Patent Application
20090269367
In surveys of global health, infectious disease often accounts for as many as five of the top ten causes of death in lower- and middle-income countries and respiratory infections are often assigned as the fourth leading cause of death in higher-income countries. Further, pathogenic outbreaks and pandemics continue to threaten human populations from previously unknown or otherwise mutated pathogenic diseases. Previously unknown or otherwise mutated pathogenic diseases often occur when a pathogen diverges from an established host, such as pigs or chickens, into a new host, such as humans. In view of this phenomenon, new strategies are continually needed for mitigating pathogenic outbreaks from previously-known or previously-unknown pathogens. Several such threatening pathogenic diseases include malaria, influenza, West Nile virus, foot-and-mouth disease, and other threats to global health in both humans and animals. Therapies or methods of treatment that are useful across different pathogenic strains or even across pathogenic groups are especially helpful in improving the fight against mutable pathogenic disease and outbreaks of previously-unknown pathogens.
Among the most threatening of global infectious diseases is malaria. Malaria kills a million or more people each year in tropical and sub-tropical environments. Malaria is most commonly and seriously caused by the trypanosome Plasmodium falciparum, which is reportedly responsible for ninety percent of malarial deaths. The majority of death from malarial infection is recorded in young children. P. falciparum is vectored by female Anopheles mosquitoes. Once in the blood stream of a human, the trypanosome multiplies rapidly within red blood cells causing anemia, flu-like symptoms, and sometimes coma and death. Partly effective vaccines are only now beginning to be marketed for malaria and no wholly-effective vaccine has yet been registered for sale in industrialized countries. As such, there continues to be a need in the art for improved methods of predicting and identifying increases in virulence, morbidity, and mortality in and from malaria.
Another threat to public health is West Nile virus (WNV), which causes encephalitis and other serious neuroinvasive diseases in a small percentage of human infections. In about four percent of reported cases, the resulting neuroinvasive disease results in death. WNV is flaviviridae virus, first observed in North America in 1999 and now considered endemic in the United States. The virus is spread to humans through mosquito (and related insect) bites. 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 and for therapies for preventing, mitigating, and treating WNV infections.
Influenza is an acute respiratory illness of global importance. Virulent and lethal outbreaks of influenza continue to threaten world health. Researchers, government officials, and medical practitioners are increasingly aware of the continuing threat of a pandemic of virulent and lethal influenza requiring new methods of treatment and novel therapeutic compounds. Researchers, government officials, and medical practitioners likewise recognize that the continuing threat of pandemic influenza requires new and more effective methods of predicting and tracking lethal outbreaks of influenza.
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 infectious threat. 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 the discovery of Replikin chemistry in the virus genome structure, the only basis for annual classification of influenza virus as present or absent in a given year was identification by serological testing of the hemagglutinin and neuraminidase proteins in an isolate of virus. The activity of a strain of influenza was, therefore, only recorded after the fact of the occurrence of the outbreak, never in advance.
There is a continuing need in the art for quantitative methods of tracking and predicting increases in virulence and lethality of influenza prior to outbreaks. There is likewise a need in the art for quantitative 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.
Replikin peptides are a family of small peptides that have been correlated with the phenomenon of rapid replication in malaria, influenza, West Nile virus, foot and mouth disease, and many other pathogens. Replikin peptides have likewise been correlated with the phenomenon of rapid replication in viruses and organisms generally.
Identification of Replikin peptides has provided targets for detection and treatment of pathogens, including vaccine development against virulent pathogens such as malaria, influenza virus, West Nile virus, and foot and mouth disease virus. In general, knowledge of and identification of this family of peptides enables development of effective therapies and vaccines for any pathogen that harbors Replikins. 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. Pat. No. 7,442,761, and U.S. application Ser. No. 11/355,120. Both Replikin concentration (number of Replikins per 100 amino acids) and Replikin composition have been correlated with the functional phenomenon of rapid replication.
There continues to be a need in the art, however, for improved methods of predicting and identifying increases in virulence, morbidity, and lethality and expansions of and outbreaks of virulent pathogens. There is likewise a need in the art for improved methods of preventing and treating outbreaks and expansions of virulent pathogens using Replikin sequences identified with increases in virulence, morbidity, and lethality of expanding pathogenic populations.
The present invention provides a quantitative cyclic structure comprising Replikin peptide concentrations identified in a strain of microorganism through time, wherein said cyclic structure correlates in time with the expansion and/or contraction of a population of said strain of microorganism, the infectivity of said strain of microorganism, and/or the lethality of said strain of microorganism.
Further, the present invention provides methods of preventing, mitigating, and treating outbreaks of a pathogen comprising predicting an expansion of a population of a strain of pathogen or an increase in the virulence, morbidity, and/or lethality of a strain of pathogen as compared to another strain of the same or a related pathogen and administering to an animal or patient a compound comprising an isolated or synthesized portion of the structure or genome of the pathogen to mitigate, prevent, or treat the predicted outbreak of the pathogen.
The present invention further provides methods of predicting an expansion of a strain of pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen comprising identifying a cycle in the Replikin Count in a protein fragment, protein, genome fragment, or genome of a pathogen and predicting an increase in the virulence, morbidity, and/or mortality of said pathogen within the identified cycle in Replikin Count. The present invention further provides Replikin peptides identified within a pathogen predicted to be expanding or to have an increase in virulence, morbidity, and/or mortality as diagnostic, therapeutic, or preventive agents against an outbreak of the pathogen.
A first non-limiting aspect of the invention provides a method of preventing, mitigating, or treating an outbreak of a pathogen predicted to have an expansion of population comprising predicting an expansion of the population of a first pathogen comprising
A further embodiment of the first aspect of the invention provides a method of preventing, mitigating, or treating an outbreak of pathogen comprising
In a non-limiting embodiment of the first aspect of the invention, the pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, taura syndrome virus, white spot syndrome virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, Helicobacter pylori, Entamoeba invadens, L. legionella, S. aureus, maize streak virus, bovine herpes virus, feline immunodeficiency virus, human immunodeficiency virus, rous sarcoma virus, avian sarcoma virus, sindbis virus, hepatitis virus, b. anthracis, or any other infectious agent. In a non-limiting embodiment, the influenza virus is an H1N1, H2N2, H3N2, H5N1, H3N8, or H9N2 strain of influenza virus.
In a further non-limiting embodiment of the first aspect of the invention, said expansion of a strain of pathogen or increase in virulence, morbidity, and/or mortality of an isolate or plurality of isolates of a strain of pathogen comprises identifying a second cycle in the Replikin concentration of a plurality of isolates of a second strain of pathogen that shares synchrony with said first cycle in the Replikin concentration of said plurality of isolates of said first strain of pathogen and identifying a first peak in the Replikin concentration within the identified first cycle at a first time point or time period and identifying a first peak in the Replikin concentration within the identified second cycle of said second strain of pathogen at a second time point or time period that is similar to said first time point or time period and predicting an increase in the virulence of said first strain of pathogen following the first time point or time period. In a non-limiting embodiment, the pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or any other infectious agent.
In a non-limiting embodiment of the first aspect of the invention, said pathogen is an influenza virus. In a further non-limiting embodiment, said first strain of influenza is any strain different from said second strain of influenza. In another non-limiting embodiment, said first strain of influenza is H5N1 and said second strain of influenza is H9N2, or vice versa.
In a further non-limiting embodiment of the first aspect of the invention, said isolated or synthesized portion of the structure or genome of the at least one isolate of a pathogen is a protein or protein fragment comprising a Replikin peptide and/or a Replikin Peak Gene, a Replikin peptide identified within a Replikin Peak Gene, or any structure or portion of the structure of said pathogen. In another embodiment, said isolated or synthesized portion of the structure or genome is a nucleic acid encoding a Replikin Peak Gene, a Replikin peptide or a plurality of Replikin peptides within a Replikin Peak Gene, or a Replikin peptide or plurality of Replikin peptides.
In another non-limiting embodiment of the first aspect of the present invention, the second time point or time period is up to three years after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about one year after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about six months after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of a pathogen following the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of influenza following the first time point or first time period. In a further non-limiting embodiment, the next influenza season is the next winter season in a geographic region following the first time point or time period. In another non-limiting embodiment, the second time point or time period is the next season of malaria following the first time point or first time period. In a further non-limiting embodiment, the next season of malaria is the next rainy season. In another non-limiting embodiment, the second time point or time period is the next season of West Nile virus. In a further non-limiting embodiment, the next season of West Nile virus is a summer season.
In another non-limiting embodiment of the first aspect of the present invention, the identified peak in the cycle of Replikin concentration has a higher Replikin concentration than a chronologically earlier peak in the cycle of Replikin concentration. In a further non-limiting embodiment of the invention, the identified peak in the cycle of Replikin concentration is significantly higher than the earlier peak. In a further non-limiting embodiment the identified peak is significantly higher than the earlier peak with a p value less than 0.01. In a further non-limiting embodiment the identified peak is significantly higher than the earlier peak with a p value less than 0.001.
A second non-limiting aspect of the invention provides a method of predicting an expansion of the population of a first pathogen comprising identifying at least one cycle of Replikin concentration in isolates of the pathogen and predicting that an expansion of the population of the first pathogen will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration, wherein the at least one cycle is cycle A.
In a further embodiment of the second aspect of the invention, the rising portion comprises a peak wherein said expansion of the population of the first pathogen is predicted after the occurrence of the peak. In a further embodiment, the cycle comprises at least a first rising portion and a second rising portion, wherein said first rising portion occurs prior in time to said second rising portion. In a further embodiment, the cycle comprises at least three rising portions, wherein the at least three rising portions are at least rising portion A′, rising portion B′ and rising portion C′. In a further embodiment, the rising portion B′ comprises a peak and the rising portion A′ comprises a peak, and the peak of rising portion B′ has a greater Replikin concentration than the peak of rising portion A′. In a further non-limiting embodiment, the method of prediction further comprises processing the method on a computer. In a further non-limiting embodiment, the cycle comprises more than one cycle including, for example, from peak to trough to peak to trough or from trough to peak to trough to peak. In a further non-limiting embodiment, the cycle comprises three peaks or three troughs or more.
In a further embodiment of the second aspect of the present invention, the method of prediction comprises identifying at least one other cycle of Replikin concentration in isolates of at least one other strain of pathogen, wherein the at least one other cycle is cycle B, and wherein cycle B shares synchrony with cycle A; and predicting that an expansion of the population of the first pathogen will occur after the occurrence of a rising portion in cycle A that corresponds to a rising portion in cycle B. In a further embodiment, the first pathogen is a first strain of influenza virus and the one other pathogen is a different strain of influenza virus. In a further embodiment, the first pathogen is an H5N1 strain of influenza virus and the one other strain of pathogen is an H9N2 strain of influenza virus. In a further embodiment, the expansion of the population of the first pathogen is predicted within three years after the peak. In a further embodiment, the expansion of the population of the first pathogen is predicted within one year after said peak. In a further embodiment, the expansion of the population of the first pathogen is predicted after the next virulence season of the pathogen.
A further embodiment of the second aspect of the invention provides a method of predicting an expansion of a population of a pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen relative to the population or the virulence, morbidity, and/or mortality of another pathogen of the same species or of another pathogen of a related species comprising: (1) identifying a cycle in the Replikin concentration of isolates of a plurality of the pathogen, (2) identifying a first peak in the Replikin concentration of isolates of a plurality of said pathogen within the identified cycle at a first time point or time period, and (3) predicting an expansion of the population of a pathogen of the same or a related species or an increase in the virulence, morbidity, and/or mortality of a pathogen of the same or a related species isolated at a second time point or time period subsequent to the first time point or time period.
In a non-limiting embodiment of the second aspect of the invention, the pathogen may be, but is not limited to, a malarial trypanosome, West Nile virus, influenza virus, equine influenza virus, coronavirus, foot and mouth disease virus, taura syndrome virus, white spot syndrome virus, or other pathogen or infectious agent.
A non-limiting embodiment of the second aspect of the present invention, the pathogen is a malarial trypanosome. In another non-limiting embodiment, the trypanosome is P. falciparum, P. vivax, P. ovale, or P. malariae. In a further non-limiting embodiment, the trypanosome is P. falciparum. In a further non-limiting embodiment, the method predicts an increase in mortality from malarial infection.
In another embodiment of the second aspect of the present invention, the identified Replikin cycle represents Replikin concentrations identified in a histidine rich protein of P. falciparum. In another non-limiting embodiment of the present invention, the identified Replikin cycle represents Replikin concentrations identified in the histidine-rich protein of P. falciparum.
In another non-limiting embodiment of the second aspect of the present invention, the pathogen is a West Nile virus. In a further embodiment, the identified Replikin cycle represents concentration identified in the envelope protein of West Nile virus. In another non-limiting embodiment, the pathogen is a foot and mouth disease virus. In a further embodiment, the identified Replikin cycle represents concentrations identified in the VP1 protein of foot and mouth disease virus. In another non-limiting embodiment, the pathogen is an influenza virus. In a further embodiment, the identified Replikin cycle represents concentrations identified in the pB1 gene area of influenza virus. In another non-limiting embodiment, the influenza virus is an H1N1, H2N2, H3N2, H3N8, H5N1, or H9N2 strain of influenza virus.
In another non-limiting embodiment of the second aspect of the present invention, the second time point or time period is up to three years after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about one year after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about six months after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of a pathogen following the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of influenza following the first time point or first time period. In a further non-limiting embodiment, the next influenza season is the next winter season in a geographic region following the first time point or time period. In a further non-limiting embodiment, the second time point or time period is following the next dry season after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is the next season of malaria following the first time point or first time period. In a further non-limiting embodiment, the next season is the next rainy season.
In another non-limiting embodiment of the second aspect of the present invention, the identified peak in the cycle of Replikin concentration has a higher Replikin concentration than a chronologically earlier peak in the cycle of Replikin concentration. In a further non-limiting embodiment of the invention, the identified peak in the cycle of Replikin concentration is significantly higher than the earlier peak. In a further non-limiting embodiment the identified peak is significantly higher than the earlier peak with a p value less than 0.01. In a further non-limiting embodiment the identified peak is significantly higher than the earlier peak with a p value less than 0.001.
In a further non-limiting embodiment of the second aspect of the invention, predicting said expansion of population or said increase in virulence, morbidity, and/or mortality of an isolate of a pathogen comprises identifying a second cycle in the Replikin concentration of a plurality of isolates of a second strain or related strain of pathogen that shares synchrony with said first cycle in the Replikin concentration of said plurality of isolates of said first strain of pathogen and identifying a first peak in the Replikin concentration within the identified first cycle at a first time point or time period and identifying a first peak in the Replikin concentration within the identified second cycle of said second strain of pathogen or related strain of pathogen at a second time point or time period that is similar to said first time point or time period and predicting an expansion of the population or an increase in the virulence, morbidity, and/or mortality of said first strain of pathogen following the first time point or time period. In a non-limiting embodiment, the pathogen is a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or any other infectious agent.
In a non-limiting embodiment, said pathogen is an influenza virus. In a further non-limiting embodiment, said first strain of influenza is any strain different from said second strain of influenza. In another non-limiting embodiment, said first strain of influenza is H5N1 and said second strain of influenza is H9N2, or vice versa. In another embodiment, the strain is any influenza strain and the related strain is any other strain wherein a relationship with said first strain is determined by comparing the Replikin cycles of said strain and said related strain. In another embodiment, the strains are related because the Replikin cycles share synchrony.
A further non-limiting embodiment of the second aspect of the invention provides a method of predicting an expanding population of a pathogen or an increase in virulence, morbidity, and/or mortality in a pathogen comprising: (1) determining the mean Replikin Count in a plurality of isolates of at least two strains of pathogen at a plurality of successive time points; (2) comparing the mean Replikin Count at least four successive time points for each strain and identifying at least one cycle of increasing mean Replikin Counts over the at least four time points for each of the at least two strains; (3) identifying at least partial synchrony between the at least one cycle of increasing mean Replikin Counts for each of the at least two strains; and (4) predicting an increase in virulence following in time the increase in mean Replikin Count in the at least one cycle in said at least two strains wherein said at least one cycle in said at least two strains occurs at a corresponding time period. In a further non-limiting embodiment, step-wise cycles are identified between successive time points. In a further non-limiting embodiment, specific conserved Replikin sequences are identified within the step-wise cycles. In a further non-limiting embodiment, Replikin sequences are identified at the peak of a stepwise cycle. The Replikin sequences identified at the peak of a stepwise cycle are useful for developing a vaccine or therapeutic composition of an isolated or synthesized Replikin peptide for use in preventing or treating outbreaks of malaria with relatively higher mortality. In a further embodiment, the pathogen is influenza. In a further embodiment, the at least two strains of influenza are H9N2 and H5N1.
Another non-limiting embodiment of the second aspect of the invention provides a method of predicting a contraction or failure of a population of a strain of pathogen, wherein an isolate of said pathogen is isolated at a time point or time period subsequent to a decreasing portion of a Replikin cycle.
A further non-limiting embodiment of the second aspect of the invention provides a method for making a vaccine comprising predicting an expanding population of a pathogen or related strain of pathogen or an increase in virulence, morbidity, and/or mortality of a pathogen or a related strain of pathogen and identifying a portion of the structure or genome of said isolated influenza virus to be comprised in a vaccine.
A further non-limiting embodiment of the second aspect of the present invention provides an isolated or synthesized portion of the structure or genome of a pathogen wherein said pathogen is predicted to have an expansion of the population of the pathogen. In a further embodiment, the isolated or synthesized portion is a protein, protein fragment, or peptide comprising a Replikin peptide or a Replikin Peak Gene. In a further non-limiting embodiment, the isolated or synthesized portion of the structure or genome of a pathogen consists of one or more Replikin peptides and/or one or more Replikin Peak Genes. In a further non-limiting embodiment, the one or more Replikin peptides are conserved during a cycle in Replikin concentration at least two successive time points or time periods in the cycle.
Another non-limiting embodiment of the second aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified as conserved in an isolate of said pathogen from among a plurality of isolates of said pathogen, wherein said isolates are isolated during a cycle in Replikin concentration at least two successive time points or time periods, and the cycle preferably includes at least two peaks or two troughs.
In a further non-limiting embodiment of the second aspect of the invention, the pathogen is an influenza virus. In a further non-limiting embodiment, the Replikin peptide is at least one of HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1), KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4), HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12).
In a further non-limiting embodiment of the second aspect of the invention, the pathogen is a West Nile virus. In a further non-limiting embodiment, the Replikin peptide is at least one of KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK (SEQ ID NO: 15), or HNDKRADPAFVCK (SEQ ID NO: 16).
In a further non-limiting embodiment, the pathogen is a foot and mouth disease virus. In a further non-limiting embodiment, the Replikin peptide is at least one of HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18).
In a further non-limiting embodiment, the pathogen is malaria. In a further non-limiting embodiment, the Replikin peptide is at least one of a Replikin peptide identified from at least one of the following accession numbers: ABU43157, CAD49281, CAD49281, or XP001349534.
Any of the above-listed or herein identified Replikin peptides may be comprised in an immunogenic compound of the invention.
A further non-limiting embodiment of the second aspect of the invention provides a computer readable medium having stored thereon instructions which, when executed, cause a processor to perform a method of predicting an expansion of a strain of pathogen or an increase in virulence, morbidity, and/or mortality of a pathogen. In a further embodiment, the processor reports a prediction to a display, user, researcher, or other machine or person. In a further embodiment, the processor identifies to a display, user, researcher, or other machine or person, a portion of a pathogen predicted to be an expanding pathogen or predicted to increase in virulence, morbidity, and/or mortality, wherein said portion may be employed as a therapeutic or diagnostic compound. Said portion may be a Replikin peptide or plurality of Replikin peptides or any other structure or portion of said genome of said pathogen including a Replikin Peak Gene.
A third non-limiting aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified in an isolate of a pathogen, wherein said isolate is isolated during a rising portion of a cycle in Replikin concentration from among a plurality of isolates of the pathogen, or is isolated at a peak in a cycle in Replikin concentration from among a plurality of isolates of a pathogen, or isolated subsequent to a peak in a cycle in Replikin concentration from among a plurality of isolates of a pathogen.
Another non-limiting embodiment of the third aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified as conserved in an isolate of a pathogen from among a plurality of isolates said pathogen, wherein said isolates are isolated during a cycle in Replikin concentration at least two successive time points or time periods, and the cycle includes at least two peaks or two troughs.
In a non-limiting embodiment of the third aspect of the present invention, the pathogen is a malarial trypanosome. In a further non-limiting embodiment, the identified cycle is in the histidine rich protein of P. falciparum. In another non-limiting embodiment, the identified cycle is in the ATP-ase protein of P. falciparum. In another non-limiting embodiment, the identified cycle is in a Replikin Peak Gene of a trypanosome that causes malaria.
In another non-limiting embodiment of the third aspect of the present invention, the pathogen is a West Nile virus. In a further non-limiting embodiment, the identified cycle is in the envelope protein of West Nile virus. In another non-limiting embodiment, the pathogen is a foot and mouth disease virus. In a further non-limiting embodiment, the identified cycle is in a VP1 protein of a foot and mouth disease virus.
In another non-limiting embodiment of the third aspect of the present invention, the pathogen is an influenza virus. In another non-limiting embodiment, the influenza virus is an H1N1, H2N2, H3N2, H3N8, H5N1, or H9N2 influenza virus. In a further non-limiting embodiment, the identified cycle is in the neuraminidase or hemagglutinin protein of an influenza virus.
A fourth non-limiting aspect of the present invention provides an immunogenic composition comprising a Replikin peptide identified in an isolate of a pathogen, wherein said isolate is isolated during a rising portion of a cycle in Replikin concentration from among a plurality of isolates of said pathogen, or is isolated at a peak in the identified cycle in Replikin concentration from among a plurality of isolates of the pathogen, or is isolated subsequent to a peak in the identified cycle in Replikin concentration from among a plurality of isolates of the pathogen.
In another non-limiting embodiment of the fourth aspect of the present invention, the immunogenic composition is a vaccine for prevention or treatment of an infection of a pathogen. Another non-limiting embodiment of the present invention provides an antibody to a Replikin peptide identified in an isolate of the pathogen, wherein said isolate is identified during a rising portion of a cycle in Replikin concentration, or is identified at a peak in a cycle in Replikin concentration, or is identified subsequent to a peak in a cycle in Replikin concentration. In another non-limiting embodiment, the pathogen is a West Nile virus. In another non-limiting embodiment, the pathogen is a foot and mouth disease virus.
A fifth non-limiting aspect of the invention provides a method of preventing, mitigating, or treating an outbreak of a pathogen comprising
In a non-limiting embodiment of the fifth aspect of the invention, said pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or any other kind of infectious agent.
In a non-limiting embodiment of the fifth aspect of the invention, said first time period is one year and said first geographic region is a country. In a further embodiment, said second time period is one year. In a further embodiment, said second geographic region is a country. In a further embodiment, where the pathogen is influenza virus, said first geographic region is China. In a further embodiment, where the pathogen is a malarial trypanosome, said first geographic region is India. In a further embodiment, where the pathogen is West Nile virus, said first geographic region is a state within the United States.
In another non-limiting embodiment of the fifth aspect of the invention, said plurality of isolates of a strain of pathogen for a first time period in a first geographic region is a plurality of isolates from all publicly available sequences in said first time period in said first geographic region. In another non-limiting embodiment, said plurality of isolates is all isolates from a species of animal. In another non-limiting embodiment, said plurality of isolates is all isolates from a particular species of bird such as swans, chickens, falcons, turkeys, ducks, or other domestic or wild birds.
In a further non-limiting embodiment of the fifth aspect of the invention, said isolated or synthesized portion of the structure or genome of the at least one isolate of pathogen is a protein or protein fragment comprising a Replikin peptide. In a further embodiment, said protein or protein fragment is a Replikin peptide. In another embodiment, said protein or protein fragment comprises a Replikin Peak Gene. In a further embodiment, said protein or protein fragment is a Replikin Peak Gene. In a further embodiment, said protein or protein fragment is a Replikin peptide identified within a Replikin Peak Gene. In another embodiment, said isolated or synthesized portion of the structure or genome is a nucleic acid encoding a Replikin Peak Gene, a nucleic acid encoding a Replikin peptide or plurality of Replikin peptides within a Replikin Peak Gene, or a nucleic acid encoding a Replikin peptide.
In another non-limiting embodiment of the fifth aspect of the invention, the at least one isolate of the same strain of pathogen from a second time period and/or second geographic region is a plurality of isolates from said second time period and/or second geographic region and the Replikin Count of each isolate of the plurality of isolates from said second time period and/or second geographic region is compared separately to said one standard deviation of said mean Replikin Count.
In a further non-limiting embodiment of the fifth aspect of the present invention, an expansion of said strain of pathogen isolated in said second time period and/or second geographic region is predicted if the number of Replikin Counts of said plurality of isolates from said second period and/or said second geographic region that is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period in said first geographic region, is greater than the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is less than said one standard deviation of the mean.
In a further non-limiting embodiment of the fifth aspect of the invention, the Replikin Count is the concentration of Replikin peptides identified encoded in the genome of an isolate of the pathogen. In a further embodiment, the Replikin Count is the concentration of Replikin peptides identified in the expressed proteins of an isolate of the pathogen. In a further embodiment, the Replikin Count is the concentration of Replikin peptides identified in at least one protein or gene area of an isolate of the pathogen. In a further embodiment, the gene area is the pB1 gene area of the genome of influenza virus, the histidine-rich protein gene area of a malarial trypanosome, the VP1 gene area of foot and mouth disease virus, or the envelope protein gene area of West Nile virus. In another embodiment, the Replikin Count is the concentration of Replikin peptides identified in at least one protein fragment of an isolate of the pathogen. In a further embodiment, the Replikin Count is the concentration of Replikin peptides identified in a Replikin Peak Gene of an isolate of the pathogen. In a further embodiment, the Replikin Peak Gene is identified in the polymerase area of an influenza virus genome. In a further embodiment, the Replikin Peak Gene is identified in the pB1 area of an influenza virus genome. In a further embodiment, the Replikin Peak Gene is identified in the histidine-rich protein area of a malarial trypanosome, the VP1 area of a foot and mouth disease virus, or the envelope protein of a West Nile virus.
A sixth non-limiting aspect of the present invention provides a method of predicting an expansion of a strain of pathogen comprising
In a non-limiting embodiment the method of predicting further comprises processing the method on a computer.
A non-limiting embodiment of the sixth aspect of the invention contemplates that the pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or any other kind of infectious agent.
A further non-limiting embodiment of the sixth aspect of the invention provides a method for making a vaccine comprising predicting an expansion of said strain of pathogen isolated in said second time period and/or second geographic region and identifying a portion of the structure or genome of said isolated influenza virus to comprise a vaccine.
In a further non-limiting embodiment of the sixth aspect of the invention, the at least one isolate of the same strain of pathogen from a second time period and/or second geographic region is a plurality of isolates from said second time period and/or second geographic region. In a further non-limiting embodiment, the Replikin Count of each isolate of the plurality of isolates from said second time period and/or second geographic region is compared separately to said one standard deviation of the mean.
In another non-limiting embodiment of the sixth aspect of the invention, an expansion of a strain of pathogen isolated in said second time period and/or said second geographic region is predicted if the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period in said first geographic region, is greater than the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is less than said one standard deviation of the mean. In a further non-limiting embodiment, an expansion of a strain of influenza virus isolated in said second time period and/or second geographic region is predicted if the ratio of the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is greater than said one standard deviation of the mean, divided by the number of Replikin Counts of said plurality of isolates from said second time period and/or said second geographic region that is less than said one standard deviation of the mean, is greater than one.
A further non-limiting embodiment of the sixth aspect of the present invention provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified in an isolate of a pathogen predicted to have an expanding population. In another non-limiting embodiment, the Replikin peptides for diagnostic, therapeutic, and/or preventive purposes are conserved over time or across geographic regions.
Another non-limiting embodiment of the sixth aspect of the invention provides a method of predicting a contraction or failure of a population of a strain of pathogen, wherein a Replikin Count of at least one isolate of a strain of pathogen from a first time period and/or first geographic region is less than one standard deviation of the mean of the Replikin Count of a plurality of isolates of influenza from a second time period and second geographic region. Another non-limiting embodiment provides a method of predicting a contraction or failure of a population of a strain of pathogen, wherein the number of Replikin Counts of a plurality of isolates from a first time period and/or a first geographic region greater than one standard deviation of the mean of the Replikin Count of a plurality of isolates from a second time period in a second geographic region, is less than the number of Replikin Counts of the plurality of isolates from the first time period and/or the first geographic region that is less than said one standard deviation of the mean. In a further non-limiting embodiment, said contraction or failure is predicted if the ratio of the number of Replikin Counts of said plurality of isolates from said first time period and/or said first geographic region that are greater than said standard deviation of the mean, divided by the number of Replikin Counts of said plurality of isolates from said first time period and/or said first geographic region that are less than said standard deviation of the mean, is less than one.
A further non-limiting embodiment of the sixth aspect of the invention provides a computer readable medium having stored thereon instructions which, when executed, cause a processor to perform a method of predicting an expansion of a strain of pathogen or the expansion of a virus or organism. In a further embodiment, the processor reports a prediction to a display, user, researcher, or other machine or person. In a further embodiment, the processor identifies to a display, user, researcher, or other machine or person, a portion of a pathogen predicted to be an expanding pathogen, wherein said portion may be employed as a therapeutic or diagnostic compound. Said portion may be a Replikin peptide or plurality of Replikin peptides or any other structure or portion of said genome of said pathogen including a Replikin Peak Gene.
A seventh non-limiting aspect of the present invention provides an immunogenic composition comprising a portion of the structure or genome of an isolate of a pathogen, wherein said isolate of said pathogen is (1) an isolate having a Replikin Count greater than one standard deviation of a mean Replikin Count of a plurality of isolates of pathogen isolated in a different time period and/or in a different geographical region, (2) an isolate from a first time period and/or geographical region wherein the number of a plurality of isolates from the first time period and/or geographical region having a Replikin Count greater than said one standard deviation of the mean is greater than the number of isolates having a Replikin Count less than said one standard deviation of the mean, (3) isolated during a rising portion of a cycle or a set of two or more synchronous cycles in Replikin concentration from among a plurality of isolates of influenza, and/or (4) isolated at a peak in the identified cycle or set of synchronous cycles in Replikin concentration from among a plurality of isolates of influenza.
In another non-limiting embodiment the seventh aspect of the present invention, the immunogenic composition is a vaccine for prevention or treatment of an infection of a pathogen. Another non-limiting embodiment provides an antibody to a Replikin peptide identified in an isolate of pathogen predicted to have an increase in virulence, morbidity, and/or lethality or expansion of its population.
As used herein, a “Replikin cycle” or “a cycle of Replikin concentration” or “a cycle of Replikin Count” means Replikin concentrations of a plurality of isolates of a species of virus or organism wherein at least four of said plurality of isolates are isolated at successive time points or in successive time periods, wherein a Replikin concentration of a second individual isolate or second mean of a plurality of isolates at a second time point or time period is higher than a Replikin concentration of a first individual isolate or a first mean of a plurality of isolates at a first time point or time period, a Replikin concentration of a third individual isolate or a third mean of a plurality of isolates at a third time point or time period is lower than the Replikin concentration at a second time point or time period, and a Replikin concentration of a fourth individual isolate or fourth mean of a plurality of isolates at a fourth time point or time period is higher than the Replikin concentration at a third time point or time period; or wherein a Replikin concentration of a second individual isolate or second mean of a plurality of isolates at a second time point or time period is lower than a Replikin concentration of a first individual isolate or a first mean of a plurality of isolates at a first time point or time period, a Replikin concentration of a third individual isolate or a third mean of a plurality of isolates at a third time point or time period is higher than the Replikin concentration at a second time point or time period, and a Replikin concentration of a fourth individual isolate or fourth mean of a plurality of isolates at a fourth time point or time period is lower than the Replikin concentration at a third time point or time period. Within the Replikin cycle, cycle of Replikin concentration, or cycle of Replikin Count, the second time point or time period must be later in time than the first time point or time period, the third time point or time period must be later in time than the second time point or time period, and the fourth time point or time period must be later in time than the third time point or time period. Within a Replikin cycle, any rising portion is predictive of an expansion in population or an increase in virulence, morbidity, and/or mortality of a pathogen in hosts and any decreasing portion is predictive of a contracting population or a decrease in virulence, morbidity, and/or mortality of pathogen in hosts. A cycle need not be complete to be predictive, a decreasing portion followed by a rising portion is predictive of an expanding population or an increase in virulence, morbidity, and/or mortality. Likewise, a rising portion followed by a decreasing portion followed by a rising portion is predictive of an expanding population or an increase in virulence, morbidity, and/or mortality. As such, cycles need not be complete cycles to provide predictive capacity concerning an expansion or contraction (or change in virulence, morbidity, and/or mortality) of a pathogen in hosts.
As used herein, a “step-wise” cycle is any set of cycles wherein a first Replikin cycle peak in time is lower than a second Replikin cycle peak in time or a first Replikin cycle peak in time is higher than a second Replikin cycle peak in time. A step-wise cycle also occurs when successive peaks are observed to move lower. A step-wise cycle may also be observed if successive troughs move higher or lower. Step-wise cycles provide additional predictive capacity for predictions of expansion or contraction of a population.
As used herein a Replikin cycle that is “synchronous,” shares “synchrony,” or any other related word, with another Replikin cycle means a cycle having a period or phase or any portion of the cycle that is similar to some period, phase, or portion of the cycle wherein said similarity may be determined visually, mathematically, statistically, or by any other method known or hereinafter known by one of skill in the art. Synchronous cycles do not necessarily share portions that arise or occur at exactly the same time. Synchronous cycles in related pathogens will at times be shifted by some measure of time from one another and may shift in time from one another in any portion of either cycle. A portion of a Replikin cycle “corresponds” in time with another Replikin cycle if there is a similarity between the portions of the cycle. Any correspondence need not be exact.
As used herein, a “rising portion” of a Replikin cycle means the Replikin concentration of an isolate or the mean Replikin concentration of a plurality of isolates, wherein the isolate or isolates were isolated at a time point or time period of the Replikin cycle where the trend of Replikin concentration in the Replikin cycle is increasing from at least a first time point or time period to at least a second time point or time period. Additionally, the rising portion may include a peak.
As used herein, a “decreasing portion” of a Replikin cycles means the opposite of a rising portion, wherein a decreasing portion may include a trough.
As used herein, a “peak” in a Replikin cycle means a second time point or time period within a Replikin cycle, wherein the Replikin concentration at a first time point or time period sequentially preceding the second time point or time period is lower than the Replikin concentration at the second time point or time period, and the Replikin concentration at a third time point or time period sequentially following the second time point or time period is lower than the Replikin concentration at the second time point or time period. One of skill in the art will understand that because of the variability of biological systems, a peak may include a general region of a cycle that is generally higher than a sequentially preceding region and generally higher than a sequentially following region rather than an exact time point or time period.
As used herein, a “trough” in a Replikin cycle means the opposite of a peak in a Replikin cycle.
As used herein, a “Replikin Count Virus Expansion Index” or “RCVE Index” or a “Replikin Count Expansion Index” or “RCE Index” is the number of Replikin Counts of a plurality of isolates from a first time period and/or first geographic region that are greater than one standard deviation of the mean of the Replikin Count of a plurality of isolates isolated in a second time period and in a second geographic region, divided by the number of Replikin Counts of said plurality of isolates from said first time period and/or said first geographic region that are less than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said second time period in said second geographic region. An RCE or RCVE Index predicts the expansion of a pathogen in a particular region and/or time period if the ratio of the RCE or RCVE Index is greater than one. An RCE or RCVE Index predicts the contraction, retraction, reduction, or failure of a pathogen in a particular region and/or time period if the ratio of the RCE or RCVE Index is less than one. An RCE or RCVE Index predicts equilibrium between expansion and contraction in the pathogen population if the ratio of the RCVE Index is equal to one.
As used herein, a “related pathogen” means a first pathogen that is of the same species, genus, or family as a second pathogen for which a relationship is known now or hereafter by one of skill in the art. A related pathogen may be a first pathogen that is of the same species but a different strain from a second pathogen. A related pathogen may be a first pathogen that is the same or different species from a second pathogen and shares a host, reservoir, or vector with the second pathogen. Even if a first pathogen is not of the same species, genus, or family as a second pathogen, the first pathogen is related to the second pathogen if the first pathogen has a Replikin cycle that is synchronous with the Replikin cycle of the second pathogen. One of skill in the art will recognize the many ways that a first pathogen may be related to a second pathogen. A related pathogen may be within the same family as a first pathogen. A related pathogen may be within the same genus as a first pathogen. A related pathogen may be within the same species as a first pathogen. A related pathogen may be within the same strain as a first pathogen.
As used herein, different “time periods” or different “time points” are any two time periods or time points that may be differentiated from each other. For example, an isolate of an organism or virus isolated during the year 2004 may be considered to be isolated in a different time period than an isolate of the same organism or virus isolated during the year 2005. Likewise, an isolate of an organism or virus isolated in May 2004 may be considered to be isolated in a different time period than an isolate of the same organism or virus isolated in June 2004. When comparing Replikin concentrations of different isolates, one may use comparable time periods. For example, an isolate from 2004 may be compared to at least one other isolate from some other year such as 2002 or 2005. Likewise, an isolate from May 2004 may be compared to at least one isolate from some other month of some year, for example, an isolate from December 2003 or from June 2004.
As used herein, an “isolate” is any virus or organism isolated from a natural source wherein a natural source includes, but is not limited to, a reservoir of an organism or virus, a vector of an organism or virus, or a host of an organism or virus. “Obtaining,” “isolating,” or “identifying” an isolate is any action by which an amino acid or nucleic acid sequence within an isolate is obtained including, but not limited to, isolating an isolate and sequencing any portion of the genome or protein sequences of the isolate, obtaining any nucleic acid sequence or amino acid sequence of an isolate 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 or within a time period.
As used herein, “an earlier-arising” virus or organism or a virus or organism isolated at “an earlier time point” or during “an earlier time period” is a specimen of a virus or organism collected from a natural source of the virus or organism on a date prior to the date on which another specimen of the virus or organism was collected from a natural source. A “later-arising” virus or organism or a virus or organism isolated at a “later time point” or during a “later time period” is a specimen of a virus or organism collected from a natural source of the virus (including, but not limited to, a reservoir, a vector, or a host) or a natural source of the organism on a date subsequent to the date on which another specimen of the virus or organism was collected from a natural source.
As used herein, the “next virulence season” of a pathogen is a time period in which an increase in morbidity of a pathogen is expected based on seasonal changes, such as a change from summer to winter or a change from a wet season to a dry season, wherein the pathogen was experiencing less morbidity in a previous sequential time period prior to the time period in which the increase in morbidity is expected to occur.
As used herein, the term “dry season” or “winter season” with respect to malaria describes a season in any geographical region wherein mosquito activity (including feeding and reproduction) is significantly less than during other times of the year. A peak in a Replikin cycle before a dry season or winter season predicts an increase in virulence, morbidity, and/or mortality in malaria in the following rainy season or summer season when mosquito activity is greatest.
As used herein “trypanosome that causes malaria,” “malarial trypanosome” or “trypanosome” in singular or plural means any Plasmodium species or other species known now or hereafter to cause malaria. Malarial trypanosomes include but are not limited to Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.
As used herein, a “Replikin Peak Gene (RPG)” (or sometimes a Replikin Peak Gene Area-RPGA) means a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has a highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) when compared to other segments or named genes of the genome. 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 of 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, 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, “outbreak” is an increase in virulence, morbidity, and/or mortality in a pathogenic disease or an expansion in the population of pathogen as compared to a baseline of an earlier occurring epidemiological pattern of infection in the same disease. 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 none or some cases were present in a geographic region in the immediate past. The past, in epidemiological terms, may mean more than one year and can mean several years or more as understood by one of ordinary skill in the art. The past may also mean less than one year as determined by one of ordinary skill in the art. In the case of annually-recurrent common influenza and seasonal malaria and West Nile virus, for example, the baseline often reflects an annual recurrence or expansion and contraction of these diseases.
As used herein, “expansion” of a pathogen or a population of pathogen and “expanding” pathogen or population of pathogen means an increase in virulence, morbidity, and/or lethality of a pathogen (e.g., strain of P. falciparum, a strain of influenza virus, etc.) and/or an expansion of the population of a pathogen (e.g., strain of P. falciparum, a strain of influenza virus, etc.) wherein said expansion includes an increase in the occurrence of the pathogen in a given geographic region or in a given time period or both, or a spreading of the occurrence of the pathogen to another geographic region.
As used herein, an increase or decrease in “virulence” includes an increase or decrease in virulence, morbidity, lethality, host mortality, and/or expansion of a pathogen, such as an influenza virus.
As used herein, “geographic region” or similar term is an area differentiated from another area by space. For example, China is a geographic region that may be differentiated from the geographic region of India. Likewise a geographic region may be a town, or city, or continent or any area differentiable from another area. A geographic region may encompass the entire earth if an isolate or plurality of isolates from a given time period is compared to isolates from another time period over the entire earth and no geographic differentiation is undertaken for the comparison.
As used herein, “conserved” or “conservation” refers to conservation of particular amino acids due to lack of substitution.
As used herein, “Replikin Count” or “Replikin Concentration” refers to the number of Replikin sequences per 100 amino acids in a protein, protein fragment, virus, or organism. A higher Replikin concentration in a first strain of a virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier-arising or later-arising strain of the virus or organism having a lower Replikin concentration. Replikin concentration is determined by counting the number of Replikin sequences in a given sequence wherein a Replikin sequence is a peptide of 7 to about 50 amino acid residues with a lysine residue on one end and a lysine residue or a histidine residue on the other end wherein the peptide comprises (1) a lysine residue six to ten residues from another lysine residue, (2) a histidine residue, (3) and 6% or more lysine residues, or wherein a Replikin sequence is a nucleic acid that encodes a Replikin peptide sequence.
As used herein, the term “continuous Replikin sequences” means a series of two or more Replikin sequences that are overlapped and/or are directly covalently linked.
The present invention provides methods of preventing, mitigating, and treating outbreaks of a pathogen by predicting an expansion of a strain of pathogen or an increase in the virulence, morbidity, and/or lethality of a strain of pathogen as compared to another strain of the same pathogen and administering to an animal or patient a compound comprising an isolated or synthesized portion of the structure or genome of the pathogen to mitigate, prevent, or treat the predicted outbreak of the pathogen. The present invention further provides methods of predicting an expanding population of a pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen comprising identifying a cycle in the Replikin Count in a protein fragment, protein, genome fragment, or genome of a pathogen and predicting an expansion of the population of the pathogen or an increase in the virulence, morbidity, and/or mortality of the pathogen within the identified cycle in Replikin Count.
An increase in the virulence, morbidity, or mortality of a pathogen relative to the virulence, morbidity, and/or mortality of another pathogen of the same species may be predicted by identifying a peak in a cycle or cycles in the concentration of Replikin sequences in the pathogen and predicting an expansion of the population of the pathogen or an increase in the virulence, morbidity, and/or mortality of a pathogen of the same or a related species isolated subsequent to the peak. A Replikin cycle is a cycle in the concentration of Replikin sequences identified in at least four isolates of a species of virus or organism isolated at successive times where (1) the concentration in the first isolate-in-time is higher than the concentration in the second isolate-in-time, the concentration in the third isolate-in-time is higher than the concentration in the second isolate-in-time, and the concentration of the fourth isolate-in-time is lower than the concentration in the third isolate-in-time, or (2) the concentration in the first isolate-in-time is lower than the concentration in the second isolate-in-time, the concentration in the third isolate-in-time is lower than the concentration in the second isolate-in-time, and the concentration of the fourth isolate-in-time is higher than the concentration in the third isolate-in-time. Within a Replikin cycle, an increase in virulence, morbidity, and/or mortality of a pathogen may be predicted for a pathogen arising during a rising portion of the cycle or subsequent to the peak of a cycle. An expanding population may represent an increase in population in a region or expansion from one region into another region. In determining a Replikin cycle, Replikin Counts may represent individual isolates, or mean Replikin Counts of groups of isolates from a given region and/or time period.
In a further non-limiting embodiment, step-wise cycles may be identified between successive time points. In a further embodiment, specific conserved Replikin sequences are identified within the step-wise cycles.
An increase in virulence, morbidity, or mortality of a pathogen may be determined using the methods of the invention in any pathogen or infectious agent where a concentration of Replikins may be determined in the genome, a genome fragment, another nucleic acid sequence, a protein, a protein fragment, or other amino acid sequence from the pathogen. A pathogen may be malaria, West Nile virus, foot and mouth disease virus, porcine circovirus, porcine respiratory and reproductive syndrome virus, taura syndrome virus, white spot syndrome virus, tomato leaf curl virus, bacillus anthracis, small pox virus, human immunodeficiency virus, sindbis virus, hepatitis virus, staphylococcus, legionella, human papilloma virus, Helicobacter, Acetobacter, Aerobacter, Brivebacterium, Clostridium, Erinia, Esheria, Klebsiealla, Maemophilus, Mycoplasma, Psuedomonas, Salmonella, Candida, Entamoeba, or any other form of infectious agent including viruses, bacteria, protozoa, fungi, or other infectious agent.
Any Replikin sequence, Replikin Peak Gene, or protein fragment containing a Replikin sequence or Replikin Peak Gene identified in a strain of pathogen that is predicted to have an increase in virulence, morbidity, or mortality may be isolated and/or synthesized as a diagnostic, therapeutic, or prophylactic agent to mitigate the predicted outbreak of the pathogen.
A cycle of Replikin concentration or “Replikin cycle” of a trypanosome may be seen in
A peak in a Replikin cycle is identified within the cycle at a second time point or time period within a Replikin cycle, wherein the Replikin concentration at a first time point or time period sequentially preceding the second time point or time period is lower than the Replikin concentration at the second time point or time period, and the Replikin concentration at a third time point or time period sequentially following the second time point or time period is lower than the Replikin concentration at the second time point or time period. One of skill in the art will understand that because of the variability of biological systems, a peak may include a general region of a cycle that is generally higher than a sequentially preceding region and generally higher than a sequentially following region rather than an exact time point or time period.
A trough in a Replikin cycle is identified within the cycle is identified within the cycle at a second time point or time period within a Replikin cycle, wherein the Replikin concentration at a first time point or time period sequentially preceding the second time point or time period is higher than the Replikin concentration at the second time point or time period, and the Replikin concentration at a third time point or time period sequentially following the second time point or time period is higher than the Replikin concentration at the second time point or time period. Once again, one of skill in the art will recognize that troughs may be identified as a general region of a cycle that is generally lower than a sequentially preceding region and generally lower than a sequentially following region rather than an exact time point or time period.
Replikin peptides of the invention identified at a peak of the Replikin cycle include Replikin peptides identified at or near the peak of the Replikin cycles including prior to and subsequent to the precise point of the peak. A rising portion of a Replikin cycle is any point at which the trend of Replikin concentration in the Replikin cycle is increasing from at least a first time point or time period to at least a second time point or time period and can include a peak. As may be seen in
In the past, it had been understood that outbreaks of pathogens correlated with increases in Replikin Count and that contractions of pathogenic populations correlated with decreases in Replikin Count. It was not understood, however, that cycles in morbidity, mortality, virulence or population expansion could be directly correlated with cycles in Replikin Count. With the new data presented in the present application, the ordinary skilled artisan will now understand, and it is contemplated by the present invention, that entire Replikin cycles from peak to trough to peak to trough and/or from trough to peak to trough to peak correlate with pathogenic cycles in virulence, morbidity, mortality, and expansion into new regions or hosts. As such, the invention now provides methods of tracking and predicting tracks of pathogens as they increase in virulence, expand in population within a region or into a region, or increase in morbidity or mortality by monitoring changes in Replikin concentration. In the past, it was not possible until months after an event to predict or track the course of pathogens as they increase in virulence, expand in population within region or into a region, or increase in morbidity or mortality where epidemiological data was collected and analyzed post hoc. Replikins analysis provides the skilled artisan with information on population expansion, and increases in virulence, morbidity, and mortality months before or at the very beginning of an outbreak. This information is clearly important for the time needed to organize public health responses, including the testing and administration of specific vaccines. The importance of prior information concerning pathogenic outbreaks may be analogized to the savings of life and property that have resulted from advance warning of hurricanes since information from weather satellites has become available.
For example, in
The present invention also provides a method of predicting an expansion of a strain of pathogen by (1) determining a mean Replikin Count and a standard deviation of the mean Replikin Count for a plurality of isolates of a strain of pathogen for a first time period in a first geographic region; (2) determining a Replikin Count of at least one isolate of the same or a related strain of pathogen from a second time period and/or second geographic region wherein the second time period is different from the first time period and/or the second geographic region is different from the first geographic region; and (3) predicting an expansion of the strain of pathogen isolated in the second time period and/or second geographic region, if the Replikin Count of the at least one isolate is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in the first time period and in the first geographic region.
In the above-described method, at least one isolate of the same or related strain of pathogen from a second time period and/or second geographic region may be a plurality of isolates from the second time period and/or second geographic region. In this case, the Replikin Count of each isolate of the plurality of isolates from the second time period and/or second geographic region is compared separately to one standard deviation of the mean. An expansion of pathogen isolated in the second time period and/or second geographic region may also be predicted if the number of Replikin Counts of a plurality of isolates from the second period and/or second geographic region that is greater than one standard deviation of the mean is greater than the number of Replikin Counts of said plurality of isolates from the second period and/or second geographic region that is less than one standard deviation of the mean.
The method may also employ a ratio of the number of Replikin Counts that are greater than one standard deviation of the mean divided by the number of Replikin Counts that are less than one standard deviation of the mean. The ratio is called a Replikin Count Expansion Index (RCE Index). Another way to determine the RCE Index is to divide the percent of Replikin Counts in a plurality of isolates of influenza virus grouped by time and/or region that are higher than one standard deviation of the mean by the percent of Replikin Counts that are lower than one standard deviation of the mean. An RCE Index may be used to quantify the future risk of an outbreak of pathogen by tracking Replikin Counts in strains of pathogen over time.
In determining a RCE Index, the mean Replikin Count of the plurality of isolates from the first time period and geographic region may be considered a control. A control population preferably has a relatively large number of isolates with a relatively small variability in the Replikin Count of the isolates but any population may be deemed a control when a comparison between the control and a related isolate or plurality of isolates is desired. A control may be related to the population that is being studied. For example, if an infection in a bird species, such as swans, is being studied, the control may be something closely related, such as chickens, wherein isolates from chickens may be relatively numerous (if available) and relatively stable (if possible) wherein stability in Replikin Count through the population demonstrates a level of equilibrium between the expansion and contraction of the strain or a related strain of influenza virus in chickens. A control may reflect a highest number of isolates reported in a year or in several years in a geographic area.
An expansion of a strain of pathogen may be determined using the methods of the invention in any pathogen or infectious agent where a concentration of Replikins may be determined in the genome, a genome fragment, another nucleic acid sequence, a protein, a protein fragment, or other amino acid sequence from the pathogen. A pathogen may be malaria, West Nile virus, foot and mouth disease virus, influenza virus, porcine circovirus, porcine respiratory and reproductive syndrome virus, taura syndrome virus, white spot syndrome virus, tomato leaf curl virus, bacillus anthracis, small pox virus, human immunodeficiency virus, sindbis virus, hepatitis virus, staphylococcus, legionella, or any other form of infectious agent including viruses, bacteria, protozoa, fungi, or other infectious agent.
Any Replikin sequence, Replikin Peak Gene, or protein fragment containing a Replikin sequence or Replikin Peak Gene identified in a strain of pathogen that is predicted to have an increase in virulence, morbidity, or mortality may be isolated and/or synthesized as a diagnostic, therapeutic, or prophylactic agent to mitigate the predicted outbreak of the pathogen.
The present invention further provides the opportunity to identify Replikin sequences (including nucleic acid sequences and peptide sequences) for diagnostic, therapeutic, or preventive purposes (such as the construction of vaccines and other pharmaceuticals). The present invention contemplates, for example, Replikin peptides identified within a pathogen where the pathogen is predicted to have an expanding population or a higher virulence, morbidity, and/or mortality than another pathogen of the same or a related species based on the predictive methods of the invention. Replikin peptides identified in an isolate of a pathogen, wherein said isolate is isolated during a rising portion of a cycle in Replikin concentration among a plurality of isolates of the pathogen or is isolated at a peak in a cycle in Replikin concentration among a plurality of isolates of the pathogen, are useful for diagnostic, therapeutic, and preventive purposes. For example, a Replikin peptide identified in the genome of an isolate identified in a rising portion of a cycle in Replikin concentration or identified at a peak in a cycle in Replikin concentration is useful as a peptide to stimulate the immune system of a human or animal to produce an immune response against infection by the pathogen or to produce antibodies against a pathogen predicted to have higher virulence, morbidity, and/or mortality. One of ordinary skill in the art will recognize that antibodies against these pathogens are useful for diagnosing the more highly virulent or mortal disease in a subject or useful as therapies against the infection either as a prophylactic or after onset of the infection.
Additionally, Replikin peptides identified during a rising portion in Replikin concentration in a Replikin cycle or identified at or near a peak in Replikin concentration in a Replikin cycle that are conserved during the rising portion of the Replikin cycle are useful as compounds for diagnostic, therapeutic, and preventive purposes. Conservation of the Replikin peptides during a rise in virulence, morbidity, and/or mortality provides targets that are more constant and likely more involved in the mechanisms of rapid replication that provide the predicted increase in virulence, morbidity, and/or mortality. As such, these conserved Replikin peptides are of use as compounds or in compositions for stimulating the immune system of a subject to produce an immune response, an antibody response, and/or a protective effect in the subject.
Replikin peptides identified and isolated using the methods of the invention include influenza peptides such as HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1), KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4), HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12), West Nile virus peptides such as KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK (SEQ ID NO: 15), or HNDKRADPAFVCK (SEQ ID NO: 16), and foot and mouth disease peptides such as HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18).
Identification of portions of a pathogen (such as Replikin Peak Genes or Replikin peptides) predicted to expand in population provide unique compounds for diagnostics and treatment of expanding pathogens, wherein the unique compounds would otherwise not be identifiable but for the methods of the invention and the compounds disclosed herein.
The invention further contemplates use of the Replikin peptide as immunogenic compositions and contemplates the immunogenic compositions as vaccines, including vaccines that provide an immune response, vaccines that provide a humoral immune response, vaccines that provide an antigenic immune response, and vaccines that provide a protective effect. The invention additionally contemplates an antibody to the Replikin peptides of the invention.
High Replikin Counts and RPGs have been shown to be related to rapid replication, viral outbreaks, epidemics, morbidity and host mortality, for example, in influenza virus strains, including H5N1, in SARS coronavirus, in shrimp taura syndrome virus, and in foot and mouth disease virus. Replikin sequences identified at or near the peak of the Replikin cycle or during a rising portion of the Replikin cycle in a pathogen are appropriate peptides for diagnostics, vaccines, and other treatments.
Because Replikin sequences are chemically defined, the sequences may be synthesized by organic chemistry rather than biological techniques, and thus are potentially more specific, more reproducible and more reliable. The chemically defined Replikin sequences identified by Applicants are likewise potentially freer from adverse reactions that are characteristic of biologically derived vaccines and antibodies.
Mitigating and Treating Outbreaks of Pathogen with Cyclic Replikin Counts
One aspect of the present invention provides methods of preventing, mitigating, or treating pathogenic outbreaks predicted through analysis of cycles of Replikin Counts or through analysis of controls using mean Replikin Counts and standard deviation (e.g., Replikin Count Expansion Index). For example, advance information concerning Replikin peptides and Replikin Peak Genes in expanding strains of a pathogen allows for the rapid production of specific effective synthetic vaccines using one, or a combination, of Replikin peptides or using Replikin Peak Genes. Such synthetic vaccines have been demonstrated in rabbits, chickens, and shrimp. See, e.g., Examples 6 and 7 of U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 and Example 2 of U.S. application Ser. No. 12/108,458, filed Apr. 23, 2008. For example, a mixture of Replikin peptides administered orally to shrimp provided up to a 91% protective effect for shrimp challenged with taura syndrome virus. Taura syndrome virus is an often lethal rapidly-replicating pathogen that has a significant negative impact on the shrimp industry.
Synthetic Replikin vaccines have also been demonstrated in the H5N1 strain of influenza virus in chickens. For example, in a test of chickens administered a mixture of twelve H5N1 Replikin peptides from the hemagglutinin and pB1 gene areas intranasally, intraocularly, and by spray inhalation and challenged with low pathogenic H5N1 influenza isolated from a black duck in the state of North Carolina in the United States, a protective effect was observed at both the entry site of influenza (diminished antibody production in the mucus was observed as compared to a control) and at excretion sites of influenza (influenza virus was not observed excreted in feces or saliva from treated chickens as compared to a control). See Example 10 below.
Administration of Replikin peptides in both shrimp and chickens appears to have provided a notable measure of mucosal immunity. For example, in Example 2 of U.S. application Ser. No. 12/108,458, a mixture of Replikin peptides was administered by mouth to shrimp later challenged with taura syndrome virus. The 91% protective effect of the vaccine is expected to have been a result, at least in part, of a mucosal immune-like responses in the gut of the shrimp.
Likewise, in chickens, the administration of a mixture of Replikin peptides provided a protective effect against entry of the H5N1 virus. For example, as may be seen in Example 10 below, three of six vaccinated chickens, when inoculated with H5N1 virus, produced no measurable amount of antibodies against H5N1 in their serum. Instead, the virus was apparently blocked by mucosal immunity from even entering the chickens' blood stream. For those three chickens in which a serum immune response was measured (that is, virus entered the host and was presented to antibody generating cells), the vaccine additionally provided a protective effect against replication of the virus in the chickens' system (no virus was excreted in the feces or saliva of the chickens). As such, mucosal immunity, in addition to other immunities, is an important aspect of the immunity imparted by Replikin-based vaccines.
Cyclic increases in Replikin concentration in the genome can be a mechanism of expansion of a pathogen into a territory. The Replikin concentration in each Replikin Peak Gene of each Replikin cycle in an expanding population apparently may build on the previous one. Timely, repeated analyses of cyclic changes in a virus' Replikin structure is useful to bring current the targets for the chemical synthesis of Replikin vaccines having a best fit for emerging pathogens having increased virulence, morbidity, and/or lethality. These strain-specific vaccines may be manufactured in seven days as have been demonstrated with a 91% protection of shrimp against the lethal taura syndrome virus. See, e.g., U.S. application Ser. No. 12/108,458, filed Apr. 23, 2008 (incorporated herein in its entirety by reference).
The present invention provides methods of predicting an expansion of the population of a trypanosome that causes malaria or an increase in the virulence, morbidity, and/or mortality of a trypanosome that causes malaria as compared to another trypanosome of the same species or a related species. An expanding population or increase in virulence, morbidity, and/or mortality of a trypanosome that causes malaria may be predicted by identifying a cycle of Replikin concentration among a plurality of isolates of the species of trypanosome and identifying a rising portion or peak in that cycle. An increase in virulence, morbidity, and/or mortality is predicted following the time point or time period when the rising portion or peak is identified. An expanding population may represent an increase in population in a region or expansion from one region into another region.
A further non-limiting embodiment of one aspect of the invention provides a method of predicting an increase in morbidity and mortality in malaria comprising: (1) determining the mean Replikin Count in a plurality of isolates of a malarial trypanosome at a plurality of successive time points; (2) comparing the mean Replikin Count at least four successive time points and identifying at least one cycle of increasing mean Replikin Counts over the at least four time points; and (3) predicting an increase in morbidity and/or mortality following in time the increase in mean Replikin count in at least one of said cycles. In a further non-limiting embodiment, step-wise cycles are identified between successive time points. In a further non-limiting embodiment, specific conserved Replikin sequences are identified within the step-wise cycles. In a further non-limiting embodiment, Replikin sequences are identified at the peak of a step-wise cycle. The Replikin sequences identified at the peak of a step-wise cycle are useful for developing a vaccine or therapeutic composition of an isolated or synthesized Replikin peptide for use in preventing or treating outbreaks of malaria with relatively higher mortality.
Analysis of the mean annual Replikin Count of the histidine rich protein between 1986 and 2007 revealed cycles of Replikin Count. A first rising portion followed by a decreasing portion of the cycle was observed from 1986 to 1995. A second rising portion followed by a decreasing portion was observed from 1996 to 2005. A first peak was identified in 1987 with a mean annual Replikin Count of 38.2 and standard deviation of ±23.5. A second peak was identified in 1999 with an even higher mean annual Replikin Count of 62.9 and standard deviation of ±62.9 (overlap of Replikin sequences within an amino acid sequence generates a Replikin Count of greater than 100 Replikin sequences per 100 amino acids in some sequences). Both the 1987 peak and the 1999 peak were observed to be related to higher human mortality. Following the 1999 peak, mean annual Replikin Counts were observed to fall to a low of 7.4 in 2005 with a standard deviation of ±6.5. Mortality rates likewise fell between 2000 and 2005. A third malaria Replikin cycle appears to have begun in 2005 with the observed mean annual Replikin Count increasing from 7.4±6.5 in 2005 to 17.2±19 in 2007. The beginning of a new cycle provides a prediction that Replikin Count may continue to increase along with an increase in malaria mortality rate.
The cycling observable in
The data for
P. falciparum Replikin Count
As is seen in
Cyclic increases in Replikin concentration in the genome can be a mechanism of expansion of an infectious organism into a territory. The Replikin concentration in each Replikin Peak Gene of each Replikin cycle apparently builds on the previous one. In both the mosquito-borne West Nile Virus and mosquito-borne malaria trypanosomes, this build-up probably occurs during winter seasons, dry seasons, or otherwise dormant periods. Timely, repeated analyses of cyclic changes in the organism's Replikin structure is useful to bring current the targets for the chemical synthesis of Replikin vaccines having a best fit for emerging pathogens having increased virulence, morbidity, and/or mortality. These strain-specific vaccines may be manufactured in seven days as has been demonstrate with a 91% protection of shrimp against the lethal taura syndrome virus. See, e.g., U.S. application Ser. No. 12/108,458, filed Apr. 23, 2008 (incorporated herein in its entirety by reference).
The Replikin cycle may be identified in any trypanosome that causes malaria. For example, it may be identified in the genome of a trypanosome, including P. falciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae. The Replikin cycle may likewise be identified in the histidine rich protein or in the ATP-ase protein, including in these proteins in P. falciparum. The Replikin cycle may likewise be identified in a Replikin Peak Gene of a trypanosome that causes malaria.
Malaria trypanosomes have been found to have the highest Replikin counts seen to date in any infectious organisms—up to twenty times those in influenza and West Nile Virus. Consistent with these high counts, trypanosomes have one of the highest replication rates in nature. This property may account in part for the resistance of malaria to previous attempts at vaccination. The discovery of the relation of Replikin sequences to rapid replication offers a new approach, and means, to inhibit rapid replication in malaria.
In the data analysis reported in Tables 1 and 5, as well as
For example, Replikin peptides identified in the ABU43157 isolate in 2007 are available as a diagnostic, therapeutic, or preventive compounds or compositions of the invention because they were identified in a rising portion of a Replikin cycle. See
In a further aspect of the invention, an expanding population of West Nile virus or an increase in virulence, morbidity, or morality of West Nile virus may be predicted by identifying a cycle of Replikin concentration in isolates of West Nile virus and predicting an expanding population of virus or an increase in virulence, morbidity, and/or mortality of West Nile virus following a rising portion, or peak in the cycle of Replikin concentration. An expanding population may represent an increase in population in a region or expansion from one region into another region.
For example, using analysis of Replikin sequences in West Nile virus, including, for example, analysis of Replikin sequences in the envelope protein of West Nile virus, a correlation between virus biochemical cycles and virus virulence, morbidity, and/or mortality cycles may be identified and used to predict expansions in a virus population or increases in virulence, morbidity, and/or mortality in a virus in a host population. A non-limiting embodiment of the aspect of the invention provides a method of predicting an increase in morbidity in a viral disease such as West Nile virus comprising: (1) determining the mean Replikin Count in genomes of a plurality of isolates of a virus at a plurality of successive time points; (2) comparing the mean Replikin Count at least four successive time points and identifying at least two peaks or two troughs in the trend of Replikin Counts over the at least four time points; and (3) predicting an increase in morbidity following in time the increase in mean Replikin count within said cycles. In a further non-limiting embodiment, step-wise cycles are identified between successive time points. In a further embodiment, specific conserved Replikin sequences are identified within the step-wise cycles.
Table 2, below, provides data from analysis of envelope protein sequences in West Nile virus available at www.pubmed.com for isolates from 2000 through 2007. The data, which are illustrated in
In
Immunogenic compounds for therapeutic vaccines against West Nile virus include, for example, KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK (SEQ ID NO: 15), and HNDKRADPAFVCK (SEQ ID NO: 16). These Replikin peptide sequences are conserved within the step-wise cycles of West Nile virus in
In a further aspect of the invention, an expanding population of foot and mouth disease virus or an increase in virulence, morbidity, or mortality of West Nile virus may be predicted by identifying a cycle of Replikin concentration in isolates of foot and mouth disease virus and predicting an expanding population of virus or an increase in virulence, morbidity, and/or mortality of virus following a rising portion, or peak in the cycle of Replikin concentration. An expanding population may represent an increase in population in a region or expansion from one region into another region.
For example, using analysis of Replikin sequences in foot and mouth disease virus, including, for example, analysis of Replikin sequences in the VP1 protein of foot and mouth disease virus, a correlation between virus biochemical cycles and virus virulence, morbidity, and/or mortality cycles may be identified and used to predict expansions in a virus population or increases in virulence, morbidity, and/or mortality in a virus in a host population. A non-limiting embodiment of the aspect of the invention provides a method of predicting an increase in morbidity in a viral disease such as foot and mouth disease virus comprising: (1) determining the mean Replikin Count in genomes of a plurality of isolates of a virus at a plurality of successive time points; (2) comparing the mean Replikin Count at least four successive time points and identifying at least two peaks or two troughs in the trend of mean Replikin Counts over the at least four time points; and (4) predicting an increase in virulence and/or morbidity following in time an increase in mean Replikin count within a cycle. In a further non-limiting embodiment, step-wise cycles are identified between successive time points. In a further embodiment, specific conserved Replikin sequences are identified within the step-wise cycles.
Increased Replikin Counts provide advance warnings of Foot and Mouth Disease outbreaks and the basis of a conserved synthetic FMDV Vaccine. One aspect of the invention contemplates provision of advance warning of outbreaks of FMDV by identifying cycles in the Replikin Count of isolates of FMDV over time. As may be seen from the data in
The data in Table 3 and
Replikin peptides (1) were identified and counted automatically, with tests of statistical significance of changes, using a software program (ReplikinsForecast™ Replikins LLC, Boston, Mass.) designed to analyze the protein sequences of any organism, in this case FMDV published in PubMed. When the history of each Replikin structure in the virus was tracked for its occurrence in each virus specimen in each of the years for which virus sequence data was published, conservation of Replikin structures for decades was found. The structure of these conserved Replikins is the basis of synthetic Replikins vaccines for FMDV.
Replikin peptides conserved in FMDV over decades include HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18). Sequences identified within Replikin cycles and as conserved within Replikin cycles are particularly useful for diagnostic and therapeutic purposes. For example, the sequences identified as new and/or conserved in FMDV Replikin cycles are useful for (1) designing and chemically synthesizing vaccines that contain both older conserved Replikins as well as newer ones to provide the most accurate and maximal anti-organism immune stimulating properties, (2) designing and chemically synthesizing antibodies that contain reactive sites against both older conserved Replikins and newer ones to provide the most accurate and maximal anti-organism immune protective properties, and (3) designing and chemically synthesizing compounds that contain reactive sites against both older conserved Replikins and newer ones to provide the most accurate and maximal anti-organism protective properties.
One aspect of the present invention provides methods of predicting an outbreak of influenza by predicting an increase in the virulence, morbidity, and/or lethality of a strain of influenza virus or an expansion of the population of a strain of influenza virus using a Replikin Count Virus Expansion Index. In this aspect of the invention, an increase in virulence, morbidity, and/or lethality or an expansion of a strain of influenza virus is predicted by (1) determining a mean Replikin Count and a standard deviation from the mean Replikin Count for a plurality of isolates of a strain of influenza virus for a first time period in a first geographic region, (2) determining a Replikin Count of at least one isolate of the same or a related strain of influenza virus from a second time period and/or a second geographic region different from the first time period and/or the second geographic region, and (3) predicting an increase in virulence, morbidity, and/or lethality or an expansion of the strain of influenza isolated in the second time period and/or second geographic region, if the Replikin Count of the at least one isolate from a second time period and/or a second geographic region is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in the first time period in the first geographic region.
In the above-described method, at least one isolate of the same or related strain of influenza virus from a second time period and/or second geographic region may be a plurality of isolates from the second time period and/or second geographic region. In this case, the Replikin Count of each isolate of the plurality of isolates from the second time period and/or second geographic region is compared separately to one standard deviation of the mean.
An expansion of influenza isolated in the second time period and/or second geographic region may also be predicted if the number of Replikin Counts of a plurality of isolates from the second period and/or second geographic region that is greater than one standard deviation of the mean is greater than the number of Replikin Counts of said plurality of isolates from the second period and/or second geographic region that is less than one standard deviation of the mean.
The method may also employ a ratio of the number of Replikin Counts that are greater than one standard deviation of the mean divided by the number of Replikin Counts that are less than one standard deviation of the mean. The ratio is called a Replikin Count Virus Expansion Index (RCVE Index). Another way to determine the RCVE Index is to divide the percent of Replikin Counts in a plurality of isolates of influenza virus grouped by time and/or region that are higher than one standard deviation of the mean by the percent of Replikin Counts that are lower than one standard deviation of the mean. An RCVE Index may be used to quantify the future risk of an outbreak of influenza by tracking Replikin Counts in strains of influenza over time.
In determining a RCVE Index, the mean Replikin Count of the plurality of isolates from the first time period and first geographic region is considered a control. A control population preferably has a relatively large number of isolates with a relatively small variability in the Replikin Count of the isolates, but any population may be deemed a control when a comparison between the control and a related isolate or plurality of isolates is desired. A control may be related to the population that is being studied. For example, if influenza infection in a bird species, such as swans, is being studied, the control may be something closely related such as chickens, wherein isolates from chickens may be relatively numerous (if available) and relatively stable (if possible) and wherein stability in Replikin Count through the population demonstrates a level of equilibrium between the expansion and contraction of the strain or related strain of influenza virus in chickens. A control may reflect a highest number of isolates reported in a year or in several years in a geographic area. As may be seen in
In determining an RCVE index, any measure of Replikin concentration may be used in influenza or in other pathogens. Replikin Count may reflect the concentration of Replikin peptides identified encoded in the genome of an isolate. Replikin Count may also reflect the concentration of Replikin peptides identified in the expressed proteins of an isolate or in at least one protein or protein fragment of an isolate. Replikin Count may also reflect the concentration of Replikin peptides identified in a Replikin Peak Gene of an isolate. The Replikin Peak Gene of an influenza virus may be any segment of the genome or of any expressed protein or protein fragment having the highest concentration of continuous and/or overlapping Replikin peptides identified.
In many influenza isolates the Replikin Peak Gene is identified in the polymerase area of the influenza virus genome. Within the polymerase area, the Replikin Peak Gene is often identified in the pB1 gene area. Replikin Counts within the pB1 gene may also be used.
Any Replikin peptide, Replikin Peak Gene, protein, protein fragment, or nucleic acid sequence encoding any Replikin peptide, Replikin Peak Gene, protein, or protein fragment in an isolate predicted by the methods of the invention to be expanding may be used for diagnostic, therapeutic, and/or preventive purposes. Further, a vaccine may be manufactured by identifying a portion of the structure or genome of an influenza isolate predicted to expand in population and using that portion in a vaccine composition.
Methods of the invention also provide methods of predicting a decrease in virulence, morbidity, and/or lethality of a strain of influenza and/or predicting a contraction or failure of a strain of influenza wherein a Replikin Count of at least one isolate of a strain of influenza from a second time period and/or second geographic region is less than one standard deviation of the mean of the Replikin Count of a plurality of isolates of influenza from a first time period and first geographic region. A decrease may also be predicted where the number of Replikin Counts of a plurality of isolates from a second period and/or a second geographic region that are greater than one standard deviation of the mean is less than the number of Replikin Counts less than one standard deviation of the mean. A decrease, contraction, or failure is predicted if the ratio of the Replikin Count Virus Expansion Index is less than one.
When a population contains isolates with Replikin Counts above one standard deviation of the mean of a control and does not contain isolates with Replikin Counts below one standard deviation of the mean of the control, the ratio of the RCVE Index is considered to have a denominator of one to avoid an index of infinity.
In determining a Replikin Count Virus Expansion Index, Replikin Counts from Replikin Peak Genes may be analyzed from regions (such as all reporting countries) in a given time period (such as a year) for a range of species. Within a country in a year, there may be a range of values over a range of species. The ordinary skilled artisan may select a mean Replikin Count as a control from the range of values, a time, a region, a species, or any combination thereof (such as a time, a region, and a species, e.g., 2004, China, and chicken). For example, in Example 7 below, the mean Replikin Count of all H5N1 isolates from chickens in China in 2004 was selected as an initial control against which Replikin Counts from swans in China in 2004 were compared. When comparing a control to the Replikin Count of an individual isolate or related group of isolates, a control that shares some similarity with the isolate or group of isolates may be used. For example, a control of all isolates from chicken in China in 2004 may be compared with other isolates from 2004. Likewise, a control of swans from 2005 in Japan may be compared to future isolates from swans in Japan. The ordinary skilled artisan will understand when a control shares similarity with an isolate or group of related isolates such that the control may be used in comparison with the isolate or group of related isolates.
When comparing the Replikin Count of an individual isolate or related group of isolates to a control, all Replikin Count values within the group of related isolates that fall within one standard deviation of the mean may be treated as a group. Additionally, all values that fall outside the range of one standard deviation from the mean may be treated as two outlying groups. A first group is the group of Replikin Counts that are greater than the mean plus one standard deviation. A second group is the group of Replikin Counts that are less than the mean minus one standard deviation. Because higher Replikin Counts are associated with future outbreaks or an expanding virus population and lower Replikin Counts are associated with cessation of outbreaks or decrease or failure of the virus population, the ratio of the percent of isolates having Replikin Counts above mean plus one standard deviation to the percent of isolates having Replikin Counts below the mean minus one standard deviation provides a quantitative index of the viability and expansion of the virus. The index provides a snapshot of current status of the virus population and the propensity for change in that population. If the ratio is greater than one, the RCVE Index predicts an expanding population. If the ratio is less than one, the RCVE Index predicts a contracting or failing virus population.
One aspect of the present invention provides methods of preventing or treating outbreaks of influenza virus by predicting an expansion of a strain of influenza virus using a Replikin Count Virus Expansion Index and administering therapies comprising an isolated or synthesized portion of the structure or genome of the influenza virus identified using the RCVE Index to prevent, mitigate, or treat the outbreak of influenza virus. A prediction of an outbreak may be made by (1) determining a mean Replikin Count with standard deviation for a group of isolates of a strain of influenza isolated during a first time period in a first geographic region, (2) determining a Replikin Count of at least one isolate of the same strain of influenza virus from a second time period and/or second geographic region that is different from the first time period and/or is different from the second geographic region, and (3) predicting an expansion of the strain of influenza isolated in said second time period and/or second geographic region if the Replikin Count of the isolate from a second time period and/or second geographic region is greater than one standard deviation from the mean of the Replikin Count of the plurality of isolates isolated in the first time period and in the first geographic region. An outbreak may be prevented, mitigated, or treated by administering a pharmaceutical compound that includes all or some portion of the structure or genome of the at least one isolate of influenza virus.
The at least one isolate of influenza from a second time period and/or geographic region may be a plurality of isolates from the second time period and/or second geographic region wherein the Replikin Count of each isolate of the plurality of isolates is compared separately to one standard deviation from the mean. Additionally, an outbreak of influenza may be predicted if the number of Replikin Counts of the plurality of isolates from a second period and/or a second geographic region that is greater than one standard deviation of the mean is greater than the number of Replikin Counts less than one standard deviation of the mean.
The portion of the structure or genome may be isolated from an influenza isolate or may be synthesized based on sequences or other structure elucidated from the influenza isolate as well understood by the ordinary skilled artisan. The structure may be a protein or protein fragment that comprises a Replikin peptide or that consists of a Replikin peptide. The structure may comprise or consist of a Replikin Peak Gene or a fragment of a Replikin Peak Gene or may consist of a Replikin peptide identified within a Replikin Peak Gene. The structure may also be a nucleic acid including but not limited to a nucleic acid encoding a Replikin Peak Gene, a Replikin peptide or plurality of Replikin peptides within a Replikin Peak Gene, or a Replikin peptide or plurality or Replikin peptides.
A peptide or mixture of peptides may be comprised in an immunogenic compound for influenza and may include at least one of HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1), KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4), HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12).
Another aspect of the invention provides methods of predicting an increase in the virulence, morbidity, and/or lethality or an expansion of the population of an isolate of a strain of influenza virus as compared to another isolate or group of isolates of the same or a related strain. Such an increase may be predicted by identifying a cycle of Replikin concentration among a plurality of isolates of influenza and identifying a peak in that cycle. An increase is predicted following the time point or time period when the peak is identified or following a rising portion of the cycle. An increase may likewise be predicted following the time point or time period when a peak is identified in two synchronous cycles wherein a first cycle is the cycle of a strain of influenza and the second cycle is a cycle of a different strain of influenza. The increase is predicted following the time period in which the peaks of the synchronous cycles are identified or in a rising portion identified in both synchronous cycles.
A cycle of Replikin concentration or “Replikin cycle” of H9N2 may be seen in
In
The data for
As illustrated in
In
The H9N2 sequences analyzed and reported as mean Replikin Count in Table 4 and in
Two Replikin Count expansion rising portions of cycles are seen in
Additionally, the standard deviations for H9N2 as illustrated in
The data in Table 4 and
The observations of specific Replikins and their concentration in malaria, West Nile virus, foot and mouth disease virus, and influenza virus proteins provides specific quantitative early chemical correlates of outbreaks and increases in mortality 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. Synthesis of these vaccines may be accomplished in seven days or less, which allows for administration of vaccines that are a best fit for a particular virulent strain of virus or organisms including malarial trypanosomes, West Nile virus, foot and mouth disease virus, and influenza virus.
By analyzing the protein sequences of isolates of a virus or other pathogen for the presence, concentration and/or conservation of Replikins, pandemics, epidemics, and other changes in virulence and mortality can be predicted and treatments developed. Furthermore, the severity of such outbreaks can be significantly lessened by administering a peptide vaccine based on the Replikin sequences found to be most abundant or shown to be on the rise in virus isolates over a given time period, such as about one to about three years.
A peptide vaccine of the invention may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in particular virus strains. However, a vaccine may include a conserved Replikin peptide(s) in combination with a new Replikin(s) peptide or may be based on new Replikin peptide sequences. The Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences, although vaccines based on peptides containing only Replikin sequences are preferred. Preferably, vaccine compositions of the invention also contain a pharmaceutically acceptable carrier and/or adjuvant. Among the Replikin peptides for use in a virus or pathogen vaccine are those Replikins observed to “re-emerge” after an absence from the amino acid sequence for one or more years.
The vaccines of 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 shrimp, 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 a virus or organism or a particular strain of virus or organism.
The Replikin peptides of the invention, alone or in various combinations are administered to a subject, in a non-limited embodiment by i.v., intramuscular injection, by mouth, or by spray inhalation, intranasal administration, or intraocular administration. The peptides are administered 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 0.01 μg to about 500 mg, about 0.05 μg to about 200 mg, or about 0.075 μg to about 30 mg, from about 0.09 μg to about 20 mg, from about 0.1 μg to about 10 mg, from 10 μg to about 1 mg, and 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.
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 or for diagnostics. See, e.g., U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 and U.S. application Ser. No. 12/010,027, filed Jan. 18, 2008 (each incorporated herein by reference in their entirety).
Publicly available sequences of isolates of P. falciparum at www.pubmed.com were analyzed using proprietary search tool software (ReplikinForecast™ available in the United States from REPLIKINS LLC, Boston, Mass.) for years 1986 to 2007 to determine the mean Replikin Count for the histidine-rich protein of all isolates available in each of those years. Mean annual Replikin Counts for each year were then compared with changes in mortality as reported by the World Health Organization.
A list of the accession numbers analyzed for the presence and concentration of Replikin sequences is provided in Table 5 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.
Analysis of the annual mean Replikin Count of the histidine rich protein between 1986 and 2007 revealed cycles of Replikin Count. The beginning of a new cycle provides a prediction that Replikin Count may continue to increase along with an increase in malaria mortality rate. The data is graphically illustrated in
Replikin peptides in an isolate identified at a peak or in a rising portion of the Replikin cycles revealed in
Applicants analyzed publicly available sequences of the ATP-ase enzyme of isolates of P. falciparum at www.pubmed.com. The data is summarized below in Table 6 and illustrated in
Envelope protein sequences from isolates of West Nile virus isolated between 2000 and 2008 that were publicly available at www.pubmed.com were analyzed for Replikin sequences and a mean annual Replikin Count was determined. The data are contained in Table 7 below and illustrated in
2000 to 2003: The standard deviation of the mean of the Replikin Count of the envelope protein increases markedly from 2000 to 2001 (p<0.001). This change has been observed in all common strains of influenza virus (not the same virus genus as WNV) to signal rapid replication and expansion of the range of the Replikin Count, thus virus population expands with Replikin Count and precedes virus outbreak. The increase in the mean Replikin Count from 2000 to 2003 appears to accompany, or precede, the increase in the number of human WNV cases recorded independently and published by the Center for Disease Control (CDC). The same relationship of Replikin Count to morbidity has been shown in influenza strains, for example H5N1 to human mortality, and in H3N8 equine encephalitis to horse morbidity, and in the trypanosome Plasmodium falciparum (malaria) to human morbidity, and to mortality rate in shrimp with shrimp taura syndrome virus. Since the relationship has already been demonstrated in several species, including crustaceans, horses, and humans, it appears to be a broadly distributed general principle. 2004 to 2007: In 2004 and 2005, there was a decrease from 2003 in both the Replikin Count and the number of human cases of WNV. In 2006, there was an increase in the Replikin Count followed by an increase in 2007 of the number of human cases.
In
The rising numbers for both the Replikin Count and the number of cases in the second rising portion of the cycle, 2004-2008, when compared to the first rising portion of the cycle, suggests an increased or ‘improved’ infective efficiency accompanying an increased Replikin Count in the second rising portion, compared to the first. The drop in efficacy of the virus is probably due to the generation of resistance in the host; the subsequent rise in infectivity in the second rising portion of the cycle is related to the appearance of new Replikins identified in WNV. Once again, the close relationship of Replikins to infectivity is demonstrated; both literally rise and fall together.
Thus the present data provide direct quantitative evidence of the relationship of Replikins to infectivity at a more accurate level than previously available. For example, in the case of H5N1 influenza, the cycle began in 1996, with the Hong Kong outbreak. It was temporarily ended in 1998 by the complete culling of chickens in Hong Kong. The H5N1 clinical ‘sub-cycle’ resumed in 2000, continued to the present, and was predicted prospectively each year by the Replikin Count. In this case, occurring mostly in East Asian countries, H5N1 was not as subject to exact epidemiological reports by the WHO of morbidity and mortality as in the case of West Nile Virus in the U.S. as here presented, since the CDC keeps much more accurate surveillance records of the morbidity and mortality.
While not wishing to be limited by theory, the close relationship of Replikin Count to morbidity and mortality, and other evidence, has led to the hypothesis that Replikins, in addition to being closely involved in the biochemistry of rapid replication, are in fact infective units, that the viruses and trypanosomes are merely carriers of the Replikin infective units, but that other virus or trypanosome structures are needed to produce infectivity in the host.
Accession numbers, number of isolates, mean Replikin Count, standard deviation and significance for accession numbers available for West Nile virus envelope protein from www.pubmed.com are contained below in Table 7. Specific conserved Replikin sequences identified within the step-wise cycles of West Nile virus in
All protein sequences from isolates taken between 1999 and 2008 that were publicly available at www.pubmed.com were analyzed for Replikin sequences and a mean annual Replikin Count was determined. The data are contained in Table 3 above and illustrated in
To provide the data in
The following Replikin peptide sequences were identified for vaccines: HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18). These sequences have been observed to be conserved in the Replikin cycles illustrated in
The two above-listed conserved Replikin peptides have been identified and tracked annually in publicly available sequences in foot and mouth disease virus at www.pubmed.com from 1934 through 2008. The sequence HKQKIIAPAK (SEQ ID NO: 17) is observed to be conserved 100% of the time in the publicly available sequences from isolates from 1934 through 2008. The sequence HKQKIVAPVK (SEQ ID NO: 18) is observed also to be conserved in 100% of isolates from 1934 through 2007 with the exception of two substitutions, namely a valine at residue 6 in the peptide and a valine at residue 9 in the peptide.
Table 8 provides the accession numbers at www.pubmed.com wherein sequence HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18) were conserved over time. The residue at which the peptide begins in the sequence disclosed in the accession number is noted.
As discussed above in Example 3, Applicants analyzed the Replikin concentration of West Nile virus envelope protein isolates publicly available in accession numbers of www.pubmed.com. As seen in
For example, as mean annual Replikin concentration increased between 2000 and 2003, West Nile virus morbidity expanded initially from New York and certain contiguous states in 2000, to the Northeast and Southeast in 2001, to most states except the Mountain states and Northwest in 2002, and to all states but the Northwest in 2003. See, e.g., annual maps available from the CDC at http://www.cdc.gov/ncidod/dvbid/westnile/surv&control.htm#maps. When the mean annual Replikin concentration began to fall in 2004, West Nile Virus was present in all continental U.S. states but with a much lower rate of morbidity. In 2005, West Nile virus infections were observed to retreat from certain parts of the U.S. and infections were not observed in Washington State, northern New England, or West Virginia. However, as annual mean Replikin concentrations began to increase again in 2006, West Nile virus morbidity again spread to all states except northern New England.
A review of the progression of West Nile virus across the United States from its first observation in New York reveals that monitoring changes in Replikin concentration provides evidence of geographic expansion of West Nile Virus. An aspect of the invention, therefore, is the prediction of an expansion into a geographic region or contraction from a geographic region based on a determination of the progression of mean annual Replikin concentrations in a graph of a cycle or series of cycles of Replikin concentration including observed step-wise cycles. For example, a peak in Replikin concentration in a cycle of Replikin concentration of a plurality of isolates from a given region provides evidence of expansion beyond the geographical area of that region into other contiguous or nearby geographical areas. Furthermore a second, still higher, peak provides even greater evidence of a pathogen that is poised for expansion.
The phenomenon of geographical expansion also applies to malaria and other pathogens. Analysis of the Replikin concentration of a Replikin Peak Gene, histidine-rich protein, or ATP-ase of P. falciparum demonstrates that Replikin concentration cycles may provide a prediction of an expansion of P. falciparum mortality and/or morbidity. For example, if a Replikin concentration cycle based on isolates from a particular region demonstrates a prolonged rise in mean annual Replikin Count or a peak following a rise in mean annual Replikin Count, the significant rise or peak predicts an expansion of the mortality rate or morbidity rate of that isolate into contiguous or nearby regions that until the significant rise or peak in Replikin Count did not experience the mortality rate or morbidity rate of the particular region.
For example, a cycle of Replikin concentration is established in the Sahel region of Africa with two peaks at years 2 and 7. The second peak at year 7 is significantly higher than the first peak at year 2 with a p value of 0.01. The Sahel region between years 0 and 7 has experienced a higher rate of mortality than more southerly regions. Based on the higher peak at year 7, it is predicted that the mortality from malaria will increase in the region contiguous to the south of the Sahel. A plurality of Replikin sequences are isolated from year 7 isolates. Replikins that have been conserved between years 0 and 7 are selected as vaccines for malaria in the Sahel and contiguous regions to the south. Replikins that are new in year 7 are likewise selected as vaccines. A mixture of these Replikin sequences is combined with a pharmaceutically acceptable carrier and/or adjuvant and administered to a subject to produce an immune response to treat and/or protect against malaria predicted to have a higher mortality rate following the dry season in year 8 in the Sahel and in its contiguous regions to the south.
Applicants analyzed all amino acid sequences of the pB1 gene area of isolates of H5N1 strains of influenza virus publicly available at www.pubmed.com for specimens isolated between 2004 and 2008. Isolates were grouped by species of bird within countries for each year in which sequences were available.
The concentration of continuous and overlapping Replikin peptides in the pB1 gene area was determined for each isolate (the Replikin Count of the Replikin Peak Gene). Within each year in each country a mean Replikin Count with standard deviation was determined. China was found to have the largest number of isolates for each year from 2004 to 2008 and the mean Replikin Count (with standard deviation) of all H5N1 isolates from chicken in China in each year was chosen as a control against which other Replikin Counts would be determined (China was chosen as a control because of a limited variability in Replikin Count among a very large number of isolates available for analysis).
The Replikin Count for each individual isolate in a given country in a given year was compared to one standard deviation from the mean Replikin Count for all isolates from chicken in China in that year. Within each country, the number of Replikin Counts greater than one standard deviation of the mean and the number of Replikin Counts less than one standard deviation of the mean were determined. For each country in each year, the percent of Replikin Counts greater than one standard deviation of the mean was then divided by the percent of Replikin Counts less than one standard deviation of the mean to provide a ratio, or Replikin Count Virus Expansion (RCVE) Index. In countries having an RCVE Index of greater than one, an expansion of H5N1 was predicted for the following year or years. In countries having a RCVE Index of less than one, a contraction or viral failure was predicted for the following year or years.
Five sets of RCVE Indices are calculated and reported below as examples for the ordinary skilled artisan. The ordinary skilled artisan will understand how to repeat the predictive methods for data from any region, time, or species and will understand from the disclosure herein how to practice methods of prevention, mitigation, and treatment for outbreaks predicted by the RCVE Indices including therapeutic compounds identified in isolates predicted to be expanding in population.
In Tables 9-13 below, individual Replikin Counts that are above the reported standard deviation of the mean of the control are bolded. Individual Replikin Counts that are below the reported standard deviation of the mean of the control are italicized and bolded. The RCVE Index ratio is reported for each group of isolates as compared to the control.
In Table 9, Replikin Counts for individual H5N1 isolates from swans in China for 2004 are compared to a control of the annual mean Replikin Count for all chicken H5N1 isolates from China in 2004.
The RCVE Index for swans in China in 2004 is 12.5/0. Because zero is set as 1 when it is in the denominator, the index returns a ratio of 12.5, which predicts an expanding population. This predicted expansion is seen below in Table 11 in an expanding population in swans in China in 2006.
In Table 10, Replikin Counts for individual isolates from swans in Mongolia, Russia, and Japan in 2005 are compared to a control of the annual mean Replikin Count for all H5N1 chicken isolates from China in 2005.
3.6, 3.7, 3.7, 3.7, 3.7,
, 2.0, 1.7,
3.7, 3.1, 1.7, 1.8, 7.1,
, 3.1, 7.1, 2.1, 3.1,
,
,
,
, 2.3,
The RCVE Index for swans in Mongolia in 2005 is 1.3, which predicts an expansion of the H5N1 population in Mongolia, because the RCVE Index is greater than 1. This predicted expansion from Mongolia is seen below in European countries, such as Sweden and Denmark, known to be in the flight path for swans and other birds from Mongolia.
In Table 11, Replikin Counts for individual isolates from a variety of bird species in eight different countries are compared to a control of the annual mean Replikin Count for all H5N1 chicken isolates from China in 2006. In Denmark, duck, swan, and falcon isolates are reported. In Czech Republic, turkey and falcon isolates are reported. All other non-control isolates are from swans.
3.8
3.6
4.3
3.6
3.7
3.6
4.3
3.6, 3.7
4.3
3.7
3.6, 3.7
22.2
4.3
3.9
17.8
17.8
3.9
3.7
3.7
17.8
3.9
The RCVE Index predicts expansion in Germany, Slovenia, Scotland, Czech Republic, and China. The Index predicts contraction or failure in Sweden, Denmark, and Mongolia. It is noteworthy that the index predicts contraction or failure of the H5N1 influenza population in swans in Mongolia in 2006 while in 2005 the index of 1.3 predicted expansion. In 2007, as predicted in 2006, no H5N1 isolates were reported in Mongolia. See Table 12 below.
In Table 12, Replikin Counts for individual isolates from swans in Japan for 2007 are compared to a control of the annual mean Replikin Count for all chicken H5N1 isolates from China in 2007.
The RCVE Index for swans in Japan in 2007 is 50/0. Because zero is set as 1 when it is in the denominator, the index returns a ratio of 50, which predicts an expanding population. So despite a small sample size, the index predicts expansion, which is seen below in Table 13 in an expanding population in swans in Japan.
In Table 13, Replikin Counts for individual isolates from swans in Japan for 2008 are compared to a control of the annual mean Replikin Count for all chicken H5N1 isolates from China in 2008. Only 3 isolates from chicken in 2008 were reported and available for analysis.
3.7, 3.7, 3.7, 3.7, 3.8, 3.8, 3.8, 3.8, 3.8,
3.8, 3.8, 3.8, 4.5, 17.8, 17.8, 17.8,
,
,
,
,
,
,
,
,
The RCVE Index for swans in Japan in 2008 is 1.8, which predicts future expansion of influenza in swans in Japan.
The RCVE Indices as described above may be practiced by one of ordinary skill in the art as a measure of the current survival and expansion status or contracting/failing status of a population of pathogen engaged in an outbreak. The ordinary skilled artisan may isolate in silico the Replikin Peak Gene, may measure the Replikin Count of the Replikin Peak Gene, and may compare the Replikin Count data of related strains of virus in other geographic regions in the same and previous time periods to understand the severity of the outbreak, the direction of the outbreak, and the attendant risk to neighboring geographic regions. Like identifying and tracking a hurricane, the appreciable advantage to the ordinary skilled artisan is time to develop therapies and to institute public health measures known now or hereafter such as isolation and culling of poultry, vaccination, and other measures. The methods disclosed herein further provide the ordinary skilled artisan with time to manufacture the synthetic Replikin vaccines disclosed herein.
All publicly available sequences of the histidine rich protein gene of isolates P. falciparum from 2004 through 2008 are analyzed for Replikin concentration. Isolates are grouped by region.
Within each year and in each region a mean Replikin Count with standard deviation is determined. The region having the largest number of isolates or the least variability among Replikin Count in isolates (or both) for each year from 2004 to 2008 is chosen as a control against which other Replikin Counts are analyzed. The Replikin Count for each individual isolate in a given region in a given year is compared to one standard deviation from the mean Replikin Count for all isolates from the control region. Within each region, the number of Replikin Counts greater than one standard deviation of the mean and the number of Replikin Counts less than one standard deviation of the mean is determined. For each region in each year, the percent of Replikin Counts greater than one standard deviation of the mean is then divided by the percent of Replikin Counts less than one standard deviation of the mean to provide a ratio, or Replikin Count Expansion (RCE) Index. In regions having an RCE Index of greater than one, an expansion of malaria is predicted for the following year or years. In regions having an RCE Index of less than one, a contraction of malaria is predicted for the following year or years.
In regions wherein malaria is predicted to expand, a Replikin Peak Gene is identified in an isolate having a Replikin Count that is higher than the mean Replikin Count for the region. The Replikin Peak Gene and/or a Replikin peptide (or plurality of Replikin peptides) within the Replikin Peak Gene is selected as an immunogenic compound for diagnostic and/or therapeutic purposes. A vaccine against the expanding population is manufactured comprising the immunogenic compound. The vaccine is administered to mitigate the expanding malarial population.
All publicly available sequences of the VP1 gene of isolates of Foot and Mouth Disease Virus Type O from 2004 through 2008 are analyzed for Replikin concentration. Isolates are grouped by region.
Within each year and in each region, a mean Replikin Count with standard deviation is determined. The region having the largest number of isolates or the least variability among Replikin Count in isolates (or both) for each year from 2000 to 2008 is chosen as a control against which other Replikin Counts are analyzed. The Replikin Count for each individual isolate in a given region in a given year is compared to one standard deviation from the mean Replikin Count for all isolates from the control region. Within each region, the number of Replikin Counts greater than one standard deviation of the mean and the number of Replikin Counts less than one standard deviation of the mean is determined. For each region in each year, the percent of Replikin Counts greater than one standard deviation of the mean is then divided by the percent of Replikin Counts less than one standard deviation of the mean to provide a ratio, or Replikin Count Virus Expansion (RCVE) Index. In regions having a RCVE Index of greater than one, an expansion of foot and mouth disease is predicted for the following year or years. In regions having a RCVE Index of less than one, a contraction of foot and mouth disease is predicted for the following year or years.
In regions wherein foot and mouth disease is predicted to expand, a Replikin Peak Gene is identified in an isolate having a Replikin Count that is higher than the mean Replikin Count for the region. The Replikin Peak Gene and/or a Replikin peptide (or plurality of Replikin peptides) within the Replikin Peak Gene is selected as an immunogenic compound for diagnostic and/or therapeutic purposes. A vaccine against the expanding population is manufactured comprising the immunogenic compound. The vaccine is administered to mitigate the expanding foot and mouth disease virus population.
A synthetic Replikin vaccine containing an approximately equal-parts-by-weight mixture of twelve H5N1 Replikin peptides was tested in chickens against a low pathogenic strain of H5N1 isolated from a black duck in North Carolina, USA. Low-Path H5N1 strains infect migratory birds and impair health and productivity of commercial flocks of U.S. chickens, usually with little mortality in the commercial flocks. These Low-Path H5N1 strains are very closely related in virus structure to their more lethal High-Path H5N1 relatives in Eurasia. A mutation from a Low-Path to a High-Path strain has so far not been observed but mutations of this type over time may be expected by one of skill in the art.
The tested vaccine was engineered to block both the entry site of H5N1 virus and the replication site of those H5N1 viruses that manage to enter into host cells. As such, the vaccine is called the TWO-PUNCH vaccine. As demonstrated below, evidence from the described test of the TWO-PUNCH vaccine in chickens suggests that both mechanisms for which the vaccine was designed were effective: (1) virus entry into inoculated chickens was diminished by immunity from the vaccine and (2) virus replication within infected cells was sufficiently limited by immunity from the vaccine to block excretion of the virus in feces of tested birds.
The TWO-PUNCH Replikins Vaccine is based on influenza Replikin peptides shared between influenza strains and conserved for decades within influenza strains. The vaccine was engineered as a mixture of twelve Replikin peptides identified as expressed from the genome of H5N1 virus. Six of the Replikin peptides are synthesized according to sequences isolated from the hemagglutinin protein of H5N1, which is involved in attachment and entry of influenza virus into a cell. Six of the Replikin peptides are synthesized according to sequences isolated from the pB1 gene area of H5N1, which has been identified as involved in replication of influenza virus in a host cell.
The following six Replikin sequences contained in the vaccine were isolated from the hemagglutinin protein:
The following six Replikin sequences contained in the vaccine were isolated from the pB1 gene area:
The vaccine comprises an approximate equal-parts-by-weight mixture of the twelve peptides. The following peptide amounts were combined to create an initial mixture of the vaccine:
The total amount of the mixture was 2237.1 mg.
The peptide mixture was then divided into three equal parts for administration of the vaccine on three different days (days 1, 7, and 28). After dissolution with water, the three equal parts were administered to individual birds in two groups of 20 birds each for a total administration at each day of 40 birds. The total amount of active peptide ingredient administered to each bird at the time of administration (either intranasally and intraocularly or via spray inhalation) was about 18.6 mg per bird per administration.
The vaccine solution was administered to chickens intranasally at a first administration on day 1 after hatch, intraocularly at a second administration on day 7 after hatch, and via fine spray inhalation at a third administration on day 14 after hatch.
Chickens on the first day of life were separated into four groups with twenty chickens per group. The first group was a control group not vaccinated and not challenged with Low-Path H5N1. The second group was vaccinated and not challenged with Low-Path H5N1. The third group was vaccinated and subsequently challenged with Low-Path H5N1. The fourth group was not vaccinated and was challenged with Low-Path H5N1.
For those chickens that were vaccinated, the synthetic H5N1 Replikins Vaccine was administered intranasally on day 1 after hatch, administered intraocularly on day 7 after hatch, and administered by fine spray inhalation on day 14 after hatch. The groups of challenged chickens were than challenged with Low-Path H5N1 virus on day 28 of the life of the chicken. Serum from selected chickens was analyzed in all groups for antibodies against the H5N1 virus on days 7, 14, and 21 following challenge. PCR for virus fecal excretion was also analyzed for all groups.
Unvaccinated control chickens demonstrated both an expected high virus entry (as indicated by a high titer of antibodies against H5N1) and an expected high virus replication (as indicated by high fecal and salival excretion of the virus detected by PCR). In contrast, the vaccinated chickens demonstrated lower virus entry (as indicated by a low titer of antibodies against H5N1 or by the observation of no antibodies against H5N1 in serum) and an absence of fecal or saliva excretion of virus indicating low or no virus replication in the vaccinated chickens. The data suggest, therefore, that the virus was partially blocked on entry by the chickens' immune response to the vaccine and the limited amount of virus that did enter the chicken's system was blocked from sufficient replication in the chickens' host cells to excrete virus in the feces or saliva.
The data in Table 14 below provide the numbers of chickens tested in each of the four groups (Negative Control, Vaccinated, Vaccinated and Challenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (not vaccinated)) on a particular test day and the numbers of chickens in which production of antibodies to H5N1 was detected with a serum titer.
The data in Table 15 below provide the number of chickens tested for H5N1 virus in their saliva and feces in each of the four groups (Negative Control, Vaccinated, Vaccinated and Challenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (not vaccinated)) on a particular test day and the numbers of chickens in which H5N1 was detected in their feces and saliva based on PCR analysis.
The data in Tables 14 and 15, demonstrate the effectiveness of the double-protective mechanism of the TWO-PUNCH vaccine. First, while several non-vaccinated chickens challenged with H5N1 excreted virus in their feces and saliva, no vaccinated chickens challenged with H5N1 excreted virus in their feces or saliva. See Table 15. These data demonstrate that the vaccine provided a protective effect against replication of the virus. Second, while four of seven unvaccinated chickens challenged with H5N1 were producing serum antibodies against H5N1 on day 7, seven of nine unvaccinated chickens challenged with H5N1 were producing serum antibodies against H5N1 on day 14, and three of nine unvaccinated chickens challenged with H5N1 were producing serum antibodies against H5N1 on day 28, only one of seven vaccinated and challenged chickens was producing serum antibodies against H5N1 on day 7, only three of six vaccinated and challenged chickens were producing serum antibodies against H5N1 on day 14, and only two of seven vaccinated and challenged chickens were producing serum antibodies against H5N1 on day 21. See Table 14. These data demonstrate that for some of the vaccinated chickens, the H5N1 virus challenge was stopped prior to entry into the chicken's system (likely by antibodies produced at the mucus membranes). These data further demonstrate that for those vaccinated and challenged chickens in which the virus entered the system (resulting in production of serum antibodies), the virus was nonetheless not excreted in feces or saliva.
As may be seen from the data in Table 14, almost all of the non-vaccinated challenged birds seroconverted (producing detectable antibody). This demonstrates infection of the non-vaccinated birds. On the other hand, only some of the vaccinated challenged birds seroconverted. Further, for those vaccinated birds that did seroconvert, the antibody titers were low. Additionally, the negative control group had no seroconversion. These data demonstrate a protective effect of the vaccine on the birds.
Additionally, Table 15 demonstrates the absence of detectable influenza in the feces and saliva of vaccinated birds. That viral excretion was blocked by this influenza Replikins vaccine is particularly significant because it is generally acknowledged that the maintenance of reservoirs of H5N1 virus in flocks of migratory birds and domestic chickens in both Asia and the U.S. (and the regional spread of H5N1 virus from these reservoirs) is dependent on viral excretions picked up by neighboring chickens and birds. Regardless of the level of lethality of a strain of H5N1 virus, absent excretion of virus, there is expected to be no spread of the virus.
As such, data observed from administration of the TWO-PUNCH Replikin peptide vaccine in chickens demonstrates the efficacy of the vaccine as (1) a barrier to entry of the virus, (2) a block of replication of the virus, and (3) a block of fecal spread of the virus.
This application claims priority to U.S. Provisional Appln. Ser. No. 61/143,618, filed Jan. 9, 2009, U.S. Provisional Appln. Ser. No. 61/087,354, filed Aug. 8, 2008, U.S. Provisional Appln. Ser. No. 61/054,010, filed May 16, 2008, U.S. application Ser. No. 12/108,458, filed Apr. 23, 2008, and PCT/US2008/61336, filed Apr. 23, 2008, each of which is incorporated herein by reference in its entirety. This application additionally incorporates herein by reference: U.S. application Ser. No. 12/010,027, filed Jan. 18, 2008, 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, U.S. Provisional Appln. Ser. No. 60/880,966, filed Jan. 18, 2007, 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, now U.S. Pat. No. 7,442,761, U.S. application Ser. No. 10/189,437, filed Jul. 8, 2002, now U.S. Pat. No. 7,452,963, 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, now U.S. Pat. No. 7,420,028, 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 | |
|---|---|---|---|
| 61054010 | May 2008 | US | |
| 61087354 | Aug 2008 | US | |
| 61143618 | Jan 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2008/061336 | Apr 2008 | US |
| Child | 12429044 | US | |
| Parent | 12108458 | Apr 2008 | US |
| Child | PCT/US2008/061336 | US |
