from the genomic information of the pB1 gene area of an H5N1 strain of Influenza A virus isolated in 2006 from Indonesia. The inventors analyzed the pB1-F2 gene area (SEQ ID NO: 1700). Upon analysis of SEQ ID NO: 1700, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 15 (histidine) and continue through residue 85 (lysine) (SEQ ID NO: 1701).
The inventors isolated the RPG (SEQ ID NO: 1701) in silico for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of influenza. Sixteen Replikin sequences (SEQ ID NOS: 1702-1717) were identified in the RPG of SEQ ID NO: 1701 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 1702-1703 were identified in the amino-terminal of the sequence of SEQ ID NO: 1701, Replikin sequences SEQ ID NOS: 1704-1717 were identified in the mid-molecule of the sequence, and no Replikin sequences were identified in the carboxy-terminal.
The Replikin Count of the whole pB1-F2 gene area sequence (SEQ ID NO: 1700) was 16 Replikin sequences in 90 total amino acids for a Replikin Count of 17.8. The Replikin Count of the RPG pB1-F2 subgene area (SEQ ID NO: 1701) was 16 Replikin sequences in 71 total amino acids for a Replikin Count 22.57.
The inventors queried Accession No. ABM90520 at www.pubmed.com. Accession No. ABM90520 discloses an amino acid sequence deduced from the genomic information of the pB1 gene area of an H5N1 strain of Influenza A virus isolated in 2007 in Indonesia. The inventors analyzed the pB1 gene area (SEQ ID NO: 1722). Upon analysis of SEQ ID NO: 1722, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 15 (histidine) and continue through residue 85 (lysine) in the pB1-F2 gene area (SEQ ID NO: 1723)
The inventors isolated the RPG (SEQ ID NO: 1723) in silico. SEQ ID NO: 1723 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of influenza. Sixteen Replikin sequences (SEQ ID NOS: 1724-1739) were identified in the RPG (or pB1-F2 gene subarea) of SEQ ID NO: 1723 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 1724-1725 were identified in the amino-terminal of the sequence of SEQ ID NO: 1723, Replikin sequences SEQ ID NOS: 1726-1739 were identified in the mid-molecule of the sequence, and no Replikin sequences were identified in the carboxy-terminal.
The Replikin Count of the whole pB1-F2 area sequence (SEQ ID NO: 1722) was 16 Replikin sequences in 90 total amino acids for a Replikin Count of 17.8. The Replikin Count of the RPG (SEQ ID NO: 1723) was 16 Replikin sequences in 71 total amino acids for a Replikin Count 22.5.
The inventors queried Accession No. ABS71678 at www.pubmed.com. Accession No. ABS71678 discloses an amino acid sequence deduced from the genomic information of the hemagglutinin gene area of an H1N1 strain of Influenza A virus isolated in 2007 in Thailand. The inventors analyzed the amino acid sequence provided at ABS71678 (SEQ ID NO: 1995). Upon analysis of SEQ ID NO: 1995, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 143 (histidine) and continue through residue 235 (lysine) (SEQ ID NO: 1996). A Replikin Scaffold, knglypnlsksyannkekevlvlwgvhh (SEQ ID NO: 2011) was observed within the RPG.
The inventors isolated the RPG (SEQ ID NO: 1996) in silico. SEQ ID NO: 1996 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of influenza. Twenty-six Replikin sequences (SEQ ID NOS: 1999-2024) were identified in the RPG of SEQ ID NO: 1996 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 1997-2016 were identified in the amino-terminal of the sequence of SEQ ID NO: 1995, Replikin sequences SEQ ID NOS: 2017-2029 were identified in the mid-molecule of the sequence, and SEQ ID NOS: 2030-2042 were identified in the carboxy-terminal. The Replikin sequences were isolated for diagnostic, therapeutic and predictive uses.
The Replikin Count of the whole hemagglutinin sequence (SEQ ID NO: 1995) was 46 Replikin sequences in 564 total amino acids for a Replikin Count of 8.1. The Replikin Count of the RPG area (SEQ ID NO: 1996) was 26 Replikin sequences in 93 total amino acids for a Replikin Count of 28.
Applicants reviewed Replikin sequences publicly available at www.pubmed.com to determine the Replikin Peak Gene Area of available isolates. A Replikin Peak Gene was identified in the pB1-F2 gene area of the virus in Accession No. ABS89395 at www.pubmed.com. The following example provides determination of the Replikin Peak Gene in a 2005 isolate of a Maryland strain of H3N8 serotype Influenza A virus.
The inventors queried Accession No. ABS89395 and analyzed the amino acid sequence provided (SEQ ID NO: 545). Upon analysis of the sequence, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 15 (histidine) and continue through residue 85 (lysine) (SEQ ID NO: 546).
The inventors isolated the RPG (SEQ ID NO: 546) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of tuberculosis. Sixteen Replikin sequences (SEQ ID NOS: 547-562) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 547-548 were identified in the amino-terminal of the sequence, Replikin sequences SEQ ID NOS: 549-562 were identified in the mid-molecule of the sequence, and no Replikins were identified in the carboxy-terminal.
The Replikin Count of the whole pB1-F2 sequence (SEQ ID NO: 545) was 16 Replikin sequences in 90 total amino acids for a Replikin Count of 17.8. The Replikin Count of the RPG area (SEQ ID NO: 546) was 16 Replikin sequences in 71 total amino acids for a Replikin Count of 22.5.
Applicants reviewed Replikin sequences publicly available at www.pubmed.com to determine the Replikin Peak Gene of an available West Nile Virus (WNV) isolate. The entire envelope protein of WNV was reported at Accession No. ABA54585. A Replikin Peak Gene was identified in the 3,433 amino acid polyprotein sequence of the WNV envelope protein. A Replikin Peak Gene was identified beginning at amino acid residue 2797 extending through amino acid residue 2836 (a total of 40 amino acid residues). The number of Replikin sequences in this section was 12. The Replikin Count (Replikins per 100 amino acids) was 30. The Replikin Peak Gene (RPG) of the envelope protein of WNV is SEQ ID NO: 258 and the RPG contains 12 uninterrupted Replikins (SEQ ID NOS: 246-257).
Applicants reviewed Replikin sequences publicly available at www.pubmed.com to determine the Replikin Peak Gene Area of available PRRSV isolates. A Replikin Peak Gene was identified in Accession No. AA043261 from mRNA encoding a reported nucleocapsid protein of a PRRSV isolate from Mexico in 2003. The inventors analyzed the amino acid sequence provided in Accession No. AA043261, which is reported with 123 amino acids within ORF 7 of the virus genome. Upon analysis of the sequence, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 7 (lysine) and continue through residue 66 (histidine).
The inventors isolated the RPG (SEQ ID NO: 394) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PRRSV. Seven Replikin sequences (SEQ ID NOS: 395-401) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NO: 395 was identified in the amino-terminal portion of the sequence and SEQ ID NOS: 396-401 were identified in the mid-molecule portion of the sequence.
The Replikin Count of the whole nucleocapsid sequence at Accession No. AA043261 was 7 Replikin sequence in 123 amino acid residues or 5.7. The Replikin Count of the RPG area (SEQ ID NO: 394) was 7 Replikin sequences in 60 total amino acids for a Replikin Count of 11.7.
The asparagine and methionine residues at positions 45 and 46 of the RPG (SEQ ID NO: 394) were identified by the inventors as non-conserved positions within the RPG as compared to other reported nucleocapsid sequences such as Accession No. ABF19568 discussed immediately below. Non-conserved positions within an RPG that are correlated with changes in lethality and/or virulence are particularly useful in methods of the invention to predict outbreaks. The presence of these point mutations in other PRRSV nucleocapsid RPG sequences provides evidence of greater virulence and/or lethality.
A Replikin Peak Gene was also identified in Accession No. ABF19568 from a nucleic acid sequence of a PRRSV 2006 isolate from Mexico. The reported sequence has 99 amino acid residues. The RPG (SEQ ID NO: 402) was isolated in silico and identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PRRSV. The total length of the RPG is 29 amino acids. The Replikin Count is 41.4. The Replikin sequences of SEQ ID NOS: 403-414 were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 403-414 were identified in the amino-terminal of the sequence. No Replikin sequences were identified in the mid-molecule or carboxy-terminus.
The glycine, proline and glycine residues at positions 14 through 16 and the asparagine, arginine, lysine, arginine and asparagine residues at positions 21 through 25 were identified within the RPG (SEQ ID NO: 507) as non-conserved positions as compared to other reported nucleocapsid sequences such as Accession No. AA043261 above. Further, as compared to the RPG in Accession No. AA043261 above, the RPG identified in the 2006 Mexico isolate demonstrates a shortening of the RPG and noteworthy condensation of Replikin sequences within the shorter RPG. The result is a remarkable increase in Replikin Count between 2003 and 2006 corresponding to a severe outbreak of PRRSV in Mexico in 2006 with an increase in mortality rate.
Applicants likewise analyzed Accession Nos. AAM18565, AAP81809, ABL60920 having sequences of isolates from China in 2000, 2003, and 2006, respectively, to determine the Replikin Peak Gene of the isolates (SEQ ID NOS: 341, 342, and 343).
A Replikin Peak Gene was identified in Accession No. AAM18565 between residue 7 (lysine) and residue 66 (histidine). The inventors isolated the RPG (SEQ ID NO: 353) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PRRSV. Thirteen Replikin sequences (SEQ ID NOS: 354-366) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 354-357 were identified in the amino-terminal portion of the sequence and SEQ ID NOS: 358-366 were identified in the mid-molecule portion of the sequence.
The Replikin Count of the whole sequence at Accession No. AAM18565 was 13 Replikin sequences within 123 amino acid residues or 10.6. The Replikin Count of the RPG area (SEQ ID NO: 353) was 13 Replikin sequences in 60 total amino acids for a Replikin Count of 21.7.
A Replikin Peak Gene was identified in Accession No. AAP81809 between residue 7 (lysine) and residue 66 (histidine). The inventors isolated the RPG (SEQ ID NO: 367) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PRRSV. Thirteen Replikin sequences (SEQ ID NOS: 368-380) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 368-371 were identified in the amino-terminal portion of the sequence and SEQ ID NOS: 372-380 were identified in the mid-molecule portion of the sequence.
The Replikin Count of the whole sequence at Accession No. AAP81809 was 13 Replikin sequences within 123 amino acid residues or 10.6. The Replikin Count of the RPG area (SEQ ID NO: 367) was 13 Replikin sequences in 60 total amino acids for a Replikin Count of 21.7.
A Replikin Peak Gene was identified in Accession No. ABL60920 between residue 7 (lysine) and residue 66 (histidine). The inventors isolated the RPG (SEQ ID NO: 382) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PRRSV. Ten Replikin sequences (SEQ ID NOS: 384-393) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 384-387 were identified in the amino-terminal portion of the sequence and SEQ ID NOS: 388-393 were identified in the mid-molecule portion of the sequence.
The Replikin Count of the whole sequence at Accession No. ABL60920 was 10 Replikin sequences within 123 amino acid residues or 8.1. The Replikin Count of the RPG area (SEQ ID NO: 367) was 10 Replikin sequences in 60 total amino acids for a Replikin Count of 16.7.
Applicants reviewed Replikin sequences publicly available at www.pubmed.com to determine the Replikin Peak Gene of available isolates of PCV. The inventors identified and compared a Replikin Peak Gene (RPG) of a protein fragment at Accession No. AAC59472 of a strain of PCV isolated from infected pigs in Manitoba, Canada in 1997 and a RPG of a putative truncated replicase protein at Accession No. ABP68657 of a strain of PCV isolated from infected pigs in China in 2007. The AAC59472 fragment was identified from nucleic acid encoding a predicted 1.8 kDa protein in open reading frame 11 of the isolate. The ABP68657 putative truncated replicase protein was identified in open reading frame 1 of the isolate.
In Accession No. AAC59472, the inventors identified an RPG (SEQ ID NO: 520) for diagnostic, therapeutic and predictive purposes as described herein. The RPG begins at residue 2 (lysine) and continues through residue 12 (lysine). Four Replikin sequences (SEQ ID NOS: 521-524) were identified for diagnostic, therapeutic and predictive uses as described in herein. The total length of the RPG is 11 amino acids. The Replikin Count is 36.4. The Replikin Count of the entire fragment is four Replikin sequences in fourteen amino acids or 28.6.
In Accession No. ABP68657, the inventors identified an RPG (SEQ ID NO: 525) for diagnostic, therapeutic and predictive purposes as described herein. Thirteen Replikin sequences (SEQ ID NOS: 526-538) were identified for diagnostic, therapeutic and predictive uses as described in herein. The total length of the RPG is 38 amino acids. The Replikin Count is 34.2. The Replikin Count of the entire putative truncated protein is 6.2.
The reported sequence at Accession No. AAC59472 has only 14 amino acid residues. Nevertheless, the high concentration of continuous, non-interrupted and overlapping Replikin sequences within the RPG (Replikin Count 36.4) is a predictor of virulence and provides sequences available as vaccines. In comparison, the RPG of the truncated replicase protein reported at Accession No. ABP68657 has 306 amino acid residues but the identified RPG has 13 Replikin sequences and a comparable Replikin Count of 34.2, which is likewise a predictor of virulence and provides sequences available as vaccines. Likewise, the high Replikin Count RPGs provide a target for production of immunogenic compounds for treatment and prevention of PCV.
A Replikin Peak Gene was identified in an isolate of PCV from 1997 publicly available at Accession No. AAC9885 between residues 4 (lysine) and 99 (histidine). The inventors isolated the RPG (SEQ ID NO: 421) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PCV. Fourteen Replikin sequences (SEQ ID NOS: 422-435) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 422-435 were identified in the amino-terminal portion of the whole sequence disclosed at the accession number and SEQ ID NOS: 436-437 were identified in the mid-molecule portion of the sequence. No Replikin sequences were identified in the carboxy-portion of the sequence.
The Replikin Count of the whole sequence at Accession No. AAC9885 was 16 Replikin sequences within 312 amino acid residues or 5.1. The Replikin Count of the RPG area (SEQ ID NO: 421) was 14 Replikin sequences in 96 total amino acid residues for a Replikin Count of 14.6.
A Replikin Peak Gene was identified in an isolate of PCV from 2001 publicly available at Accession No. AAL01075 between residues 57 (histidine) and 94 (lysine). The inventors isolated the RPG (SEQ ID NO: 438) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PCV. Twelve Replikin sequences (SEQ ID NOS: 439-450) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 439-445 were identified in the amino-terminal portion of the whole sequence disclosed at the accession number and SEQ ID NOS: 446-450 were identified in the mid-molecule portion of the sequence. No Replikin sequences were identified in the carboxy-portion of the sequence.
The Replikin Count of the whole sequence at Accession No. AAC9885 was 12 Replikin sequences within 314 amino acid residues or 3.8. The Replikin Count of the RPG area (SEQ ID NO: 438) was 12 Replikin sequences in 90 total amino acids for a Replikin Count of 13.3.
A Replikin Peak Gene was identified in an isolate of PCV from Canada in 2007 that is publicly available at Accession No. ABP68657. The RPG was identified between residues 57 (histidine) and 94 (lysine). The inventors isolated the RPG (SEQ ID NO: 462) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PCV. Fourteen Replikin sequences (SEQ ID NOS: 462-476) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 462-476 were identified in the amino-terminal portion of the whole sequence disclosed at the accession number and SEQ ID NOS: 477-481 were identified in the mid-molecule portion of the sequence. No Replikin sequences were identified in the carboxy-portion of the sequence.
The Replikin Count of the whole sequence at Accession No. ABP68657 was 19 Replikin sequences within 306 amino acid residues or 6.2. The Replikin Count of the RPG area (SEQ ID NO: 462) was 14 Replikin sequences in 38 total amino acids for a Replikin Count of 36.8.
In Applicants' review of RPGs in publicly available PCV sequences, the inventors identified an RPG from Accession No. ABQ 10608 that contained each of the highly conserved Replikin sequences discussed in Section XI.G. above, namely, kngrsgpqphk (SEQ ID NO: 345); hlqgfanfvkkqtfnk (SEQ ID NO: 346) and kkqtfnkvkwylgarch (SEQ ID NO: 347).
The RPG was identified between residues 57 (histidine) and 94 (lysine). The inventors isolated the RPG (SEQ ID NO: 498) in silico and identified the sequence for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for outbreaks of PCV. Six Replikin sequences (SEQ ID NOS: 487-492) were identified in the RPG for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 486-492 were identified in the amino-terminal portion of the whole sequence disclosed at the accession number and SEQ ID NOS: 493-497 were identified in the mid-molecule portion of the sequence for therapeutic, diagnostic and predictive purposes. No Replikin sequences were identified in the carboxy-portion of the sequence.
The Replikin Count of the whole sequence at Accession No. ABQ10608 was 12 Replikin sequences within 314 amino acid residues or 3.8. The Replikin Count of the RPG area (SEQ ID NO: 498) was six Replikin sequences in 38 total amino acids or 15.8.
The inventors queried Accession No. AAS59518 at www.pubmed.com. Accession No. AAS59518 discloses an amino acid sequence from Mycobacterium mucogenicum strain CIP 105384. The inventors analyzed the amino acid sequence provided at AAS59518 (SEQ ID NO: 2901). Upon analysis of SEQ ID NO: 2901, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 3 (histidine) and continue through residue 88 (histidine) (SEQ ID NO: 3649).
The inventors isolated the RPG (SEQ ID NO: 3659) in silico for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of tuberculosis. Twenty-four Replikin sequences (SEQ ID NOS: 2902-2925) were identified in the RPG of SEQ ID NO: 3659 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 2902-2924 were identified in the amino-terminal of the sequence of SEQ ID NO: 2901, Replikin sequences SEQ ID NO: 2925 was identified in the mid-molecule of the sequence, and no Replikins were identified in the carboxy-terminal. All were isolated for diagnostic, therapeutic and predictive purposes.
The Replikin Count of the whole hemagglutinin sequence (SEQ ID NO: 2901) was 24 Replikin sequences in 147 total amino acids for a Replikin Count of 16.3. The Replikin Count of the RPG area (SEQ ID NO: 3659) was 24 Replikin sequences in 87 total amino acids for a Replikin Count of 27.6.
Replikin concentration was determined for ribonucleotide reductase of a white spot syndrome virus (WSSV) isolate publicly available at Accession No. AAL89390. The amino acid sequence was translated from the total genome of a 2000 isolate of WSSV made publicly available at Accession No. NC 003225.1. The Replikin concentration in the protein was an unusually high 103.8 and the Replikin concentration of the Replikin Peak Gene of the protein was a yet higher 110.7.
The amino acid sequence of the protein publicly available at Accession No. AAL89390 is of particular interest because it demonstrates an overlapping of Replikin sequences that results in very high Replikin concentrations comparable to P. falciparum. The high concentrations of Replikin sequences provide a reservoir for transfer to influenza viruses.
In Accession No. AAL89390, SEQ ID NO: 668 is disclosed as a ribonucleotide reductase protein of white spot syndrome virus. Within SEQ ID NO: 668, the inventors identified a Replikin Peak Gene (SEQ ID NO: 669). The Replikin Peak Gene is observed to occupy most of the disclosed protein of SEQ ID NO: 668. The expansiveness of the Replikin Peak Gene across most of the amino acid sequence of the protein is highly unusual and creates a remarkably high Replikin concentration.
Replikin Count of SEQ ID NO: 668 was determined by dividing the number of Replikin sequences identified in the amino acid sequence of the protein, 497 Replikin sequences, by the total amino acid length of the protein, 479 amino acids, to arrive at 103.8 Replikin sequences per 100 amino acids. The Replikin Count of the RPG of SEQ ID NO: 669 was determined by dividing the number of Replikin sequences identified in the segment of the protein containing the highest concentration of continuous Replikin sequences, 497 Replikin sequences, by the total amino acid length Replikin Peak Gene, 449 amino acids, to arrive at 110.7.
Within the RPG of SEQ ID NO: 669, SEQ ID NOS: 670-1166 were identified as Replikin sequences. SEQ ID NOS: 669-866 were identified in the amino-terminus of the peptide, SEQ ID NOS: 867-1065 were identified in the middle portion, and SEQ ID NOS: 1066-1166 were identified in the carboxy-terminus.
SEQ ID NO: 669 was further observed to contain significant Replikin Scaffold sequences. SEQ ID NOS: 663-667 were identified as Replikin Scaffold repeats and were isolated for diagnostic, therapeutic and predictive uses.
Replikin Count was determined for a functionally undefined protein in the genome of a 2000 isolate WSSV at Accession No. NP 478030 (SEQ ID NO: 1167). The Replikin Count in the protein was again an unusually high 97.6 Replikin sequences per 100 amino acids determined by dividing the number of Replikin sequences identified in the amino acid sequence of the protein, 361 Replikin sequences, by the total amino acid length of the protein, 370 amino acids.
An RPG (SEQ ID NO: 1168) was identified within SEQ ID NO: 1167 between residues 22 (histidine) and 361 (lysine) and is available for diagnostic, therapeutic and predictive uses as described herein. Total Replikin sequences identified in the RPG were 361 with total amino acid residues of 361, for a Replikin Count in the RPG of 100. SEQ ID NOS: 1169-1330 were identified in the amino-terminus of the RPG. SEQ ID NOS: 1331-1465 were identified in the mid-molecule of the RPG and SEQ ID NOS: 1466-1529 were identified in the carboxy-terminus of the RPG. Each Replikin sequence is available for diagnostic, therapeutic and predictive purposes as described herein.
The amino acid sequence of Accession No. NP 478030 is of interest because, like the protein in Accession No. AAL89390, it demonstrates an overlapping of Replikin sequences that results in very high Replikin concentrations comparable to the highly-replicating P. falciparum of malaria. Overlapping Replikin sequences are exceptional targets for therapies such as immunogenic agents and vaccines and have excellent predictive capacities.
In 2006 and 2007 WSSV has been observed to be dormant in shrimp. This continued decline of WSSV into “quiescent” or “dormant” levels in 2006-2007 is demonstrated in mean Replikin Counts for viruses isolated during 2005-2007 that are very low as compared to years wherein the virus demonstrated greater virulence, such as 2001. The continued quiescence in WSSV in 2007 may be contrasted with an observed rising of Replikin concentration in taura syndrome virus Replikin during this period.
As may be seen from the analysis below, Accession Nos. ABS00973 and AAW88445 have low observed Replikin concentrations. ABS00973 contains a single Replikin sequence (SEQ ID NO: 1548) in the entire disclosed amino acid sequence of 240 residues at SEQ ID NO: 1547. The Replikin concentration of Accession No. ABS00973 is an inordinately low 0.5. AAW88445 contains a white spot syndrome virus protein of 261 amino acid residues (SEQ ID NO: 1530). An RPG of 34-105 was identified (SEQ ID NO: 1531). Within the RPG, eleven Replikin sequences were identified (SEQ ID NOS: 1532-1542). SEQ ID NOS: 1532-1542 were identified in the amino-terminus of SEQ ID NO: 1530 and SEQ ID NOS: 1543-1546 were identified in the carboxy-terminus of SEQ ID NO: 1530.
The inventors queried Accession No. AAM73766 at www.pubmed.com. Accession No. AAM73766 discloses an amino acid sequence from a 2005 isolate of TSV (SEQ ID NO: 3566). Applicants identified SEQ ID NOS: 3567-3569 as Replikin sequences in the amino-terminus of the sequence and SEQ ID NOS: 3570-3573 as Replikin sequences in the carboxy-terminus of the sequence. Each sequence was isolated in silico for diagnostic, therapeutic and predictive purposes as described herein. No Replikin sequence was identified in the mid-molecule. The Replikin Count of SEQ ID NO: 3566 was seven Replikin sequences in 1011 amino acid residues or 0.7.
The inventors queried Accession No. AAY89096 at www.pubmed.com. Accession No. AAY89096 discloses an amino acid sequence from a 2005 isolate of TSV (SEQ ID NO: 3574). Applicants identified SEQ ID NOS: 3575-3587 in the amino-terminus of the sequence. SEQ ID NOS: 3588-3634 were identified as Replikin sequences in the mid-molecule. And SEQ ID NOS: 3635-3657 were identified as Replikin sequences in the carboxy-terminus of the sequence. Each sequence was isolated in silico for diagnostic, therapeutic and predictive purposes as described herein. Replikin Count of SEQ ID NO: 3574 was 83 Replikin sequences in 2107 amino acid residues or 3.9.
The inventors queried www.pubmed.com with the software program FluForecast® available from Replikins LLC of Boston, Mass. to analyze all amino acid sequences from the pB1-F2 gene area of all isolates of Influenza A available between 2002 and 2007. Table 20 provides the results of the query. The data for mean Replikin count for 2005, 2006 and 2007 suggest that the current epidemic is not over. For example, the SARS data in
The inventors queried www.pubmed.com with the software program FluForecast® available from Replikins LLC of Boston, Mass. to analyze all amino acid sequences of all isolates of H1N1 Influenza A available between 1917 and 2007. Table 21 provides the results of the query.
Applicants analyzed publicly available sequences for isolates of EIV from PubMed using proprietary search tool software (ReplikinForecast™ available in the United States from REPLIKINS LLC, Boston, Mass.). The data is contained in Table 22, below, and Table 4, above, and graphically described in
Table 22 provides the data for Replikin concentration for publicly available sequences of the pB1 gene area of the H3N8 strain of influenza virus from 1963 to 2005. Sequences were publicly available under accession numbers at www.pubmed.com. Standard deviation and significance as compared to the mean Replikin Count of the previous year and of the lowest mean Replikin Count within the data set are also provided along with the mean Replikin Count for each year. Where data was not available in a given year the year is not
As may be seen in Table 22 over the 42 year period for which sequence information is publicly available for H3N8 isolates, the cyclic nature of changes in Replikin concentration becomes evident. Where Replikin concentrations reach a high within the cycle, an epidemic occurs within about 1 to about 2 years. For example, a high of 22.2 Replikin sequences per 100 amino acids in 1979 falls to 2 Replikin sequences per 100 amino acids in 1998 and 1999 with no epidemics reported between 1995 and 2001. The Replikin concentration then appears to be on its way up again in 2001 with epidemics following in the United Kingdom and in Germany in 2002 and 2003, respectively, and then falls back to around 2 in 2003 and 2004 with a marked increase in 2005 to 18.5 that approaches the highs of 1979. Epidemics follow the 2005 increase in Australia, Italy and Japan in 2007.
Table 23 provides the data for Replikin concentration for publicly available sequences of the pB2 gene of the H3N8 strain of influenza virus from 1963 to 2005. Sequences were available under accession numbers at www.pubmed.com. Standard deviation and significance as compared to the mean Replikin Count of the previous year and of the lowest mean Replikin Count within the data set are also provided along with the mean Replikin Count for each year
A review of the Replikin concentrations of available sequences for the pB2 gene area of the H3N8 strain of influenza virus reveals much less variability in the Replikin concentration through the years. The pB2 Replikin concentration can be considered control data that validate the location of the most significant Replikin Peak Gene for the present isolates of virus in the pB1 gene area. Because the pB2 gene is right next to the pB1 gene, the difference in variability in Replikin Count between these neighboring areas is remarkable.
Data from a review of the Replikin Count of available sequences for the pA gene area of the H3N8 strain of influenza virus may be seen in
Applicants analyzed publicly available sequences for isolates of EIV from years 1942 to 2007 and determined the mean whole genome Replikin Count for all isolates having genomic sequences in each year for which they were available.
A list of the accession numbers analyzed by FluForecast® (REPLIKINS LLC, Boston, Mass.) for the presence and concentration of Replikin sequences is provided in Table 24 below. The mean Replikin concentration for each year is provided following the list of accession numbers from isolates in each corresponding year. Standard deviation and significance as compared to the mean Replikin concentration of the previous year and of the lowest mean Replikin concentration within the data set are also provided along with the mean Replikin concentration for each year.
Applicants analyzed publicly available sequences for isolates of PCV from www.pubmed.com using proprietary search tool software (ReplikinForecast™ available in the United States from REPLIKINS LLC, Boston, Mass.) from years 1997 to 2007 and determined the mean Replikin Count for all isolates in each of years 1997 through 2007. Applicants then compared the mean Replikin Count for each year with qualitative changes in infection rates and mortality in pigs in Canada.
A list of the accession numbers analyzed for the presence and concentration of Replikin sequences is provided in Table 25 below. The mean Replikin Count for each year is provided following the list of accession numbers from isolates in each corresponding year. Standard deviation and significance as compared to the mean Replikin Count of the previous year and of the lowest mean Replikin Count within the data set are also provided along with the mean Replikin Count for each year.
Publicly available amino acid sequences at Accession Nos: Q9NS56 and 117607067, for non-small lung cancer and tobacco mosaic virus, respectively, were analyzed for Replikin Peak Genes. The inventors queried Accession No. Q9NS56 at www.pubmed.com. Accession No. Q9NS56 discloses the amino acid sequence of E3 ubiquitin-protein ligase Topors from human chromosome 9 of non-small cell lung cancer (SEQ ID NO: 1740). Upon analysis of SEQ ID NO: 1740, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 880 (lysine) and continue through residue 897 (histidine).
The inventors isolated the RPG (SEQ ID NO: 1741) in silico. SEQ ID NO: 1741 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethality. Fifty-two Replikin sequences (SEQ ID NOS: 1886-1937) were identified in the RPG of SEQ ID NO: 1741 for diagnostic, therapeutic and predictive uses as described herein. SEQ ID NOS: 1742-1747 were identified in the amino-terminal of the sequence disclosed in Accession No. Q9NS56 (SEQ ID NO: 1741), SEQ ID NOS: 1748-1780 were identified in the mid-molecule of the sequence, and SEQ ID NOS: 1781-1885 were identified in the carboxy-terminal of the sequence.
The Replikin Count of the amino acid sequence (SEQ ID NO: 1740) disclosed at Q9NS56 was 144 Replikin sequences in 1045 total amino acids for a Replikin Count of 13.8. The Replikin Count of the RPG (SEQ ID NO: 1741) was 52 Replikin sequences in 18 total amino acids for a Replikin Count of 289, the highest count yet observed.
Within the Replikin sequences identified in the RPG (SEQ ID NO: 1741), the KHKK signature was observed 57 times within 52 Replikin sequences. This high concentration of lethal signatures corresponds to the high lethality of non-small cell lung malignancies.
The inventors queried Accession No. 117607067 at www.pubmed.com. Accession No. 117607067 discloses the amino acid sequence of a hot pepper 26S proteasome subunit RPN7 induced by tobacco mosaic virus (SEQ ID NO: 1938). Upon analysis of SEQ ID NO: 1938, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 91 (histidine) and continue through residue 175 (lysine).
The inventors isolated the RPG (SEQ ID NO: 1939) in silico. SEQ ID NO: 1939 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of tobacco mosaic virus. Fifty-four Replikin sequences (SEQ ID NOS: 1941-1994) were identified in the RPG of SEQ ID NO: 1939 for diagnostic, therapeutic and predictive uses as described herein.
SEQ ID NO: 1941 was identified in the amino-terminal of the sequence disclosed in Accession No. 117607067 (SEQ ID NO: 1938), SEQ ID NOS: 1942-1986 were identified in the mid-molecule of the sequence, and SEQ ID NOS: 1987-1994 were identified in the carboxy-terminal of the sequence. Each Replikin sequence was isolated in silico for diagnostic, therapeutic and predictive purposes as described herein including for immunogenic compositions and vaccines.
The Replikin Count of the amino acid sequence (SEQ ID NO: 1938) disclosed at Accession No. 117607067 was 55 Replikin sequences in 179 total amino acids for a Replikin Count of 30.7. The Replikin Count of the RPG (SEQ ID NO: 1939) was 54 Replikin sequences in 89 total amino acids for a Replikin Count of 61.
Within the Replikin sequences identified in 117607067 (SEQ ID NO: 1938), the KHKK (SEQ ID NO: 1584) signature was observed twenty times within 61 Replikin sequences. This high concentration of lethal signatures corresponds to the high lethality of tobacco mosaic virus and connects tobacco mosaic virus through KHKK (SEQ ID NO: 1584) signatures to lethal lung cancer.
As discussed above, repeating signatures such as a “KHKK” (SEQ ID NO: 1584) signature have been observed in Replikin sequences within RPGs of lethal malignancies, viruses and organisms. The KHKK (SEQ ID NO: 1584) signature has been observed eleven times within the RPG of the protozoa that causes most malaria, P. falciparum, 20 times within the RPG of tobacco mosaic virus, which caused exacerbated cell death induced by tobacco mosaic virus, and 57 times in non-small cell lung carcinoma within 52 Replikins observed within the 18 amino acid RPG identified in chromosome 9 of a non-small cell lung carcinoma. The presence of such a high number of KHKK (SEQ ID NO: 1584) signatures within the 18 amino acid RPG of the non-small cell lung carcinoma is explained by overlapping of the signatures. Overlapping of Replikin sequences and repeated signatures such as KHKK (SEQ ID NO: 1584) has now been associated with lethality, virulence and rapid replication. Together, these data indicate that a Replikin gene is quantitatively associated with lethal functions, and may be a mobile agent of lethality transferring between strains and species.
The inventors queried Accession No. P13817 at www.pubmed.com. Accession No. P13817 discloses an amino acid sequence from Plasmodium falciparum. The inventors analyzed the amino acid sequence provided at P13817 (SEQ ID NO: 2043). Upon analysis of SEQ ID NO: 2043, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 323 (histidine) and continue through residue 473 (lysine) (SEQ ID NO: 3659).
The inventors isolated the RPG (SEQ ID NO: 3659) in silico. SEQ ID NO: 3659 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of malaria. Two hundred and thirty-one Replikin sequences (SEQ ID NOS: 2312-2315 and 2317-2544) were identified in the RPG of SEQ ID NO: 3659 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 2044-2077 were identified in the amino-terminal of the sequence of SEQ ID NO: 2043, Replikin sequences SEQ ID NOS: 2079-2080 were identified in the mid-molecule of the sequence, and Replikin sequence SEQ ID NOS: 2081-2315 were identified in the carboxy-terminal.
The Replikin Count of the whole sequence (SEQ ID NO: 2043) was 268 Replikin sequences in 473 total amino acids for a Replikin Count of 56.7. The Replikin Count of the RPG area (SEQ ID NO: 3659) was 231 Replikin sequences in 151 total amino acids for a Replikin Count of 153.
The inventors queried Accession No. A44396 at www.pubmed.com. Accession No. A44396 discloses an amino acid sequence from an ATP-ase-like molecule of P. falciparum isolated in 1993. The inventors analyzed the amino acid sequence provided at A44396 (SEQ ID NO: 2926). Upon analysis of SEQ ID NO: 2926, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 1297 (histidine) and continue through residue 1333 (histidine).
The inventors isolated the RPG (SEQ ID NO: 3661) in silico. SEQ ID NO: 3661 was identified for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of malaria. Seventeen Replikin sequences (SEQ ID NOS: 3282-3285, 3287-3291, 3293, 3295, 3299-3300, 3302, 3304, 3306, 3308, 3310-3313 and 3663) were identified in the RPG of SEQ ID NO: 3661 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 2546-2632 were identified in the amino-terminal of the sequence of SEQ ID NO: 2926, Replikin sequences SEQ ID NO: 2633-2720 were identified in the mid-molecule of the sequence, and SEQ ID NOS: 2721-2900 were identified in the carboxy-terminus.
The Replikin Count of the whole ATP-ase sequence (SEQ ID NO: 2926) was 355 Replikin sequences in 1984 total amino acids for a Replikin Count of 17.9. The Replikin Count of the RPG area (SEQ ID NO: 3661) was 15 Replikin sequences in 37 total amino acids for a Replikin Count of 41.
Eleven signature repeat KHKK (SEQ ID NO: 1584) sequences were noted in the Replikin sequences of the RPG. The eleven signature repeats, namely, SEQ ID NOS: 3286, 3292, 3294, 3296, 3298, 3662, 3301, 3303, 3305, 3307, and 3309 are respectfully found within noted Replikin sequences SEQ ID NOS: 3286, 3291, 3293, 3295, 3297, 3299, 3300, 3302, 3204, and 3206.
The presence of such a high number of KHKK (SEQ ID NO: 1584) signatures within the fifteen Replikin sequences in the 37 amino acid RPG of the P. falciparum is explained by overlapping of the signatures. Overlapping of Replikin sequences and repeated signatures such as KHKK (SEQ ID NO: 1584) has now been associated with lethality, virulence and rapid replication as in malaria, which has an exceptionally high rate of replication within its lifecycle.
To test further the relationship of Replikins to virulence, the relationship of Replikin count of shrimp viruses to mortality in shrimp was examined in a controlled situation. Based on the hypothesis that the Replikin count of a virus is related to virulence of the virus and the percent mortality of the host, as developed from the evidence on H5N1 virus infections in humans, Applicants tested whether it would be possible to predict solely from the Replikin Count of the amino acid sequence of the whole genome what the order of virulence would be of four strains of the virus. Taura syndrome shrimp virus (TSV), which kills most host shrimp within a few days of infection, was chosen to be studied. The amino acid sequences of four strains of taura syndrome virus (Belize, Thailand, Hawaii, and Venezuela) were analyzed with the FluForecast® software of REPLIKINS LLC, Boston, Mass. and the results held in confidence until the laboratory challenge experiments with the virus were completed, then compared with the percent mortality produced by each strain.
In the laboratory, there was a significant linear correlation between the mortality rates of the host shrimp challenged with each of the four virus strains and the mortality rates predicted earlier by only the Replikin counts of each strain. These data support the conclusion that virus Replikin peptide concentration, in addition to predicting virus outbreaks, relates quantitatively to host mortality rate and to the increase in virulence over time observed.
A. Replikin Analysis
Visual Replikin analysis was performed on the sequence information for the taura syndrome virus isolates from Belize, Thailand, Hawaii, and Venezuela by applying the algorithm defining Replikins with computer access to protein and genomic sequences freely available on PubMed or other public databases. The specific defining algorithm follows: a Replikin is a peptide sequence in a protein or genome, 7 to 50 amino acids long having a terminal lysine and a terminal lysine or histidine, containing at least 2 lysine groups 6 to 10 amino acids apart, at least 1 histidine group, and at least 6% lysine. Overlapping Replikins are common and are counted separately. The quantitative correlations with rapid replication and epidemics and lethality require all components of the algorithm to be in place for each Replikin. Thus for example, if the length and lysine requirements are present but there is no histidine present, the peptide is not a Replikin. Automated Replikin analysis was performed with the FluForecast® software service of Replikins Ltd., Boston, Mass.
B. Identification of the Replikin Peak Gene
The Replikin count was used to identify that area of the genome which had the highest concentration of Replikins, and this area called the Replikin Peak Gene (RPG) area. The further two to eight-fold increase in the Replikin count of the RPG which occurred with outbreaks was further used to confirm the identity of this gene. The function of the gene was therefore used to identify it or isolate it “in silico”.
C. Shrimp Virus Laboratory Methods
At the Aquaculture Pathology Laboratory, Department of Veterinary Science and Microbiology, University of Arizona, Tucson Ariz., small juveniles of specific-pathogen-free Litopenaeus vannamei shrimp per tank, mean weight: 1.8 g, were fed minced TSV-infected tissues (infected separately with each of the 4 isolates originating from Belize, Thailand, Venezuela and Hawaii) for 3 days at 5% of their body weight. These shrimp were maintained with pelleted ration (Rangen 35%) for the following 12 days. Each challenge bioassay of a specific isolate was done in triplicate. During the bioassay period, all tanks were checked daily for dead or moribund shrimp. All mortalities were removed from the tank and frozen. One to three moribund shrimp from each isolate were preserved in Davidson's AFA fixative and processed for routine histology to confirm viral infection. For each isolate, six moribund shrimp were collected during the acute phase infection and total RNA was extracted from their gill tissues with a High Pure RNA tissue kit (Roche). The extracted RNA was analyzed for the presence of TSV by real-time RT-PCR. All tanks were outfitted with an acclimated biological filter and aeration, and were covered with plastic to contain aerosols. The average salinity of the water was 23 ppt and the water temperature was 28° C. The challenge study was terminated after 15 days with live animals counted as survivors.
D. Comparison of Virulence
First mortality was seen on day 2 after exposure to TSV in all 4 isolates. For Belize isolate, most (83%) of shrimp died by day 4 and had a 0% survival at day 11 (
The correlation of the virulence observed for each of the TSV isolates with the predicted virulence by Replikin Count alone are shown in
1TSV lesions = Presence of TSV pathognomonic lesions in the gills, mouth, stomach, intecumental cuticular epithelium, and appendages.
2LOS = presence of lymphoid organ spheroids within the lymphoid organ.
Belize TSV: Acute lesions of diagnostic TSV infection were found in one representative shrimp sample (06-407J/1) at a severity grade of G4. Nuclear pyknosis and karyorrhexis were observed in the cuticular epithelium of the general body surface, appendages, gills, stomach and esophagus. Lymphoid organ spheroids were also found at severity grade G4. Thailand TSV: Severe (G4) TSV infection was detected in 2 out 3 shrimp (06-407D/1, F/1), another shrimp (06-407E/1) showed a moderate to high grade (G3) of infection. Lymphoid organ spheroids were found at severities of G2 and G3. Hawaii TSV: Moderate level (G2) of TSV infection was detected in 2 shrimp (06-407A/1, C/1) collected at day 4. Lymphoid organ spheroids were found at severities of G3 and G4. Venezuela TSV: Severe (G4) TSV infection was detected in one representative shrimp (06-407H/1) sampled at day 4. Lymphoid organ spheroids were found at severity of G2.
The real-time TSV RT-PCR assay was designed specifically for Hawaii TSV and thus a high level (107 copies/μl RNA) of TSV was detected in the Hawaii-TSV challenged shrimp Table 28). The target sequence in 3 other isolates has 2 mis-matched nucleotides with the primers/TaqMan probe. Thus, there is 10 times less quantity of TSV (106 copies/μl RNA) detected in Belize and Thailand samples. The Venezuela samples were detected with 100-100,000 times less: 102-105 copies/μl RNA; this may be due to both the effect of mismatches and a lower level of infection in the samples analyzed. Nevertheless, all 24 samples (6 from each isolates) were all positive for TSV infection. This confirms that the mortalities observed from bioassays are from TSV infection. The real-time TSV RT-PCR assay data is found below in the Table 28.
E. Laboratory Mortality Results Correlated With Replikin Counts
Virulence of 4 TSV isolates (Hawaii, Belize, Thailand and Venezuela) was compared through a per os laboratory infection in juvenile Litopenaeus vannamei (Kona stock, Oceanic Institute, Hawaii). The results showed that the Belize isolate is the most virulent, Thailand is the second, followed by the Hawaii isolate, and the Venezuela isolate is the least virulent. This is based on the analyses of cumulative survivals at the end of the bioassay (p<0.047) and the time when 50% mortality was occurred (p<0.001). That the mortality of the shrimp was caused by TSV infection was confirmed by positive reactions in RT-PCR detection and by the appearance of characteristic lesions observed in histological analysis
F. Laboratory Mortality Results Correlated With Replikin Counts
Experimentally, Replikin Counts alone prospectively correctly predicted: (1) blind in controlled experiments in the laboratory, the order of lethality in shrimp of four strains of taura syndrome virus (
Shrimp cultured using the Challenge Methods described in Example 18 above were exposed in a first experiment for two weeks to synthetic Replikins per os mixed in their feed. The Replikins were peptides specific to Replikin sequences present in the TSV Hawaii strain isolate with which the shrimp were challenged.
In the experiment, mortality was reduced by 50% compared to a control group. The control group was given feed not containing synthetic Replikin sequences. A second control group was fed Replikin sequences synthesized with the covalent binding of additional amino acids to the same synthetic Replikins fed to the shrimp. The covalently “blocked” Replikins did not increase shrimp resistance to the virus in the same experiment demonstrating that the increase in host resistance was specific to the Replikin peptide structure.
Because little is known about the details of the immune system of the shrimp (shrimp appear not to produce antibodies), the phenomenon of “resistance” to infection appears to be based in a “primitive immune system” perhaps similar to the “toll receptor” and related systems. Thus the term “increased resistance” is used for the observed phenomenon and Replikin feed is used rather than “vaccine” for the administered substance which increases resistance.
The surviving shrimp of the first challenge were then set up in a fresh culture, fed for an additional two weeks with feed containing Replikin sequences, then again challenged with the Hawaii strain of taura syndrome virus. The Replikin sequence supplemented feed was maintained while the survivors were again challenged repeatedly by the same virus, in repeated cycles, until 100% of the shrimp survived the TSV challenge.
The inventors queried Accession No. ABQ42711 at www.pubmed.com. Accession No. ABQ42711 discloses an amino acid sequence from a glycoprotein in hemorrhagic septicemia virus. Hemorrhagic septicemia virus is a cause of hemorrhagic disease in fish. The inventors analyzed the amino acid sequence provided at ABQ42711 (SEQ ID NO: 3787). Upon analysis, the inventors observed a Replikin Peak Gene having continuous Replikin sequences that begin at residue 81 (histidine) and continue through residue 204 (histidine).
The inventors isolated the RPG in silico for diagnostic and therapeutic uses in, for example, an immunogenic compound and a therapeutic vaccine compound and as a predictive sequence for lethal outbreaks of hemorrhagic disease in fish. Thirty-six Replikin sequences (SEQ ID NOS: 3788-3823) were identified in SEQ ID NO: 3787 for diagnostic, therapeutic and predictive uses as described herein. Replikin sequences SEQ ID NOS: 3788-3795 were identified in the amino-terminal, Replikin sequences SEQ ID NOS: 3796-3815 were identified in the mid-molecule of the sequence, and Replikin sequences 3816-3823 were identified in the carboxy-terminal. All were isolated for diagnostic, therapeutic and predictive purposes.
The Replikin Count of the whole sequence (SEQ ID NO: 3787) was 36 Replikin sequences in 222 total amino acids for a Replikin Count of 16. The highest Replikin Count of an identified RPG area in hemorrhagic septicemia virus was 73 Replikin sequences in 123 total amino acids for a Replikin Count of 59.
The inventors queried publicly available sequences from isolates of hemorrhagic viral disease syndrome in fish from 1990 through 2007. The following table provides the accession numbers queried. The highest Replikin Count of an identified RPG area in hemorrhagic septicemia virus was 73 Replikin sequences in 123 total amino acids for a Replikin Count of 59.
The inventors queried all sequences for hemorrhagic viral disease in fish publicly available at www.pubmed.com between 1990 and 2007 Using FluForecast® (Replikins LLC, Boston, Mass.), the inventors determined the mean Replikin Count in each year from 1990-2007. The data is provided in Table 29. The table does not included years in which no data was available.
The instant application contains a “lengthy” Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. Said CD-R, recorded on Jan. 17, 2008, are labeled CRF, “Copy 1” and “Copy 2”, respectively, and each contains only one identical 1.36 Mb file (27129301.txt). This application claims priority to U.S. Provisional Appln. Ser. No. 60/991,676, filed Nov. 30, 2007, U.S. application Ser. No. 11/923,559, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No. 60/982,336, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No. 60/982,333, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No. 60/982,338, filed Oct. 24, 2007, U.S. Provisional Appln. Ser. No. 60/935,816, filed Aug. 31, 2007, U.S. Provisional Appln. Ser. No. 60/935,499 filed Aug. 16, 2007, U.S. Provisional Appln. Ser. No. 60/954,743, filed Aug. 8, 2007, U.S. application Ser. No. 11/755,597, filed May 30, 2007, U.S. Provisional Appln. Ser. No. 60/898,097, filed Jan. 30, 2007, and U.S. Provisional Appln. Ser. No. 60/880,966, filed Jan. 18, 2007, each of which is incorporated herein by reference in its entirety. This application additionally incorporates herein by reference: U.S. Provisional Appln. Ser. No. 60/853,744, filed Oct. 24, 2006, U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006, U.S. application Ser. No. 11/116,203, filed Apr. 28, 2005, U.S. application Ser. No. 10/860,050, filed Jun. 4, 2004, U.S. application Ser. No. 10/189,437, filed Jul. 8, 2002, U.S. application Ser. No. 10/105,232, filed Mar. 26, 2002, now U.S. Pat. No. 7,189,800, U.S. application Ser. No. 09/984,057, filed Oct. 26, 2001, and U.S. application Ser. No. 09/984,056, filed Oct. 26, 2001, now U.S. Pat. No. 7,176,275, each in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60991676 | Nov 2007 | US | |
| 60982336 | Oct 2007 | US | |
| 60982333 | Oct 2007 | US | |
| 60982338 | Oct 2007 | US | |
| 60935816 | Aug 2007 | US | |
| 60935499 | Aug 2007 | US | |
| 60954743 | Aug 2007 | US | |
| 60898097 | Jan 2007 | US | |
| 60880966 | Jan 2007 | US |
