# Patent

## Title
Synthetic replikin peptides against pathogenic infection of invertebrates in aquaculture

## Abstract

The present invention provides isolated or synthesized peptides of about 7 to about 50 amino acids identified in the genome of pathogens in invertebrates in aquaculture for prevention and treatment of outbreaks of these pathogens in aquaculture and methods of preventing and treating pathogenic outbreaks using the identified peptides.

## Background
This application claims priority to 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, and U.S. application Ser. No. 11/755,597, filed May 30, 2007, each of which is incorporated herein by reference in its entirety. This application additionally incorporates herein by reference: U.S. Provisional Appln. Ser. No. 60/898,097, 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, U.S. application Ser. No. 10/189,437, filed Jul. 8, 2002, U.S. application Ser. No. 10/105,232, filed Mar. 26, 2002, now U.S. Pat. No. 7,189,800, U.S. application Ser. No. 09/984,057, filed Oct. 26, 2001, and U.S. application Ser. No. 09/984,056, filed Oct. 26, 2001, now U.S. Pat. No. 7,176,275, each in its entirety.


Aquaculture refers generally to the breeding and raising of aquatic animals for food. Aquaculture is a rapidly expanding global industry in the 21st century. The most significant cause of economic loss in the industry is disease. Meyer, F. P., J Anim Sci 1991. 69:4201-4208. Aquaculture, and in particular invertebrate aquaculture, frequently involves short-term holding of dense numbers of animals in pens or tanks before marketing or to induce molting. Id. These concentrations are stressful for the invertebrates such as shrimp, clams, oysters, lobster, scallops, abalone, etc. The stressful conditions are ideal for transmission of pathogens including bacteria, such as Vibrio, Chlamydia-like, and Rickettsia-like species, viruses, such as taura syndrome virus and white spot syndrome virus, and other microbial pathogens.
One particularly severe viral disease in aquaculture is taura syndrome, which significantly impacts the shrimp farming industry worldwide. Taura syndrome is caused by the taura syndrome virus (TSV), which is a member of the Discistroviridae family in the genus Cripavirus and has a single positive stranded genome of about 10,000 nucleotides. The genome contains two open reading frames (ORF). ORF1 reportedly contains coding for a helicase, a protease and an RNA-dependent RNA polymerase. ORF2 reportedly contains coding for three capsid proteins.
Taura Syndrome is now considered endemic in the Americas and outbreaks have been observed in Asia. Infected shrimp generally have a red tail, are anorexic and erratic in their behavior, tail muscles may become opaque, and the cutical may become soft. Mortality rates between 5% and 95% have been observed during the acute phase of the disease. Shrimp that survive outbreaks of TSV seem to be refractory to reinfection while remaining infectious.
Rapid replication is characteristic of virulence in certain bacteria, viruses and malignancies including TSV and other viral and bacterial diseases in aquaculture such as Chlamydia-like diseases and white spot syndrome, a viral disease. The inventors have found a family of conserved peptide sequences related to rapid replication, designated Replikins. The inventors have correlated an increase in the concentration of Replikin peptides in strains of influenza and other viruses with increased viral virulence. There is a need in the art for methods of preventing and treating pathogenic infections by manipulating the replicating function of Replikin sequences and for identifying molecular targets related to the replicating function of Replikin sequences for treatment of virulent infections, including vaccines and other therapies. Additionally, there is need in the art for reducing the time required for development of vaccines and other therapies to emerging pathogens that rapidly expand through a population or are highly mutable or both. The present time frame of three to twelve months for development of a vaccine often delivers vaccines after an epidemic has long since ended or, if still active, after mutations in the pathogen have rendered a vaccine less effective or useless. These problems in the art are a significant worry to human and animal health professionals and governments.
SUMMARY OF THE INVENTION
The present invention provides synthetic peptide sequences and isolated, synthetic or synthesized Replikin peptide sequences for prevention and treatment of outbreaks of pathogens in invertebrates in aquaculture, and methods of administering to the invertebrates in aquaculture one or more substances comprising or consisting of one or more of the isolated, synthetic or synthesized Replikin peptide sequences to prevent and/or treat outbreaks of pathogens in the invertebrates.
A first non-limiting aspect of the present invention provides a substance for use in aquaculture comprising at least one synthetic peptide of about seven to about fifty amino acids wherein the at least one synthetic peptide, when administered to at least one invertebrate that is capable of culture in water, increases the resistance of that invertebrate to at least one pathogen. In a non-limiting embodiment, the at least one invertebrate is a crustacean. In a further non-limiting embodiment, the at least one crustacean is a shrimp.
A further non-limiting embodiment of the first aspect of the invention provides a substance for increasing the resistance of an invertebrate in aquaculture comprising at least one isolated or synthetic peptide, which is at least one isolated or synthetic form of at least one peptide from a pathogen to said invertebrate, wherein said at least one isolated or synthetic peptide is at least one isolated or synthetic Replikin peptide consisting of 7 to about 50 amino acids comprising a Replikin motif wherein said Replikin motif comprises:

    
    
        (1) at least one lysine residue located at a first terminus of said motif and a least one lysine residue or at least one histidine residue located at a second terminus of said motif;
        (2) at least one lysine residue located six to ten residues from a second lysine residue;
        (3) at least one histidine residue; and
        (4) at least six percent lysine residues.
    
    


In a further non-limiting embodiment, the at least one isolated or synthetic Replikin peptide consists of the Replikin motif. In a further non-limiting embodiment, the Replikin motif consists of about 7 to about 10, about 10 to about 15 amino acids, about 15 to about 20 amino acids, about 20 to about 25 amino acids, about 25 to about 30 amino acids, about 30 to about 35 amino acids, about 35 to about 40 amino acids, about 40 to about 45 amino acids, or about 45 to about 50 amino acids.
In a further non-limiting embodiment of the invention, the at least one isolated or synthetic Replikin peptide comprises any one of SEQ ID NOS: 1-11, 86, 87, 103-112, and 114-198. In a further non-limiting embodiment, the substance comprises a mixture of one or more of the isolated or synthetic Replikin peptides of SEQ ID NOS: 1-11, 86, 87, 103-112, and 114-198. In a further non-limiting embodiment, the at least one isolated or synthetic Replikin peptide comprises KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), or HVEKIVDKAK (SEQ ID NO: 11). In a further non-limiting embodiment, the substance of the first aspect of the invention comprises a mixture of any two or more of the synthetic Replikin peptides. In a further non-limiting embodiment, the substance comprises an equal mixture by weight of the synthetic Replikin peptides.
A second non-limiting aspect of the invention provides a vaccine comprising a substance for use in aquaculture, comprising at least one isolated or synthetic peptide of about 7 to about 50 amino acids, wherein the at least one isolated or synthetic peptide, when administered to at least one invertebrate capable of culture in water, increases the resistance of the invertebrate to at least one pathogen.
In a non-limiting embodiment of the second aspect of the invention, the at least one isolated or synthetic peptide of the substance of the vaccine is at least one Replikin peptide wherein the Replikin peptide consists of 7 to about 50 amino acids and comprises a Replikin motif comprising:

    
    
        (1) at least one lysine residue located at a first terminus of said motif and a least one lysine residue or at least one histidine residue located at a second terminus of said motif;
        (2) at least one lysine residue located six to ten residues from a second lysine residue;
        (3) at least one histidine residue; and
        (4) at least six percent lysine residues.

In a further embodiment, the vaccine further comprises a pharmaceutically acceptable carrier.

    
    


In a further non-limiting embodiment of the second aspect of the invention, the vaccine provides protection against at least one pathogen in an invertebrate, such as a crustacean or mollusk. In a further non-limiting embodiment, the vaccine provides protection against at least one pathogen in a shrimp. In a further non-limiting embodiment, the vaccine is mixed with feed for shrimp for administration to shrimp. In a further non-limiting embodiment, the vaccine is mixed with the daily ration of feed for shrimp. In a further non-limiting embodiment, the pathogen is a virus. In a further non-limiting embodiment, the pathogen is a taura syndrome virus.
In a further non-limiting embodiment of the second aspect of the invention, at least one isolated or synthetic peptide of the vaccines is any one of SEQ ID NOS: 1-11, 86, 87, 103-112, and 114-198. In a further non-limiting embodiment, the vaccine comprises a mixture of one or more of the isolated or synthetic Replikin peptides of SEQ ID NOS: 1-11, 86, 87, 103-112, and 114-198.
In a further non-limiting embodiment of a second aspect of the invention, at least one synthetic peptide of the vaccine is KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), or HVEKIVDKAK (SEQ ID NO: 11). In a further non-limiting embodiment, the vaccine comprises a mixture of any two or more of the synthetic peptides. In a further non-limiting embodiment, the vaccine comprises a mixture of each of the synthetic peptides. In a further non-limiting embodiment, the vaccine comprises an equal mixture by weight of each of the synthetic peptides.
In a further non-limiting embodiment of the second aspect of the invention, the vaccine is administered to at least one shrimp at about 0.001 mg to about 10 mg of vaccine per gram of body weight of each treated shrimp per day. In a further non-limiting embodiment, the vaccine is administered to at least one shrimp at about 0.005 mg to about 5 mg of vaccine per gram of body weight of each treated shrimp per day. In a further non-limiting embodiment, the vaccine is administered to at least one shrimp at about 0.01 mg to about 2 mg of vaccine per gram of body weight of each treated shrimp per day. In a further non-limiting embodiment, the vaccine is administered to at least one shrimp at about 0.02 mg to about 1.5 mg of vaccine per gram of body weight of each treated shrimp per day. In a further non-limiting embodiment, the vaccine is administered to at least one shrimp at about 0.08 mg to about 1.0 mg of vaccine per gram of body weight of each treated shrimp per day. In a further non-limiting embodiment, the vaccine is administered to at least one shrimp at about 0.1 mg to about 0.9 mg of vaccine per gram of body weight of each treated shrimp per day. In a further non-limiting embodiment, the vaccine is administered to at least one shrimp at about 0.2 mg to about 0.8 mg of vaccine per gram of body weight of each treated shrimp per day. In a further non-limiting embodiment, the vaccine is administered to at least one shrimp at about 0.5 mg of vaccine per gram of body weight of each treated shrimp per day.
In a further non-limiting embodiment of the second aspect of the invention, the at least one isolated, synthesized or synthetic Replikin peptide of a vaccine of the invention is at least one isolated, synthesized or synthetic form of at least one Replikin peptide present in an emerging strain of taura syndrome virus.
In a further non-limiting embodiment of the second aspect of the invention, a vaccine of the invention is administered to shrimp as a prophylactic therapy prior to the onset of symptoms of taura syndrome virus. In a further non-limiting embodiment, the vaccine of the invention is administered to shrimp as a prophylactic therapy after the onset of symptoms of taura syndrome virus. In a further non-limiting embodiment, the vaccine is administered at sub-therapeutic concentrations. In a further non-limiting embodiment, the vaccine is administered over substantially all of the life cycle of at least one shrimp.
A third non-limiting aspect of the present invention provides a method of providing resistance in an invertebrate in aquaculture, comprising administering a substance comprising at least one synthetic peptide, wherein the at least one synthetic peptide, when administered to at least one invertebrate that is capable of culture in water, is capable of increasing resistance to at least one pathogen. In a non-limiting embodiment, the substance is administered orally, via submersion of the invertebrate in an aqueous medium containing the substance, or via injection. In a further non-limiting embodiment, the substance is administered orally. In a non-limiting embodiment, the invertebrate is a crustacean. In a further non-limiting embodiment, the crustacean is a shrimp.
A fourth non-limiting aspect of the present invention provides an isolated, synthetic or synthesized taura syndrome virus Replikin peptide consisting of 7 to about 50 amino acids wherein the Replikin peptide comprises a Replikin motif comprising:

    
    
        (1) at least one lysine residue located at a first terminus of said motif and a least one lysine residue or at least one histidine residue located at a second terminus of said motif;
        (2) at least one lysine residue located six to ten residues from a second lysine residue;
        (3) at least one histidine residue; and
        (4) at least six percent lysine residues.

In a further non-limiting embodiment of the invention the isolated, synthetic or synthesized taura syndrome virus Replikin peptide consists of the Replikin motif. A further non-limiting embodiment provides a nucleotide sequence encoding the taura syndrome virus Replikin peptide.

    
    


A fifth non-limiting aspect of the present invention provides an isolated, synthetic or synthesized taura syndrome virus Replikin Scaffold peptide consisting of about 16 to about 34 amino acids comprising:

    
    
        (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine;
        (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine;
        (3) a lysine within about 6 to about 10 amino acids from another lysine; and
        (4) at least 6% lysines.

In a further non-limiting embodiment, the isolated, synthetic or synthesized taura syndrome virus Replikin Scaffold peptide consists of about 28 to about 33 amino acids. In a further non-limiting embodiment, the peptide comprises

    
    











(SEQ ID NO: 197)



KKVQANKTRVFAASNQGLALALRRYYLSFLDH



or



 



(SEQ ID NO: 198)



KKACRNAGYKEACLHELDCKSFLLAQQGRAGAH.






A sixth non-limiting aspect of the present invention provides a method of increasing resistance of an invertebrate to a pathogen comprising:

    
    
        (1) challenging a plurality of invertebrates being of the same species as said invertebrate with said pathogen at sufficient levels to cause disease in at least one individual of the plurality of said invertebrates;
        (2) discarding at least one individual of the plurality said invertebrates that is diseased or dead;
        (3) repeating steps 1 and 2 at least once; and
        (4) at least once administering to the plurality of said invertebrates the substance of claim 1 in steps (1), (2) and/or (3) which increases the resistance of said invertebrate to the pathogen.
    
    


In a non-limiting embodiment of the sixth aspect of the present invention, the at least one isolated or synthetic peptide is at least one Replikin peptide consisting of 7 to about 50 amino acids and comprising a Replikin motif comprising:

    
    
        (1) at least one lysine residue located at a first terminus of said motif and a least one lysine residue or at least one histidine residue located at a second terminus of said motif;
        (2) at least one lysine residue located six to ten residues from a second lysine residue;
        (3) at least one histidine residue; and
        (4) at least six percent lysine residues.

In a further non-limiting embodiment, the invertebrate is a shrimp. In a further non-limiting embodiment, the pathogen is taura syndrome virus.

    
    


A seventh non-limiting aspect of the present invention provides a method of producing a vaccine comprising: (1) identifying at least one Replikin sequence in a pathogen of an invertebrate capable of culture in an aqueous medium, and (2) chemically synthesizing the at least one identified Replikin sequence as an active agent of the vaccine. In a non-limiting embodiment, the vaccine is produced in seven days or fewer from the time the at least one Replikin sequence in the pathogen is identified.
An eighth non-limiting aspect of the present invention provides animal feed comprising a substance for use in aquaculture, comprising at least one synthetic peptide of about seven to about fifty amino acids, wherein the at least one synthetic peptide, when administered to at least one invertebrate that is capable of culture in an aqueous medium, increases the resistance of that invertebrate to at least one pathogen. In a non-limiting embodiment, the animal feed is feed for a crustacean. In a further non-limiting embodiment, the animal feed is feed for a shrimp. In a further embodiment the synthetic peptide of the substance of the animal feed is a synthetic Replikin peptide. In a further embodiment, the animal feed comprises shrimp production Rangen 35 mash, 1% sodium alginate, 1% sodium hexametaphosphate, and at least one synthetic Replikin peptide that is KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), or HVEKIVDKAK (SEQ ID NO: 11).
A ninth non-limiting aspect of the present invention provides a shrimp treated with a vaccine comprising at least one Replikin peptide wherein the at least one Replikin peptide consists of 7 to about 50 amino acids and comprises a Replikin motif comprising:

    
    
        (1) at least one lysine residue located at a first terminus of said motif and a least one lysine residue or at least one histidine residue located at a second terminus of said motif;
        (2) at least one lysine residue located six to ten residues from a second lysine residue;
        (3) at least one histidine residue; and
        (4) at least six percent lysine residues.
    
    


In a further embodiment, the Replikin peptide is a taura syndrome virus Replikin peptide. In a further embodiment of the ninth aspect of the present invention, the shrimp is (1) challenged with taura syndrome virus along with a plurality of other shrimp at sufficient levels to cause disease in at least one of the plurality of shrimp; (2) the shrimp of the plurality of shrimp that are diseased or dead or both are discarded; (3) steps 1 and 2 are repeated at least once; and (4) said plurality of shrimp are administered a vaccine at least once in steps (1), (2) and/or (3), wherein the vaccine comprises at least one isolated or synthetic taura syndrome virus Replikin peptide.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates percent survival of shrimp (Penaeus vannamei) challenged with taura syndrome virus over 15 day trials. FIG. 1A illustrates shrimp vaccinated with the T1 vaccine containing Replikin sequences from taura syndrome virus (TSV). FIG. 1B illustrates non-vaccinated shrimp (a control). The data in FIG. 1 reflect the trials set forth in Example 2 below. FIG. 1A illustrates 5 trials each conducted over the same 15 day period along with controls. FIG. 1B illustrates the three control trials over the same 15 day period. After removal of one outlier from a total of six trials (as explained in Example 2 below), the vaccinated shrimp had a cumulative survival of 59% (FIG. 1A). The non-vaccinated shrimp has a cumulative survival of 25% (FIG. 1B). Cumulative survival of vaccinated shrimp was greater than cumulative survival of non-vaccinated shrimp. These data indicate that vaccination with Replikin sequences provided protection from TSV infection. The relative percent survival against TSV after vaccination was 45%.
FIG. 2 illustrates 15 day cumulative survival of shrimp (Penaeus vannamei) previously challenged (chronically infected) with taura syndrome virus (TSV) and subsequently challenged a second time with TSV after treatment with T1 vaccine, T2 inhibited or “blocked” vaccine, or no vaccine (control). An additional control study is illustrated with shrimp that had not been previously challenged with TSV (not chronically infected). This additional control was not treated with vaccine. For T1-vaccinated shrimp, there were no mortalities until day 13. This group had a 91% survival at day 15 after exposure to TSV. For shrimp fed inhibited-vaccine T2, the first mortality was observed at day 2. The group had a survival of 60%. For the non-vaccinated control group that had been subject to previous TSV challenge, the first mortality occurred on day 10, and the group had a survival of 75%. For the additional control group that had not previously been challenged with taura syndrome virus (SPF shrimp), cumulative survival was 25%. These results show that TSV-chronically-infected shrimp had higher percent survivals (60-91%) when re-challenged with TSV. In particular, T1-vaccinated P. vannamei had a highest survival and lowest viral load. The percent survival for SPF P. vannamei challenged with TSV was 25%. The trial illustrated in FIG. 2 is described in Example 3 below.
FIG. 3 illustrates a direct sequential correlation between Replikin concentration of isolates of taura syndrome virus (TSV) collected from Belize, Thailand, Hawaii and Venezuela, respectively, and mean number of days to 50% mortality in Litopenaeus vannamei shrimp challenged with the respective TSV isolates on days 1, 2, and 3. Statistical differences between the Replikin concentration for each isolate are significant at a level of p<0.001. The data illustrated in FIG. 3 are described in Example 1 below.
FIG. 4 illustrates a direct correlation between Replikin concentration in isolates of taura syndrome virus (TSV) collected from Belize, Thailand, Hawaii and Venezuela, respectively, and mean cumulative survival of Litopenaeus vannamei shrimp at 15 days after challenge with respective TSV isolates. Statistical differences between the Replikin concentrations for each isolate are significant at a level of p<0.001. The data illustrated in FIG. 4 are described in Example 1 below.
FIG. 5 illustrates a correlation between cumulative survival of Litopenaeus vannamei shrimp challenged with four different taura syndrome virus isolates over 15 days (unless 100% mortality occurred prior to 15 days) and the Replikin concentration of Open Reading Frame 1 (ORF1) of each isolate. Translated amino acid sequences of ORF1 of the genome of individual isolates of TSV from Belize, Thailand, Hawaii and Venezuela were analyzed for Replikin concentration. Replikin concentration was determined to be 3.5 for the Belize isolate, 3.4 for the Thailand isolate, 3.3 for the Hawaii isolate and 3.0 for the Venezuela isolate. Graph A illustrates observed percent survival in three trials of shrimp challenged with the Belize isolate of TSV. In one trial, total mortality was observed on day 6. In the other trials, total mortality was observed on day 11. Graphs B, C and D illustrate observed percent survival of shrimp challenged with the Thailand isolate, the Hawaii isolate and the Venezuela isolate, respectively, each in three trials over 15 days. In the Thailand isolate, a mean of 80% percent mortality was observed on day 15. In the Hawaii isolate, a mean of 78.3% mortality was observed on day 15. In the Venezuela isolate, a mean of 58.3% mortality was observed on day 15. The data illustrated in FIG. 5 are described in Example 1 below.
FIG. 6 illustrates a correlation between increased Replikin concentration in the genome of taura syndrome virus and outbreaks of the virus in 2000 and 2007 in shrimp. Taura syndrome virus peptide sequences available at www.pubmed.com were analyzed by the inventors for mean Replikin concentration in the publicly available sequences. FIG. 6 is a graph comparing mean Replikin concentration for each year in which peptide sequences were publicly available between 2000 and 2005 (with standard deviation) and dates of significant outbreaks of taura syndrome virus. The Replikin concentration data reflected in the graph is found in Table 13 below. Significant outbreaks of the disease are noted at years 2000 and 2007. It may be observed from the graph that outbreaks of the virus occur following an increase in Replikin concentration. In year 2000, TSV had a Replikin concentration of 2.7. Between 2001 and 2004, TSV had a lower mean Replikin concentration, as low as 0.6, and an identified Replikin Scaffold disappeared. In 2005 the Replikin Scaffold reappeared, with an increase in lysines and histidines, and a commensurate increase in Replikin concentration to 1.8, followed by an increase in TSV outbreaks in 2006-2007. See Replikin Scaffolds in Example 5 for TSV in 2000 and 2005.
FIG. 7 illustrates cyclic production of West Nile virus Replikins and annual human morbidity by demonstrating a correlation between annual Replikin concentration per 100 amino acids (Replikin Count) of the envelope protein of West Nile virus (Replikin Count mean is illustrated with black bars with standard deviation depicted) and the annual number of human cases in the United States as reported by the Centers for Disease Control (CDC) on a statewide basis. A review of the data reveals that the standard deviation of the mean of the Replikin Count of the envelope protein increases markedly between 2000 and 2001 (statistical significance with a p<0.001). This kind of change in standard deviation from the mean of the Replikin Count of a virus population has been observed to signal rapid replication and expansion of the range of the Replikin Count in all common strains of influenza virus (influenza virus is classified in a significantly different family of viruses than is West Nile virus). In influenza, a marked statistical change in the Replikin Count has been observed to precede viral outbreaks.
The observed increase in mean Replikin Count from 2000 to 2003 in West Nile virus precedes an increase in the number of human West Nile virus (WNV) cases recorded independently and published by CDC. The same detailed relationship of Replikin Count to morbidity has been shown in influenza strains, for example H5N1 to human mortality (see FIG. 8), and in H3N8 equine encephalitis to horse morbidity (see FIG. 12), and in the trypanosome Plasmodium falciparum (malaria) to human mortality (see FIG. 9), and to mortality rate in shrimp with taura syndrome virus (see FIGS. 3-5). Since the relationship has already been demonstrated in several species, namely crustacea, horses, and humans, and since Replikin sequences have been associated with rapid replication in plants, invertebrate animals, vertebrate animals, protozoa (such as trypanosomes), bacteria (such as anthrax) and viruses (see, e.g., U.S. Pat. Nos. 7,176,275 and 7,189,800), relating Replikin Count to morbidity appears to be a broadly distributed general principle.
The data in FIG. 7 additionally illustrate in 2004, and again in 2005, that there was a decrease in both the Replikin Count and the number of human cases of WNV. In 2006, an increase in Replikin Count was followed by an increase in the number of human cases (morbidity) in 2007. FIG. 7, therefore, illustrates two cycles of Replikin concentration (Replikin Count) and two cycles of changing WNV human morbidity. The two cycles (Virus Replikin Cycles) between 2000-2003 and 2004-2008 are observed to correlate in FIG. 7. Cycles in Replikin Count have been observed in previous influenza virus data with H1N1 and H3N2 (see, e.g., FIG. 11). The relationship is even more clearly demonstrated in the WNV data than it is influenza because the actual number of cases resulting from the H1N1 or H3N2 influenza viruses was not historically recorded for the particular strain as accurately as they are now recorded for West Nile Virus. Instead, H1N1 and H3N2 were recorded as outbreaks, epidemics, or pandemics. The stepwise increase in Replikin concentration (Replikin Count) in repeated cycles can serve as a method for predicting viral expansion. Monitoring of stepwise increases in Replikin Count provides the skilled artisan with data predicting expansion in viral population and resultant morbidity including, outbreaks, epidemics, and pandemics of viral diseases. Actual morbidity in these events was not recorded. It should be noted that data for Replikin Count in 2007 and 2008 are excluded from FIG. 7 because CDC reporting of WNV sequences was delayed as of the drafting of this application.
The data in FIG. 7 is also important because, rather than the virus revisiting a country after many previous epidemics, as for example in the case of H1N1 or H3N2, FIG. 7 describes the progress of a virus that arrived for the first time in New York City in 1999 and spread over the entire United States thereafter. The opportunity is present to examine the steps in this spread in terms of the newly recognized Replikin structure of the virus and the excellent epidemiological data provided by CDC.
FIG. 8 illustrates the relationship of Replikin Count of the Replikin Peak Gene pB1 gene area in human H5N1 to percent human mortality between 2003 and 2007 in human cases of H5N1 infection. An increase in Replikin Count in the pB1 gene area of H5N1 is observed to be quantitatively related to higher mortality in the host. In the graph, (1) light gray represents the mean Replikin Count of whole virus isolates at a given year, (2) dark gray represents the mean Replikin Count in the pB1 area of publicly available sequences of isolates of human H5N1 at a given year, (3) the white bars represent the standard deviation from the mean of Replikin Count in a given year, and (4) the black bars represent 10% of the percent mortality of identified human cases of H5N1 infection in the given year, finally reaching 86% mortality (shown in FIG. 8 as 8.6).
FIG. 9 illustrates that mortality rates per 1000 clinical cases in humans from Plasmodium falciparum correlate with Replikin Count in the P. falciparum ATP-ase enzyme. High malaria morbidity and mortality rates occurred in the late 1990s and these rates were thought to be due to adaptation of the microorganism and decreased effectiveness of anti-malarials. ATP-ase is a primary target of arteminisin treatment of malaria. With increased use of arteminisin, and improved public health measures, morbidity and mortality rates declined from 1999 to 2006. The Replikin Count of P. falciparum ATP-ase increased from 1997 to 1998 along with an increase in mortality per malaria case. The Replikin Count of P. falciparum ATP-ase decreased along with mortality rates from 1998 to 2006 (consistent mortality presently available only through 2005). Mortality rates per 1000 human cases of malaria for 1997 to 2005 were as follows: 1997 mortality rate was 17; 1998 mortality rate was 17; 1999 mortality rate was 19; 2000 mortality rate was 16; 2001 mortality rate was 13; 2002 mortality rate was 10; 2003 mortality rate was 10; 2004 mortality rate was 9; and 2005 mortality rate was 9. Mortality rates are recorded as declared by the World Health Organization. See www.who.int.
FIG. 10 illustrates a correlation between the mean Replikin Count and standard deviation of Replikin sequences observed in publicly available amino acid sequences of white spot syndrome virus (WSSV) isolated between 1995 and 2007 and a significant outbreak of WSSV in 2001 in shrimp. The Replikin concentration in 2000 of 97.6 foretells the 2001 outbreak. Furthermore, a Replikin concentration of 103.8 was observed in a ribonucleotide reductase protein sequence from a 2000 isolate of WSSV wherein a Replikin Peak Gene was identified with an even higher Replikin concentration of 110.7.
FIG. 11 illustrates total hemagglutinin Replikin Counts in the three influenza pandemics of the last century. Strain-specific high Replikin Counts accompany each of the three pandemics: 1918, 1957, and 1968. In each case, this peak is followed by a decline (likely due to immunity in the hosts), then by a recovery and a “rebound” epidemic. The probability is very low that these correlations are due to chance, since they are specific for each strain, specific for each of the three pandemic years out of the century, specific for each post-pandemic decline, and specific for each rebound epidemic.
FIG. 12 illustrates a relationship between Replikin Counts of Replikin Peak Genes identified within the pB1, pB2, and pA genomic areas of equine influenza 1977-2007 and epidemics of equine encephalitis caused by H3N8 equine influenza. Series 1 reflects the mean Replikin Count identified in the Replikin Peak Gene in the pB1 area of the genome. Series 2 reflects the standard deviation from mean Replikin Count in the pB1 gene area. Series 3 reflects the Replikin Count identified in the Replikin Peak Gene in the pA gene area of the genome, which neighbors the pB1 gene area. Series 4 reflects the Replikin Count identified in the Replikin Peak Gene in the pB2 gene area of the genome, which also neighbors the pB1 gene area. Replikin Count increases in the pB1 gene area are observed to occur one to three years before epidemic outbreaks while no increase in Replikin Count is observed in the pB2 and pA gene areas.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, “synthetic peptide” means a peptide produced by a process wherein at least one step in the process is performed outside of a living cell and the process is directly or indirectly initiated or directed by a human.
As used herein, “aquaculture” means cultivation of the natural produce of water, such as cultivation of shellfish.
As used herein, “administer,” “administration”, or related term means any introduction for therapeutic purposes of a compound to an animal or other subject wherein said introduction may be accomplished through application via the mouth, application to the skin, application through the skin, application through the gills, application via transdermal injection, or application using any other method known to one of skill in the art now and hereafter, whereby the compound is introduced either directly or indirectly into the body of the animal or other subject.
As used herein, a peptide or other compound is “immunogenic” if it is capable, either separately or in combination with other compounds, of stimulating an immune response or a resistance response in a living animal.
As used herein, a compound, treatment, or therapy that stimulates “resistance” or stimulates the development of a “resistant” invertebrate or other animal is a compound, treatment, or therapy that is capable either separately, or in combination with other compounds, treatments, or therapies, of stimulating an immune response or a resistance response in a living animal including, for example, an invertebrate.
As used herein, “therapeutic concentration” means a concentration of a therapeutic agent at which a pathogenic disease is inhibited at a statistically measurable level over at least one life-cycle of the disease.
As used herein, “subtherapeutic concentration” means a concentration of a therapeutic agent at which a pathogenic disease is not inhibited at a statistically measurable level over at least one life-cycle of the disease.
As used herein, “vaccine” means administration to the body of an animal or human a compound capable of stimulating resistance to or an immune response against a pathogen. Administration may be by mouth, gills, transdermal injection, submersion in a concentration of the vaccine or any other method of administration known to one of skill in the art now and hereafter. Details of the immune system of invertebrates such as shrimp, clams, scallops, etc. are not fully understood. For example, shrimp apparently may not produce antibodies. Nevertheless, the phenomenon of resistance to infection is established by the data herein. This resistance may be based in a “primitive immune system.” While not being limited by theory, the “primitive immune system” of shrimp and some other invertebrates has been theorized to be similar to the “toll receptor” and related systems in other animals.
As used herein, “animal” includes shrimp and any other animal.
As used herein, “invertebrate” means any animal lacking a spinal column. Invertebrates that are susceptible to aquaculture include but are not limited to mollusks, crustaceans, and echinoderms. Mollusks include but are not limited to clams, mussels, oysters, winkles, scallops, and cephalopods, such as squid, octopus, cuttlefish and terrestrial snails. Crustaceans include but are not limited to shrimp, crab, lobster, prawn, and crayfish. Echinoderms include but are not limited to sea cucumber and sea urchin.
As used herein, “crustacean” is a group of generally aquatic arthropods as understood by one of skill in the art and includes but is not limited to shrimp, crab, lobster, prawn, and crayfish.
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, an “isolated” or “purified” 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. A “synthetic” or “synthesized” peptide or biologically active portion thereof refers to a peptide that is, after synthesis, 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.
An “encoded” protein, protein sequence, protein fragment sequence or peptide sequence is a peptide sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence wherein the nucleic acids encode amino acids using any codon known to one of 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, a Replikin sequence is an amino acid sequence having 7 to about 50 amino acids comprising:

    
    
        (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
        (2) at least one histidine residue; and
        (3) at least 6% lysine residues.

A Replikin sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine. A Replikin peptide or Replikin protein is a peptide or protein consisting of a Replikin sequence. A Replikin sequence may also be described as a Replikin sequence of 7 to about 50 amino acids comprising or consisting of a Replikin motif wherein the Replikin motif comprises (1) at least one lysine residue located at a first terminus of said isolated peptide and at least one lysine residue or at least one histidine residue located at a second terminus of said isolated peptide; (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues. For the purpose of determining Replikin concentration, a Replikin sequence must have a lysine residue at one terminus and a lysine residue or a histidine residue at the other terminus.

    
    


The term “Replikin sequence” can also refer to a nucleic acid sequence encoding an amino acid sequence having 7 to about 50 amino acids comprising:

    
    
        (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
        (2) at least one histidine residue; and
        (3) at least 6% lysine residues,

wherein the amino acid sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine.

    
    


As used herein, a Replikin Peak Gene (RPG) or a Replikin Peak Gene Area (RPGA) are used interchangeably to mean a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has a highest concentration of Replikins (number of Replikin sequences per 100 amino acids) when compared to other segments or named genes of the genome having continuous, non-interrupted and overlapping Replikin sequences. Generally, the gene or gene segment associated with a whole protein or protein-expressing gene is known as the Replikin Peak Gene and the gene or gene segment associated with a protein fragment is known as a Replikin Peak Gene Area. More than one RPG or RPGA may be identified within a gene, gene segment, protein or protein fragment. An RPG or RPGA may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine. An RPG or RPGA may likewise have neither a terminal lysine nor a terminal histidine so long as it contains a Replikin sequence or Replikin sequences defined by the definition of a Replikin sequence, namely, an amino acid sequence having 7 to about 50 amino acids comprising:

    
    
        (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
        (2) at least one histidine residue; and
        (3) at least 6% lysine residues.
    
    


As used herein, “emerging strain” refers to a strain of a virus or other pathogen identified as having an increased or increasing concentration of Replikin sequences in one or more of its protein sequences relative to the concentration of Replikins in other strains of such organism. An emerging strain of virus indicates an increase in virulence or replication.
As used herein, “outbreak” is an increase in virulence, morbidity or mortality in a viral disease as compared to an earlier-arising epidemiological pattern of infection in the same viral disease.
As used herein, “Replikin Count” or “Replikin concentration” refers to the number of Replikins per 100 amino acids in a protein, protein fragment, virus or organism. A higher Replikin concentration in a first strain of virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier- or later-arising strain of the virus or organism having a lower Replikin concentration.
As used herein a “Replikin Scaffold” refers to a series of conserved Replikin peptides wherein each of said Replikin peptide sequences comprises about 16 to about 34 amino acids and preferably about 28 to about 33 amino acids and further comprises: (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine; (3) a lysine within 6 to 10 amino acid residues from another lysine; and (4) about 6% lysine. “Replikin Scaffold” also refers to an individual member or a plurality of members of a series of Replikin Scaffolds.
In a taura syndrome virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, preferably about 27 to about 33 amino acid residues and more preferably about 30 to 33 amino acid residues. In an influenza virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues and more preferably about 28 to about 30 amino acid residues. In a White Spot Syndrome Virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues and more preferably about 27 to about 29 amino acid residues.
As used herein, the “life cycle” of a shrimp extends substantially from the time when a shrimp is capable of consuming food by mouth until the shrimp is harvested.
As used herein, “time periods” or “time points” is any two time periods or points that may be differentiated one from another. For example, an isolate of virus isolated during the year 2004 is isolated in a different time period than an isolate of the same virus isolated during the year 2005. Likewise, an isolate of virus isolated in May 2004 is isolated in a different time period than an isolate of the same virus isolated in June 2004.
Replikin Sequences as Therapeutic Compounds in Invertebrates including Shrimp
The inventors have identified Replikin sequences in the genome of pathogens to invertebrates in aquaculture including Replikin sequences in the genome of taura syndrome virus (TSV) as therapeutic compounds against viral, bacterial and protozoic diseases in invertebrates such as shrimp, scallops, oysters, lobsters, etc. The identified therapeutic compounds stimulate resistance in the invertebrates challenged with the pathogen such as Chlamydia-like, Rickettsia-like, Vibrio, taura syndrome virus, white spot syndrome virus, and other pathogens. The identified sequences are isolated or synthesized Replikin peptides consisting of 7 to about 50 amino acids comprising a Replikin motif comprising (1) at least one lysine residue located at a first terminus of the motif and at least one lysine residue or at least one histidine residue located at a second terminus of the motif, (2) a first lysine residue located six to ten residues from a second lysine residue, (3) at least one histidine residue; and (4) at least 6% lysine residues wherein said isolated or synthesized peptides are isolated or synthesized by selecting the identified motif and isolating or synthesizing said peptide comprising said motif. In a further embodiment, the Replikin peptide consists of the Replikin motif. The skilled artisan will understand how to isolate and synthesize Replikin peptides using the above-described method. Any method of producing peptides of known structure may be used.
Replikin peptides have been related to rapid replication in plants, animals, bacteria, viruses, trypanosomes and other living things. See U.S. Pat. Nos. 7,176,275 and 7,189,800. Replikins have further been correlated with virulence, morbidity, and mortality in pathogens such as influenza, West Nile virus, malaria, white spot syndrome virus, and taura syndrome virus. See FIGS. 6-12. Additionally, Replikin sequences have been shown to be immunogenic and/or resistance stimulating, or both, in all tests or studies undertaken to date including in chickens, rabbits, shrimp and humans. See U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 (Examples 6 and 7), U.S. Pat. No. 6,638,505. Additionally, the concentration of Replikin sequences in the genome of a pathogen (or in an area of particular interest in the genome of a pathogen known as the Replikin Peak Gene) has been correlated with pathogenic outbreaks, morbidity and mortality. See FIGS. 3-12. In particular, Replikin concentration has been correlated with mortality in invertebrates (shrimp) in taura syndrome virus and with pathogenic outbreaks in invertebrates (in shrimp) in both taura syndrome virus and white spot syndrome virus. See FIGS. 3-6 and 10. Replikin sequences have been widely associated with virulence, morbidity, and mortality and have consistently been associated with immunogenicity and resistance including in horses, rabbits, chickens, humans, and shrimp. These wide-ranging data provide support for vaccines containing Replikin sequences including support for vaccines containing Replikin sequences from pathogens in aquaculture.
The isolated or synthesized Replikin peptides identified in a pathogen to an invertebrate in aquaculture are then fed, injected, otherwise administered to the invertebrate such as shrimp, scallops, oysters, lobsters, etc. as a prophylactic against possible outbreaks of pathogenic diseases including TSV or other pathogenic disease, prior to a predicted outbreak of the disease or during an outbreak of the disease. The peptides may be administered to the invertebrate alone or in combination with regular feed or with supplemental feed formulated for carrying the peptides. The skilled artisan will understand that any method of formulating feed may be used that provides the Replikin sequences to the invertebrate per os. A stabilizer or preservative or both may be added to a mixture of Replikin sequences or a mixture of Replikin sequences and invertebrate feed to maintain a therapeutically effective ration for feeding of and treatment of cultured invertebrates over a period of time, including shrimp, scallops, oysters, clams, crabs, abalone, lobster, etc. The skilled artisan will understand extensive options of methods of stabilizing short peptides in feed and in therapeutic mixtures.
Administration of peptides may be at therapeutic or subtherapeutic levels before or after infection occurs. Administration may also be made to chronically infected populations.
Any Replikin identified in the genome of or identified as expressed by the pathogen may be administered as a therapeutic compound to stimulate a resistance response in shrimp. The following exemplary sequences may be fed alone or in any combination to shrimp as a vaccine against taura syndrome virus: KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), or HVEKIVDKAK (SEQ ID NO: 11). The exemplary sequences, as with all taura syndrome virus Replikin sequences, including Replikin sequences and Replikin Scaffold sequences as discussed below, may be fed, or used in an immersion method of administration or injected. The sequences may be administered individually or in any combination including in an equal combination of peptides by weight. Any Replikin sequence or immunogenic sequence of about 7 to about 50 amino acids may be used as a compound for administration to an invertebrate including use as a vaccine. For example, SEQ ID NOS: 1-11, 86, 87, 103-112, 114-198 may each be used alone or in combination as a compound for administration to an invertebrate against TSV infection.
Reduction of Viral Load and Dose-Response Curves
Immunogenic or resistance-stimulating synthetic peptides may be administered to invertebrates at about 0.001 mg to about 10 mg of synthetic peptide per gram of body weight of each treated invertebrate per day, at about 0.005 mg to about 5 mg of synthetic peptide per gram of body weight of each treated invertebrate per day, at about 0.01 mg to about 2 mg of synthetic peptide per gram of body weight of each treated invertebrate per day, at about 0.02 mg to about 1.5 mg of synthetic peptide per gram of body weight of each treated invertebrate per day, at about 0.08 mg to about 1.0 mg of synthetic peptide per gram of body weight of each treated invertebrate per day, at about 0.1 mg to about 0.9 mg of synthetic peptide per gram of body weight of each treated invertebrate per day, at about 0.2 mg to about 0.8 mg of synthetic peptide per gram of body weight of each treated invertebrate per day, at about 0.5 mg of synthetic peptide per gram of body weight of each treated invertebrate per day. One of skill in the art will understand how to determine dosage of synthetic peptide that is appropriate for therapeutic or sub-therapeutic administration.
TSV vaccine doses containing a mixture of peptides by equal weight have now been tested in shrimp and shown to provide a protective effect. The following peptides have been tested: KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), and HVEKIVDKAK (SEQ ID NO: 11). The vaccine has been given in different experiments at approximately 0.02, 0.08 and 0.50 mg of Replikin vaccine per one gram of body weight of tested shrimp per day. The doses are approximate because of individual differences in individual shrimp consumption. Taken orally by shrimp weighing 1 g to 4 g, all three dose levels have been well tolerated, and all produced statistically significant lower viral loads in the shrimp as compared to unvaccinated controls. The 0.08 mg and the 0.5 mg doses provided statistically significant protection. The lowest dose of 0.02 mg, in one of two experiments, gave protection which did not reach statistical significance; nevertheless, as demonstrated above, statistically significant lower viral loads were observed. The 0.50 mg per “shrimp-gram” dose (the highest dose tested to date) provided the best statistically significant protection of 91%. Therefore, from the results to date, it appears that doses of 0.50 mg per “shrimp-gram,” and possibly higher, are preferred embodiments of an aspect of the invention.
Production of Vaccine in About Seven Days or Less
Another non-limiting aspect of the present invention provides a method of producing a vaccine wherein at least one Replikin sequence is identified in a pathogen and the at least one Replikin sequence is chemically synthesized as an active agent of the vaccine. The inventors have successfully produced such an effective vaccine in seven days or fewer from the time a pathogen is identified.
Once a pathogen is identified, its genome is determined. The artisan then surveys the genome for Replikin sequences using, for example, ReplikinsForecastTM (Replikins LLC, Boston, Mass.). Once Replikin sequences have been identified, any one or more Replikin sequence may be chosen for chemical synthesis. A preferred Replikin sequence may be a Replikin sequence identified in a Replikin Peak Gene. Chemical synthesis of the identified at least one Replikin sequence is undertaken as understood by one of skill in the art.
The synthetic peptide or peptides are then administered to a host of the pathogen. Administration of the vaccine may be orally, mixed with a food source for oral consumption, through the gills, in a concentrated emersion wherein the vaccine is absorbed into the body through the gills, skin, mouth, etc., via injection, or using any other method known to one of skill in the art now and hereafter. The vaccine may be combined with a pharmaceutically acceptable carrier, excipient, binder, or other helpful compound, adjuvant, solution or mixture known to one of skill in the art.
The process is easily accomplished in seven days or fewer based on the ease of identification of Replikin sequences in a genome of a pathogen and the ease of chemical synthesis of peptides in large volumes. This novel process of providing effective active ingredient for vaccines in seven days or fewer solves a critical problem in the art because current methods of production of vaccines generally requires three to twelve months. This delay in vaccine production may deliver vaccine after an epidemic has long since ended or, if still active, after mutations in the pathogen have rendered the vaccine less effective or useless. The long process of vaccine development is a significant worry among health professionals and government. The inventors have now provided a method for greatly reducing delay in vaccine development.
Development of Resistant Lines of Cultured Invertebrates
Another non-limiting aspect of the present invention provides a method of increasing resistance of an invertebrate to a pathogen comprising:

    
    
        (1) challenging a plurality of invertebrates being of the same species as said invertebrate with said pathogen at sufficient levels to cause disease in at least one individual of the plurality of said invertebrates;
        (2) discarding at least one individual of the plurality said invertebrates that is diseased or dead;
        (3) repeating steps (1) and (2) at least once; and
        (4) at least once administering to the plurality of said invertebrates the substance of claim 1 in steps (1), (2) and/or (3) which increases the resistance of said invertebrate to the pathogen.

The process may be repeated a number of times until a desired level of survival is observed in the cultured invertebrates including 100% survival. The cultured resistant invertebrates may then be used as breeding stock for production of resistant lines of invertebrates for aquaculture. The compound comprising an immunogenic peptide may be administered at each repetition of steps 1 and 2, may be administered in the second repetition of steps 1 and 2, or may be administered at some later repetition of steps 1 and 2. As demonstrated in Example 3 below and in FIG. 2, shrimp chronically infected with taura syndrome virus prior to administration of about 0.5 mg per gram of shrimp body weight of a compound comprising eleven taura syndrome virus immunogenic peptides by equal weight provided 91% survival of shrimp challenged with TSV. In contrast only 25% of shrimp that had not been previously challenged with TSV and had not been administered vaccine survived the challenge. Further, 75% of shrimp chronically infected with TSV not administered vaccine additionally survived the challenge.

    
    


The trials reported in Example 3 and FIG. 2 demonstrate that refractory resistance in combination with vaccine treatment has to date provided the highest observed protection against challenge from TSV. As such, one aspect of the invention provides a method of increasing resistance using cycled challenge of invertebrates with a chosen pathogen in combination with administration of a vaccine. In a preferred embodiment, the at least one isolated or synthetic peptide of the vaccine is a Replikin peptide. Replikin peptides have been demonstrated to stimulate immunogenic and/or resistance responses in vertebrates and invertebrates. For example, vaccine products against SARS Replikin sequences and H5N1 influenza virus Replikin Scaffolds have been demonstrated, see, e.g., U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 (Examples 6 and 7), all Replikin sequences tested in rabbit or chicken have induced an immune response, and the glioma Replikin sequence has been identified in cancer cells and isolated with or synthesized into peptides that induce an immune response in rabbits and react with natural antibody responses in humans. See U.S. Pat. No. 6,638,505.
Conservation of Replikin Structure Relates to Virulence and Lethality
The conservation of any structure is critical to whether that structure provides a stable invariant target to attack and destroy or to stimulate. Replikin sequences have been shown to generally be conserved. When a structure is tied in some way to a basic survival mechanism of the organism, the structures tend to be conserved. A varying structure provides an inconstant target, which is a good strategy for avoiding attackers, such as antibodies that have been generated specifically against the prior structure and thus are ineffective against the modified form. This strategy is used by influenza virus, for example, so that a previous vaccine may be quite ineffective against the current virulent virus.
Certain structures too closely related to survival functions, however, apparently cannot change constantly. An essential component of the Replikin structure is histidine (h), which is known for its frequent binding to metal groups in redox enzymes and is a probable source of energy needed for replication. Since the histidine structure remains constant, Replikin sequence structures remain all the more attractive a target for destruction or stimulation. Additionally, as demonstrated below in Tables 1 and 2, Replikin structures generally are conserved throughout virulent pathogens including pathogens to invertebrates.
In view of the conservation of Replikin structures in invertebrate pathogens, an aspect of the present invention provides an antibody or antibody fragment or anti-Replikin small molecule to at least one isolated or synthesized Replikin sequence within a protein or protein fragment of a pathogen in aquaculture or within a Replikin Peak Gene or within a protein or gene area comprising a Replikin Peak Gene in a pathogen in aquaculture. Antibodies to Replikins and anti-Replikin small molecules are useful as therapies against pathogenic outbreaks. Following identification of Replikin sequences in a pathogen, one of skill in the art knows many ways of developing antibodies or antibody fragments for therapeutic and diagnostic purposes. One of skill in the art likewise knows how to produce small molecules to bind identified Replikin sequences.
Replikin Scaffolds as Therapeutic Targets and Predictors
The inventors have established in strains of influenza, in White Spot Syndrome Virus and in taura syndrome virus, that the presence of Replikin Scaffolds is predictive of epidemics. As such, in addition to the total number of Replikins in a virus, the structure of each Replikin through time is informative and Replikin Scaffolds provide a particularly useful target for identifying and controlling rapid replication including outbreaks of pathogenic diseases in invertebrates in aquaculture, including shrimp. Table 1, below, shows a Replikin Scaffold first observed in a goose infected with influenza in 1917 (Goose Replikin). Constant length, constant lysines at the amino terminal and histidine residues at the carboxy terminal were conserved in different strains in a fixed scaffold for decades. Homologues of the Goose Replikin appeared from 1917 through 2006 in strains including each strain responsible for the three pandemics of 1918, 1957, and 1968, H1N1, H2N2 and H3N2. Homologues with further substitutions are also observed in H1N2, H7N7, H5N2 and H5N1.







TABLE 1







Replikin Scaffold showing ordered substitution in the 89 year conservation of influenza virus


Replikin peptides related to rapid replication, from a 1917 goose influenza Replikin and the 1918


human pandemic Replikin to 2006 H5N1 “Bird Flu” homologues. (SEQ ID NOS: 12-74,


respectively, in order of appearance)

























*Residues identical to Goose Replikin amino acids are unshaded; amino acid substitutions are shaded lightly and darkly to show scaffold pattern across years and strains. Substitution at position 24 in 2004 and 2006 H5N1, 1957 H2N2, 1968 H3N2 and H7N7 are boxed.






Table 1 illustrates the history, by year or smaller time period, of the existence in the protein structure of the Goose Replikin and its homologues in other influenza Replikins. Table 1 further illustrates the history of amino acid substitutions in those homologues and the conservation of certain amino acids of the Replikin structure that are essential to the definition of a Replikin and the function of rapid replication supplied by Replikins.
A review of Table 1 illustrates that if random substitution of amino acids were to occur in virulent strains of influenza from 1917 through the present, certain framework amino acids of the Goose Replikin would not be conserved from year to year in strains in which epidemics occurred. However, contrary to what would result from random substitution, virulent strains of influenza from year to year consistently contain conserved amino acids at those positions that define a Replikin. That is, if a substitution were to occur in one of the amino acids that define a Replikin, e.g. lysine or a histidine, the definition of the Replikin would be lost. Nevertheless, the Replikin sequence is conserved over more than 89 years. Thus, since there is conservation of certain amino acids over decades, substitution cannot be said to be completely at random. The fact that substitutions do occur in amino acids that are not essential to the definition of a Replikin (i.e., amino acids other than lysines or histidines) demonstrates the importance of the Replikin and the Replikin Scaffold in the pathogenicity of the strain.
It may be further noted from Table 1 that when substitutions do occur, they are seen to occur at certain apparently preferred positions of the Replikin Scaffold. Table 1 illustrates recurring substitutions at positions 1, 3-24 and 26-27. Further, while substitutions occur throughout these positions, a lysine continues to exist at a position 6 to 10 amino acids from a second lysine (which has not been substituted in these virulent strains).
As seen in Table 1, even when there is a substitution of a lysine position within the 29 amino acid stretch, as is seen in 1957, when K at position 11 shifts to position 10, that new position is maintained until 2005. Additionally, YP (at positions 6-7), SY (at positions 12-13), N (at position 15), and LVLWG (SEQ ID NO: 75) (at positions 22-26) conserve the homologous structure of the Replikin Scaffold with few exceptions.
Homologous Replikin Scaffold Sequences in Influenza, WSSV, and TSV
The inventors have further established a relationship between virulent influenza virus and TSV and white spot syndrome virus (another viral pathogen in shrimp) in the Replikin Scaffold portions of the viruses as may be seen in Table 2 below. Although there is extensive substitution, several short Replikins of the white spot syndrome virus demonstrate significant homologies to the influenza virus Replikin sequences, especially with regard to length and key lysine (k) and histidine (h) residues. Similar, but less extensive homologies are seen in taura syndrome virus. These homologies suggest that the sequences are derived from a shared reservoir and/or that similar mechanisms of Replikin production are used in both virus groups and are significant to virulence in each of the viruses in which the homologues are shared. Because Replikin structure appears to be conserved within a reservoir or within a shared mechanism, it is no surprise that the relationship of the Replikin structure to rapid replication, virulence, mortality, and morbidity appears to be conserved across a wide-range of pathogens and hosts including pathogens in invertebrates in aquaculture including shrimp, scallops, clams, crabs, mussels, etc.







TABLE 2





Shrimp White Spot and Taura Syndrome Scaffolding (SEQ


ID NOS 76-87, respectively, in order of appearance)


































Replikin and Replikin Scaffold Therapeutic Compounds

Another aspect of the invention provides a method of making a preventive or therapeutic compound comprising identifying a Replikin Scaffold comprising about 16 to about 34 amino acids and isolating or synthesizing said Replikin Scaffold for use as a preventive or therapeutic treatment against pathogens in invertebrate aquaculture wherein said Replikin Scaffold comprises: (1) a terminal lysine and a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and a histidine immediately adjacent to the terminal histidine; (3) a lysine within about 6 to about 10 amino acids from another lysine; and (4) at least 6% lysines. The Replikin Scaffold may consist of 27 to about 33 amino acids and may further consist of about 30 to about 33 amino acids. In a further non-limiting embodiment, the Replikin Scaffold consists of 32 or 33 amino acids.
In another aspect of the invention, a preventive or therapeutic compound is provided comprising at least one isolated or synthesized Replikin sequence from a pathogen of an invertebrate cultured for food in water including taura syndrome virus and white spot syndrome virus in shrimp, and Chlamydia-like and Rickettsia-like bacterial pathogens in mussels, claims, scallops, etc. In a non-limiting embodiment of the invention, a preventive or therapeutic compound is provided comprising at least one Replikin sequence or at least one Replikin Scaffold sequence isolated or synthesized from a pathogen such as TSV. In a non-limiting embodiment of the invention, the at least one isolated or synthesized Replikin sequence or at least one Replikin Scaffold sequence is present in an emerging strain of a pathogen such as TSV. In a further non-limiting embodiment of the invention, the preventive or therapeutic compound comprises two or more Replikin sequences. In a further non-limiting embodiment, the preventive or therapeutic compound comprises two or more Replikin Scaffold sequences. In a further non-limiting embodiment, the preventive or therapeutic compound comprises at least one Replikin sequence and at least one Replikin Scaffold sequence. In another non-limiting embodiment, the preventive or therapeutic compound comprises at least one Replikin sequence or at least one Replikin Scaffold sequence and a pharmaceutically acceptable carrier.
EXAMPLE 1
Comparison of the Replikin Concentration of Four Strains of Taura Syndrome Virus by an Independent Laboratory
The Replikin concentrations of the protein sequences of four taura syndrome virus (TSV) isolates from Hawaii, Belize, Thailand and Venezuela, respectively, were examined without knowledge of the exact order of virulence of the four isolates, and the virulence was ranked quantitatively in the order of the Replikin concentrations. The virulence of the four TSV isolates was compared in an independent laboratory, without knowledge of the exact order of the Replikin concentrations. The virulence was compared through a per os laboratory infection in juvenile Litopenaeus vannamei (Kona stock, Oceanic Institute, Hawaii). The results showed that the Belize isolate is the most virulent, the Thailand isolate is the second most virulent, followed by the Hawaii isolate, and the Venezuela isolate, which is the least virulent. This is based on the analyses of cumulative survivals at the end of a bioassay and based on the time of 50% mortality. TSV infection as the cause of death was confirmed by positive reactions in RT-PCR detection and by the appearance of characteristic lesions observed in histological analysis. The correlation of Replikin concentrations with virulence as indicated by Mortality Rate was quantitative and substantially linear.
CHALLENGE METHODS
Small juveniles of specific-pathogen-free Litopenaeus vannamei (20 shrimp per tank, mean weight: 1.8 g) were fed minced TSV-infected tissues (infected separately with each of the 4 isolates originating from Belize, Thailand, Venezuela and Hawaii) for 3 days at 5% of their body weight. These shrimp were maintained with pelleted ration (Rangen 35%) for the following 12 days. Each challenge bioassay of a specific isolate was triplicated. During the bioassay period, all tanks were checked daily for dead or moribund shrimp. All mortalities were removed from the tank and frozen. One to three moribund shrimp from each isolate were preserved in Davidson's AFA fixative and processed for routine histology to confirm viral infection. For each isolate, six moribund shrimp were collected during the acute phase infection and total RNA was extracted from their gill tissues with a High Pure RNA tissue kit (Roche). The extracted RNA was analyzed for the presence of TSV by real-time RT-PCR.
All tanks were outfitted with an acclimated biological filter and aeration, and were covered with plastic to contain aerosols. The average salinity of the water was 23 ppt and the water temperature was 28° C. The challenge study was terminated after 15 days with live animals counted as survivors.
RESULTS
Comparison of Virulence: Mortality in Shrimp
First mortality was seen on day 2 after exposure to TSV in all 4 isolates. For the Belize isolate, most (83%) of shrimp died by day 4 and had a 0% survival rate at day 11. For the Thailand isolate, 63% mortalities occurred by day 4 and had a 20% survival rate at the end of 15-day bioassay. For the Hawaii isolate, mortalities increased starting at day 2 and reached a peak at day 5; the final cumulative survival was 22%. For the Venezuela isolate, mortalities occurred slowly at days 2 and 3 with 22% mortality on day 4 followed by a decline in mortality, 42% of shrimp survived through to termination of the study. See FIG. 5 and Table 6. The time period for reaching 50% mortality caused by TSV infection for the isolate of Belize, Thailand, Hawaii and Venezuela were 2.8, 3.5, 4.5 and 7 days, respectively (Table 3).







TABLE 3







Results from per os TSV challenge in SPF Litopenaeus vannamei


(Kona stock)













GenBank No.
Survival
Day of 50%



TSV isolate
(ORF1)
(%) (Mean)
mortality
















Belize
AAT81157
0
2.8



Thailand
AAY56363
20
3.5



US-Hawaii
AAK72220
22
4.5



Venezuela
ABB17263
42
7.0*







*High variation was observed in Venezuela's triplicate tanks, thus the Day of 50% mortality was determined by Kaplan-Meier survival analysis with the Statistix 8 program.






Pathology

Histological analysis of the samples of L. vannamei juveniles is summarized in Table 4.







TABLE 4







Summary of histological findings















Days after
TSV




UAZ ID#
TSV Isolate
exposure
lesions1
LOS2







O6-407J/1
Belize
3
G4
G4



06-407F/1
Thailand
3
G4
G2



06-407D/1
Thailand
4
G4
G3



06-407E/1
Thailand
4
G3
G2



06-407A/1
Hawaii
4
G2
G3



06-407C/1
Hawaii
4
G2
G4



06-407H/1
Venezuela
4
G4
G2







Severity grade: G1: sign of infection; G2: moderate signs of infection; G3: moderate to high signs of infection; G4: severe infection.



1TSV lesions = Presence of TSV pathognomonic lesions in the gills, mouth, stomach, intecumental cuticular epithelium, and appendages.



2LOS = presence of lymphoid organ spheroids within the lymphoid organ.






Belize TSV.
Acute lesions of diagnostic TSV infection were found in one representative shrimp sample at a severity grade of G4. Nuclear pyknosis and karyorrhexis were observed in the cuticular epithelium of the general body surface, appendages, gills, stomach and esophagus. Lymphoid organ spheroids were also found at severity grade G4.
Thailand TSV.
Severe (G4) TSV infection was detected in 2 out of 3 shrimp, another shrimp showed a moderate to high grade (G3) of infection. Lymphoid organ spheroids were found at severities of G2 and G3.
Hawaii TSV.
Moderate level (G2) of TSV infection was detected in 2 shrimp collected at day 4. Lymphoid organ spheroids were found at severities of G3 and G4.
Venezuela TSV.
Severe (G4) TSV infection was detected in one representative shrimp sampled at day 4. Lymphoid organ spheroids were found at severity of G2.
Real-Time TSV RT-PCR
All 24 samples (6 from each isolates) were all positive for TSV infection. This confirms that the mortalities observed from bioassays are from TSV infection.







TABLE 5







Mean and range of TSV RNA in gills from shrimp challenge with TSV










TSV isolate
Mean (Range) TSV copies/:1 RNA







Belize
2.7 × 106 (4.8 × 105-4.4 × 106)



Thailand
2.7 × 106 (4.3 × 105-7.5 × 106)



Hawaii
5.2 × 107 (2.3 × 107-7.5 × 107)



Venezuela
6.5 × 105 (6.5 × 102-2.0 × 105)

















TABLE 6







Percent Mortality and Blind Replikin Concentration


The results of 4 TSV virulence (percent mortality) comparisons


with blind Replikin Count are:












Percent
Blind Replikin



Isolate
Mortality
Concentration















Belize
100
3.5



Thailand
80
3.4



Hawaii
78.3
3.3



Venezuela
58.3
3.0










The order of virulence: Belize>Thailand>(or =) Hawaii>Venezuela, is in agreement with the Replikin concentration. The differences in the Replikin concentrations appear to be small but they are statistically significant at a level of p<0.001. See FIGS. 3 and 4.
EXAMPLE 2
Increased Host Resistance to Taura Syndrome Virus by Administration of Synthetic Replikins
SUMMARY
A vaccine (T1 vaccine) comprising equal parts by weight KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), and HVEKIVDKAK (SEQ ID NO: 11) was developed in seven days by identifying eleven Replikin sequences in the genome of taura syndrome virus (Hawaii isolate) and chemically synthesizing the eleven Replikin sequences in sufficient volume for a vaccine trial in shrimp as described below.
The T1 vaccine was tested by oral administration to shrimp (Penaeus vannamei) in a laboratory bioassay. One month after vaccination, shrimp were challenged per os with TSV to determine if the vaccine induced protection. The results showed that shrimp fed with the vaccine showed resistance to TSV (P=0.0038<0.05). The vaccinated shrimp had a 59% survival and the non-vaccinated shrimp had a 25% survival. The relative percent survival was 45%. That the mortality of the shrimp was caused by TSV infection was confirmed by positive reactions in real-time RT-PCR detection or by the appearance of characteristic lesions observed in histological analysis.
MATERIALS AND METHODS
Animal and Challenge Design:
P. vannamei (120 shrimp, Kona stock, from Oceanic Institute, mean weight: 2.0 g) were stocked into 6 tanks (20 shrimp/tank) and fed vaccine T1 5% of total body weight daily for 4 weeks. At the end of 4 weeks, shrimp were exposed to TSV through feeding at 5% of total body weight for 2 days in the morning; in the afternoon, the shrimp were also fed vaccine-mixed feed. Beginning on day 3, all the shrimp were maintained on a vaccine-mixed feed for additional 2 weeks.
For non-vaccinated group, 60 shrimp (20 shrimp per tank) were fed control diet feed for 4 weeks as the positive control for virus-infected tissues.
Preparation of Vaccine-Mixed Feed:
The lyophilized vaccine T1 (Replikins, LLC, Boston, Mass.) was mixed (on ice) with shrimp production 35 mash (Rangen), 1% sodium alginate, 1% sodium hexametaphosphate (added as a binder), and 50% water. The mixed feed was extruded, freeze-dried and then packed into approximately 42 bags for each tank (for each tank: 28 bags for 4-week vaccination and 14 bags for TSV challenge; 2 g per bag) and stored at −20° C. until used. Shrimp feed for positive control was prepared as above without the addition of T1 vaccine (designated as control diet).







TABLE 7







The set up of tanks. All tanks were outfitted with an acclimated


biological filter and aeration, and were covered with plastic to


contain aerosols.










Tank #
Vaccine
Treatment
Virus exposure (90-L tank)





A1, 2, 3
T1
feed vaccine-mixed
A1: Hawaii TSV (20 shrimp)


20 shrimp

feed for 4 weeks
A2: Hawaii TSV (20 shrimp)


per tank


A3: Hawaii TSV (20 shrimp)


B1, 2, 3
T1
feed vaccine-mixed
B1: Hawaii TSV (20 shrimp)


20 shrimp

feed for 4 weeks
B2: Hawaii TSV (20 shrimp)


per tank


B3: Hawaii TSV (20 shrimp)


C1, 2, 3
Control
feed control diet for
C1: Hawaii TSV (20 shrimp)


20 shrimp
diet
4 weeks
C2: Hawaii TSV (20 shrimp)


per tank


C3: Hawaii TSV (20 shrimp)









Statistical Analysis.
The survival between vaccinated and non-vaccinated (positive control) groups were calculated as relative percent survival (RPS: 1-vaccinated group mortality/positive control group mortality)×100 (Amend DF, 1981. Potency testing of fish vaccines. In: Anderson DP, Hennessen W (Eds.) Fish Biologics: Serodiagnostics and vaccines. S. Karger, Basel. Pp. 447-454).
RESULTS
By one-way ANOVA, comparison of the survival at day 15, the percent survival was 51% for the vaccinated groups (6 tanks: combining A1-3 and B1-3), higher than the non-vaccinated group (3 tanks: C1-3), 25%. But the difference is not statistically significant (P=0.1010>0.05). However, tank B-3 in the vaccinated group was an outlier (10% survival). In this tank, severe mortalities occurred early, and the final value for this tank was so far from the others, it was thus was eliminated from the analysis.
From the 5 vaccinated groups (A1-3 and B1, 2), the first mortality was seen on day 2 after exposure to TSV (FIG. 1A). For the non-vaccinated group, the first mortality occurred on day 3, and 50% of the shrimp died by day 8.5 (FIG. 1B). The vaccinated groups had significant (5% level) resistance to Hawaii TSV infection. The probability is 0.0038 by one-way ANOVA test analysis. The vaccinated groups had a cumulative survival of 59%, higher than the 25% for non-vaccinated group, indicating that vaccination with T1 provided protection from TSV infection. The relative percent survival (RPS) against TSV after vaccination was 45%.







TABLE 8







Percent survival from TSV challenge in vaccinated Penaeus vannamei.












Survival (%)
Day of first


Tank no.
Feed
at day 15
mortality





A-1
Vaccine-mixed
45
3


A-2
Vaccine-mixed
65
2


A-3
Vaccine-mixed
65
3


B-1
Vaccine-mixed
70
4


B-2
Vaccine-mixed
50
3


B-3
Vaccine-mixed
10
3


C-1
control diet
30
3


C-2
control diet
30
5


C-3
control diet
15
5









Because little is known about the details of the immune system of the shrimp (shrimp appear not to produce antibodies) and other invertebrates, the phenomenon of “resistance” to infection appears to be based in a “primitive immune system” perhaps similar to the “toll receptor” and related systems. Thus the term “increased resistance” is used for the observed phenomenon and Replikin feed is at times used rather than “vaccine” for the administered substance that increases resistance. Nevertheless, vaccine includes the compound administered to shrimp in the trials disclosed in the Examples provided herein.
EXAMPLE 3
Increased Host Resistance to Taura Syndrome Virus by Cycled Challenges with Virus in Combination with Administration of Synthetic Replikins
SUMMARY
Pacific white shrimp (Penaeus vannamei) that survived from TSV infection were found to be more tolerant to a second TSV infection. Three groups of TSV chronically-infected shrimp were either fed (1) T1 vaccine containing equal parts by weight KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), and HVEKIVDKAK (SEQ ID NO: 11) (Replikin sequences from Taura syndrome virus-Hawaii isolate), (2) inhibited vaccine T2 containing equal parts by weight KVGSRRYKSHKKKKKHK (SEQ ID NO: 88), HFATKCFGEVPKKKKKKKHK (SEQ ID NO: 89), KAENEFWDGVKQSHKKKKKHK (SEQ ID NO: 90), KGHRKVPCEQKKKKKKHK (SEQ ID NO: 91), HRKVPCEQKKKKKKHK (SEQ ID NO: 92), KVPCEQKIWLHKKKKKHK (SEQ ID NO: 93), KIWLHQNPGKKKKKKHK (SEQ ID NO: 94), HQNPGKTQQDMKKKKKKHK (SEQ ID NO: 95), KGNTRVHVKKKKKKHK (SEQ ID NO: 96), KEHVEKIVDKKKKKKHK (SEQ ID NO: 97), and HVEKIVDKAKKKKKKHK (SEQ ID NO: 98) (the same Replikin sequences as T1 except that a seven-residue amino acid tail of KKKKKHK (SEQ ID NO: 204) was added to the C-terminus of each of the eleven Replikin sequences to investigate the effect the exact Replikin structure might have on a resistance effect, or (3) control diet (35% Rangen) for 2 weeks followed by feeding with TSV (Hawaii isolate)-infected tissues for 2 days. A fourth group of shrimp that had not been exposed to TSV infection was also fed a control diet. By day 15 after exposure to TSV, 91% of the vaccinated (T1) shrimp survived. Real-time TSV RT-PCR also showed that the T1-vaccinated shrimp had the lowest viral load of the four groups of shrimp in the trial. For shrimp fed inhibited vaccine (T2), 60% survived, and the shrimp had a slightly higher viral load. For the shrimp fed control diet, there was a 75% survival, and the shrimp contained 10 times higher viral load. The shrimp that had not been previously exposed to TSV (SPF p. vannamei) had only 25% survival after exposure to TSV. The trial set up is described in Table 9 below.







TABLE 9







The setup of tanks.










Tank no.
Number of shrimp
Vaccine
Virus challenged





A
11 surviving
T1
Hawaii TSV



shrimp1


B
10 surviving
T2
Hawaii TSV



shrimp2


C
12 surviving
not vaccinated
Hawaii TSV



shrimp3


D
20 SPF shrimp
not vaccinated
Hawaii TSV





1chronic TSV infection, shrimp were fed T1 and challenged with TSV.


2chronic TSV infection, shrimp were fed T2 and challenged with TSV.


3chronic TSV infection, shrimp were fed control diet and challenged with TSV.






MATERIALS AND METHODS
Preparation of Vaccine-Mixed Feed:
Each lyophilized vaccine (T1 and T2, provided by Replikins, LLC, Boston, Mass.) was mixed (on ice) with shrimp production 35 mash (Rangen), 1% sodium alginate, 1% sodium hexametaphosphate (added as a binder), and 50% water. The mixed feed was extruded, freeze-dried and then packed into approximately 30 bags (4 g per bag, 120 g of vaccine-mixed feed in total) and stored at −20° C. until used. Shrimp feed for the control group was prepared as above without the addition of vaccine (designated as control diet).
Vaccination Groups:
For each group (vaccine T1 and inhibited vaccine T2), 11 and 10 TSV chronically-infected P. vannamei were stocked into each of the 1000-L tanks as described in Table 9 and fed vaccine-mixed feed at 5% of their total body weight, daily, for 2 weeks. The total mass of T1 Replikin sequences administered per gram of total shrimp body weight per day was about 0.50 mg. The shrimp were subsequently fed minced TSV-infected tissues (Hawaii isolate) at 5% of their body weight daily for 2 days in the morning. In the afternoon, the shrimp were also fed vaccine-mixed feed. Beginning on day 3, all the shrimp were maintained on a vaccine-mixed feed for an additional 2 weeks.
Non-Vaccinated Group:
12 shrimp (not vaccinated, previously survived TSV infection) were fed control diet for 2 weeks. The shrimp were subsequently fed minced TSV-infected tissues at 5% of their body weight daily for 2 days. Beginning on day 3, all the shrimp were maintained on the control diet.
Histology.
After exposure to Hawaii TSV, all tanks were checked twice a day for dead or moribund shrimp. All mortalities were removed from the tank and frozen at −70° C. One moribund shrimp from tank C was preserved in Davidson's AFA fixative and processed for routine histology to evaluate the severity of TSV infection.
Real-Time TSV RT-PCR.
For each group, the surviving shrimp were sampled at day 15. Total RNA was extracted from the pleopods and gills with a High Pure RNA tissue kit (Roche). The extracted RNA was analyzed for the presence of TSV by a real-time RT-PCR described by Tang et al (J. Virol. Method 115: 109-114, 2004).
RESULTS
Cumulative survival of Penaeus vannamei after re-challenge with TSV for shrimp vaccinated with T1, shrimp fed with inhibited T2 vaccine, chronically infected TSV shrimp fed with control diet, and for shrimp not previously exposed to TSV and fed with control diet is set forth in FIG. 2. For T1-vaccinated shrimp, there were no mortalities until day 13. This group had a 91% of survival at day 15 after exposure to TSV (Table 10). For shrimp those fed inhibited-vaccine T2, the first mortality was observed at day 2. The group had a survival rate of 60%. For the non-vaccinated group, the first mortality occurred on day 10, and the group had a survival rate of 75%. The percent survival for SPF shrimp after fed Hawaii TSV was 25%.







TABLE 10







Percent survival from TSV challenge.















Day of first


Tank no.
Shrimp
Feed
Survival (%)
mortality














A
Chronic
Vaccine (T1)-
91
13




mixed


B
Chronic
Inactive
60
2




vaccine (T2)-




mixed


C
Chronic
control diet
75
10


D
SPF
control diet
25
3









Pathology

Histological analysis of the sample of P. vannamei after challenge with TSV is summarized in Table 11.







TABLE 11







Histological findings for TSV infection














Days after




UAZ ID#
Treatment
exposure
TSV lesions







07-280C
non-vaccinated
10
LOS@G3 chronic



(tank C)


stage of TSV






infection







LOS: lymphoid organ spheroids






Moderate to high (G3) level of lymphoid organ spheroids were found in the non-vaccinated group collected at day 10, indicating chronic TSV infection.
Real-Time TSV RT-PCR
The real-time TSV RT-PCR assay was used to quantify the viral load in the surviving shrimp (Table 12). The results showed that the viral load was lowest in the shrimp vaccinated with T1, 2.46×103 copies/μl RNA. The T2-inhibited vaccine fed shrimp contained higher viral loads, 8.88×103 copies/μl RNA. For the non-vaccinated group, the shrimp had the highest viral loads, 5.20×104 copies/μl RNA.







TABLE 12







Mean TSV copies per 1 RNA in TSV surviving shrimp











Mean ± S.D. TSV


Tank no.
Feed
copies/μl





A
Vaccine (T1)-mixed
2.46 × 103 ± 2.22 × 103


B
Inactive vaccine (T2)-
8.88 × 103 ± 8.88 × 103



mixed


C
Control diet
5.20 × 104 ± 9.01 × 104









These results showed that TSV chronically-infected shrimp had higher percent survivals (60-91%) when re-challenged with TSV. In particular, T1-vaccinated P. vannamei had the highest survival and lowest viral load. The percent survival for SPF P. vannamei challenged with Hawaii TSV was 25%.
EXAMPLE 4
Determination of Replikin Concentration in Publicly Available Accession Numbers for Isolates of TSV from 2000 through 2005
Mean Replikin concentrations were determined for all amino acid sequences for taura syndrome virus with accession numbers publicly available at www.pubmed.com. The amino acid sequences were scanned for Replikin sequences of 7 to 50 amino acids comprising (1) at least one lysine residue located at a first terminus of the sequence and at least one lysine residue or at least one histidine residue located at a second terminus of the sequence; (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues. The total number of Replikin sequences was determined for each available accession number. The total number of Replikin sequences in each accession number was then divided by the total number of amino acid residues disclosed in the accession number. The result was the Replikin concentration. The mean Replikin concentration was then determined for all viruses isolated and reported in a particular year. Table 13 provides the results.







TABLE 13







TSV Replikin Concentration by Year














No. of
Mean Replikin





PubMed Accession Number-Replikin
Isolates
Concentration


Year
Count
per year
per year
S.D.
Significance















2000
NP_149058 70 NP_149057 70
5
2.7
1.3
low p < 0.02



AAK72221 70 AAK72220 70 AAG44834 4


2001
AAM73766 7
1
0.7
0.0
prev p < 0.02


2002
AAN77089 2 AAN77088 2 AAN77087 2
8
0.7
0.4
low p > 0.50



AAN77086 2 AAW32934 2 AAW32932



2 AAW32930 2 AAW32929 1


2003
AAR11292 6 AAR11291 6 AAR11290 6
3
0.6
0.0
prev p < 0.20


2004
AAX07125 2 AAX07117 2 AAT81157
23
0.8
0.9
low p < 0.40,



75 AAT81158 75 AAX07127 2



prev p < 0.20



AAX07126 2 AAX07124 2 AAX07123 2



AAX07122 2 AAX07121 2 AAX07120 2



AAX07119 2 AAX07118 2 AAX07116 2



AAX07115 2 AAX07114 2 AAX07113 2



AAX07112 2 AAX35819 2 AAX35818 1



AAX35817 2 AAX35816 1 AAX35815 2


2005
AAY56364 71 AAY56363 71 AAY44822
12
1.8
1.7
low p < 0.02,



1 AAY44821 1 AAY44820 1 AAY44819



prev p < 0.05



1 AAY44818 1 AAY44817 1 AAY89097



83 AAY89096 83 ABB17263 63



ABB17264 63









EXAMPLE 5
Determination of Replikin Concentrations in 2001 and 2005 Isolates of TSV Publicly Available at Accession Nos. AAM73766 and AAY89096
The taura syndrome virus (TSV) is generally a less virulent virus than white spot syndrome virus (WSSV) and the structure of the TSV Replikin Scaffold is less closely related to influenza virus than are the structures of WSSV Replikin Scaffolds. See Replikin Scaffold Sequences below. In year 2000, TSV had a Replikin concentration of 2.7. Between 2001 and 2004, TSV had a lower mean Replikin concentration, as low as 0.6, and its Replikin Scaffold disappeared. In 2005 the Replikin Scaffold reappeared along with an increase in lysines and histidines and a commensurate increase in Replikin concentration (1.8) followed by an increase in TSV outbreaks in 2006-2007. See FIG. 6 and Replikin Scaffolds below.
Below is a comparison of the Replikin Scaffold identified in Accession no. AAK72220 in an isolate of TSV from 2000 and the Replikin Scaffold identified in Accession no. AAY89096 in an isolate of TSV from 2005. The TSV Replikin Scaffolds are also compared to two Replikin Scaffold sequences in H1N1 influenza virus in the 1918 pandemic and shrimp WSSV in 2000.
Replikin Scaffold Sequences (SEQ ID NOS 99-104, Respectively, in Order of Appearance)

























The following analysis of Accession Nos. AAM73766 and AAY89096 demonstrate Replikin concentration analysis of amino acid sequences of isolates of taura syndrome virus having publicly available accession numbers at www.pubmed.com.








PubMed Code: AAM73766



Isolated: 2001


Source: Taura Syndrome Virus


(SEQ ID NO: 105)



M1P2A3N4P5V6E7I8D9N10F11D12T13T14T15S16G17G18L19I20P21G22G23S24V25T26N27S28E29G30S31T32I33L34M35N36D37



 


I38P39I40T41N42Q43N44V45V46L47S48K49N50V51T52D53N54L55F56E57V58Q59D60Q61A62L63I64E65S66L67S68R69D70V71


 


L72L73H74N75D76S77W78T79S80S81D82D83E84I85G86T87T88M89T90Q91E92Q93L94A95T96E97F98N99Q100P101H102L103


 


Y104E105I106S107L108P109D110D111I112V113R114K115S116L117F118M119S120N121K122L123A124N125I126A127Y128


 


M129R130C131D132


 


Y133E134V135T136V137R138V139Q140A141T142P143F144L145Q146G147A148L149W150L151W152N153K154M155N156A157


 


K158


 


Q159T160S161I162I163R164R165T166L167T168E169H170L171R172S173I174T175S176F177P178G179I180E181M182N183


 


L184Q185S186


 


E187A188R189A190I191T192L193S194I195P196Y197T198S199E200F201Q202V203F204N205P206R207N208V209N210N211


 


L212N213S214


 


I215R216L217S218V219L220S221Q222L223Q224G225P226E227D228V229E230S231A232S233Y234S235I236Y237G238R239


 


L240K241N242I243K244L245Y246G247H248A249P250S251V252T253S254S255V256Y257P258S259T260Q261S262G263Y264


 


D265D266D267C268P269I270V271H272A273G274T275D276E277D278S279S280K281Q282G283I284V285S286R287V288A289


 


D290T291V292G293A294V295A296N297V298V299D300G301V302G303V304P305I306L307S208T309I310A311K312P313V314


 


S315W316V317S318G319V320V321S322N323V324


 


A325S326M327F328G329F330S331K332D333R334D335M336T337K338V339N340A341Y342E343N344L345P346G347K348G349


 


F350T351H352G353V354G355F356D357Y358G359V360P361L362S363L364F365P366N367N368A369I370D371P372T373I374


 


A375V376P377E378G379L380D381E382M383S384I385E386Y387L388A389Q390R391P392Y393M394L395N396R397Y398T399


 


I400R401G402G403D404T405P406


 


D407V408H409G410T411T412V413A414D415I416P417V418S419P420V421N422F423S424L425Y426G427K428V429I430A431


 


K432Y433


 


R434T435L436F437A438A439P440V441S442L443A444V445A446M447A448N449W450W451R452G453N454I455N456L457N458


 


L459R460F461A462K463T464Q465Y466H467Q468C469R470L471L472V473Q474Y475L476P477Y478G479S480G481V482Q483


 


P484I485E486S487


 


I488L489S490Q491I492I493D494I495S496Q497V498D499D500K501G502I503D504I505A506F507P508S509V510Y511P512


 


N513K514W515M516R517V518Y519D520P521A522K523V524G525Y526T527A528D529C530A531P532G533R534I535V536I537


 


S538V539L540N541P542


 


L543I544S545A546S547T548V549S550P551N552I553V554M555Y556P557W558V559H560W561S562N563L564E565V566A567


 


E568P569


 


G570T571L572A573K574A575A576I577G578F579N580Y581P582A583D584V585P586E587E588P589T590F591S592V593T594


 


R595A596


 


P597V598S599G600T601L602F603T604L605L606Q607D608T609K610V611S612L613G614E615A616D617G618V619F620S621


 


L622Y623


 


F624T625N626T627T628T629G630R631R632H633R634L635T636Y637A638G639L640P641G642E643L644G645S646C647E648


 


I649V650


 


K651L652P653Q654G655Q656Y657S658I659E660Y661A662A663T664S665A666P667T668L669V670L671D672R673P674I675


 


F676S677E678P679I680G681P682K683Y684V685V686T687K688V689K690N691G692D693V694V695S696I697S698E699E700


 


T701L702V703T704C705


 


G706S707M708A709A710L711G712G713A714T715V716A717L718Q719S720V721D722E723T724I725E726I727L728K729L730


 


E731S732


 


D733F734E735S736K737A738P739V740K741F742T743P744G745N746Y747T748V749V750T751E752A753S754D755V756E757


 


L758V759


 


T760N761Q762D763I764T765V766N767E768H769N770P771R772T773H774A775G776I777D778E779E780P781P782V783K784


 


R785S786


 


V787I788G789R790I791V792R793R794V795A796R797Y798P799P800N801K802L803I804R805R806I807L808R809D810L811


 


S812Q813S814P815C816I817Y818P819S820T821H822A823G824L825D826Y827S828S829S830D831T832S833T834M835L836


 


T837T838M839G840E841Q842F843V844S845L846R847M848L849T850R851R852S853S854P855V856D857I858L859R860G861


 


D862L863V864T865L866P867G868S870F871G872T873D874N875S876L877R878Q879S880L881V882N883I884I885S886Y887


 


M888Y889R890F891T892H893G894S895I896S897Y898K899I900I901P902K903N904K905G906D907L908Y909I910T911T912


 


T913S914P915D916S917I918E919T920S921T922S923A924Y925Q926


 


F927D928T929N930R931A932M933H934Y935I936N937T938S939L940N941P942M943A944Q945I946S947L948P949Y950Y951


 


S952P953


 


A954E955N956L957V958I959D960S961K962S963F964P965Q966L967S968D969L970S971I972S973N974L975E976R977T978


 


E979N980E981Y982F983V984L985A986S987A988G989D990D991H992T993F994S995Q996L997A998G999C1000P1001A1002


 


F1003T1004F1005G1006P1007A1008E1009L1010A1011


 


Replikin Sequences in Amino-Terminal Portion of Peptide


(SEQ ID NO: 106)










 (1)
H102L103Y104E105I106S107L108P109D110D111I112V113R114K115S116L117F118M119S120N121K122



 








(SEQ ID NO: 107)










 (2)
K332D333R334D335M336T337K338V339N340A341Y342E343N344L345P346G347K348G349F350T351H352



 








(SEQ ID NO: 108)










 (3)
K338V339N340A341Y342E343N344L345P346G347K348G349F350T351H352



 








Replikin Sequences in Mid-Molecule Portion of Peptide



Zero Replikins.


Replikin Sequences in Carboxy-Terminal Portion of Peptide


(SEQ ID NO: 109)










 (4)
K729L730E731S732D733F734E735S736K737A738P739V740K741F742T743P744G745N746Y747T748V749V750T751E752



 



A753S754D755V756E757L758V759T760N761Q762D763I764T765V766N767E768H769N770P771R772T773H774


 








(SEQ ID NO: 110)










 (5)
K729L730E731S732D733F734E735S736K737A738P739V740K741F742T743P744G745N746Y747T748V749V750T751E752



 



A753S754D755V756E757L758V759T760N761Q762D763I764T765V766N767E768H769


 








(SEQ ID NO: 111)










 (6)
H893G894S895I896S897Y898K899I900I901P902K903N904K905



 








(SEQ ID NO: 112)










 (7)
K899I900I901P902K903N904K905G906D907L908Y909I910T911T912T913S914P915D916S917I918E919T920S921T922




S923A924Y925Q926F927D928T929N930R931A932M933H934


 








Replikin Count = Number of Replikins per 100 amino acids = 7/1011 = 0.7



PubMed Code: AAY89096


Isolated: 2005


Source: Taura Syndrome Virus


(SEQ ID NO: 113)



M1A2S3Y4Y5L6N7I8K9T10H11N12L13R14R15T16P17G18A19H20R21A22F23Y24V25M26N27D28D29G30E31N32R33I34Y35



 


S36L37I38G39T40L41R42R43A44P45A46F47K48V49G50S51R52R53Y54K55S56H57I58P59Y60R61R62K63A64T65V66A67E68


 


L69C70


 


N71Q72L73H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88V89I90S91E92N93K94V95Q96P97D98S99M100L101K102


 


A103A104F105G106N107W108E109E110W111P112K113D114K115V116C117E118E119L120Y121S122E123C124E125C126G127


 


Y128V129G130T131C132Y133V134S135V136D137W138L139R140P141Q142A143T144K145C146N147D148C149I150L151K152


 


M153N154R155N156V157E158Y159P160Y161H162T163I164G165V166S167G168N169V170V171T172N173T174D175I176V177


 


Y178T179G180Y181A182D183V184


 


F185K186C187E188K189C190D191L192L193M194G195A196W197A198P199N200D201I202P203A204L205T206H207N208I209


 


R210S211


 


S212Q213C214V215Q216F217K218L219P220T221E222N223L224A225A226R227N228Y229V230L231L232C233E234E235I236


 


E237R238


 


E239N240I241P242V243I244F245Q246D247Y248S249E250G251N252V253F254T255C256R257I258V259S260G261D262L263


 


T264A265V266G267T268A269S270N271M272Y273T274A275R276D277V278A279S280K281S282L283L284D285Q286L287H288


 


N289T290P291N292V293H294M295H296S297L298H299S300L301P302Y303E304N305F306P307C308E309A310L311E312F313


 


A314V315E316Q317G318I319I320P321P322V323T324F325D326E327V328F329A330N331D332E333Y334V335I336T337I338


 


S339C340S341L342L343V344V345S346D347V348G349P350T351Q352A353V354A355R356E357R358A359A360K361R362F363


 


L364K365M366Y367D368Y369S370A371S372Y373P374S375T376


 


H377M378F379T380L381S382T383L384P385Q386R387S388G389E390T391L392E393L394A395N396A397T398L399N400N401


 


V402N403


 


N404V405I406D407R408H409D410E411A412I413S414N415V416R417Q418N419V420E421V422K423L424T425D426S428R429


 


Q430


 


V431G432A433M434L435P436K437V438E439T440V441I442D443D444V445S446S447T448L449S450S451F452R453G454V455


 


L456D457


 


K458I459S460A461W462M463P464S465S466N467P468K469I470I471D472L473I474K475E476T477F478V479S480L481F482


 


F483A484I485


 


L486T487K488S489L490Y491P492I493I494Q495G496I497S498S499Y500A501L502R503N504N505L506M507A508N509H510


 


L511T512A513L514S515E516W517L518M519T520L521E522Y523D524S525P526D527E528E529E530M531P532S533T534H535


 


G536F537M538D539D540


 


L541T542S543R544L545P546G547L548N549G550A551K552V553Q554A555A556T557I558Y559E560S561I562G563T564G565


 


L566C567


 


A568A569L570S571G572I573L574S575F576I577A578V579M580C581L582G583I584T585D586L587S588A589V590T591F592


 


N593K594L595L596T597Q598S599S600L601V602G603R604A605L606V607G608V609R610S611F612K613D614V615F616F617


 


G618I619W620D621Y622


 


V623D624N625Q626V627C628E629I630L631Y632G633K634S635R636K637N638L639D640L641L642K643E644Y645P646S647


 


L648D649


 


S650L651L652S653I654F655N656Y657F658H659D660T661V662D663A664N665V666L667I668S669C670N671R672A673A674


 


C675E676L677L678V679K680A681D682N683L684Y685Q686G687Y688L689D690K691S692I693T694L695M696H697R698E699


 


I700S701S702R703L704


 


K705E706A707R708N709S710V711K712D713L714I715A716K717A718Q719V720Y721L722T723C724G725D726G727S728R729


 


V730P731


 


P732V733V734V735Y736M737Y738G739D740A741G742C743G744K745T746E747L748S749M750A751L752Q753D754H755F756


 


A757T758K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774A775E776N777E778F779W780D781


 


G782V783K784Q785S786H787K788I789I790A791Y792D793D794V795L796Q797I798V799D800S801A802Q803K804P805N806


 


P807E808L809F810E811F812I813R814L815N816N817S818D819P820Y821Q822V823H824M825S826S827V828S829D830K831


 


A832N833T834F835T836A837P838S839F840


 


V841F842A843T844S845N846V847N848P849G850T851Y852V853P854K855S856I857H858S859A860D861A862F863R864R865


 


R866L867


 


D868L869C870V871Y872V873D874V875K876D877E878F879A880R881I882V883A884G885S886K887G888H889R890K891V892


 


P893C894


 


E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913E914I915V916A917G918T919


 


Y920K921


 


I922T923P924E925T926A927V928Y929E930L931H932V933D934T935T936L937G939D940A941Q942S943K944V945C946A947


 


Y948


 


D949G950L951V952S953L954I955E956Q957V958R959R960L961R962V963A964A965H966S967D968K969V970E971T972D973


 


V974P975


 


V976L977P978T979R980L981H982E983L984S985Q986E987T988F989P990N991T992H993A994G995V996G997F998Q999F1000


 


A1001T1002D1003W1004L1005G1006D1007F1008D1009R1010P1011V1012E1013A1014L1015S1016Y1017L1018N1019K1020


 


T1021L1022E1023A1024


 


H1025F1026V1027S1028R1029S1030A1031N1032D1033G1034S1035M1036F1037I1038P1039A1040S1041E1042V1043A1044


 


D1045L1046L1047C1048Q1049R1050H1051N1052N1053T1054N1055L1056N1057E1058E1059L1060V1061Y1062L1063T1064


 


W1065M1066T1067Q1068I1069


 


T1070D1071K1072E1073L1074A1075S1076S1077L1078V1079Y1080F1081T1082N1083N1084G1085M1086D1087K1088S1089


 


I1090W1091


 


K1092K1093S1094A1095E1096R1097S1098A1099Q1100A1101I1102S1103Q1104C1105K1106N1107A1108W1109T1110R1111


 


I1112N1113D1114F1115L1116K1117N1118H1119W1120I1121S1122I1123S1124A1125V1126I1127G1128S1129A1130L1131


 


L1132I1133G1134G1135V1136S1137


 


S1138A1139V1140K1141C1142A1143T1144K1145C1146R1147V1148R1149K1150I1151L1152Q1153D1154G1155G1156S1157


 


I1158M1159Q1160L1161V1162G1163V1164R1165S1166C1167M1168Y1169A1170C1171Q1172L1173C1174K1175R1176I1177


 


K1178N1179C1180D1181L1182


 


R1183L1184R1185V1186R1187N1188R1189S1190S1191G1192V1193T1194T1195F1196V1197P1198G1199D1200V1201R1202


 


R1203V1204


 


A1205R1206H1207V1208I1209S1210A1211A1212D1213F1214C1215E1216V1217P1218V1219H1220H1221S1222F1223I1224


 


Q1225S1226L1227C1228D1229E1230A1231F1232T1233V1234H1235S1236D1237K1238E1239E1240T1241F1242S1243I1244


 


L1245D1246F1247T1248P1249E1250


 


A1251K1252G1253R1254N1255S1256P1257E1258S1259A1260V1261V1262E1263S1264H1265Q1266D1267Y1268R1269V1270


 


K1271A1272


 


A1273V1274V1275E1276S1277H1278Q1279D1280F1281K1282P1283K1284D1285A1286I1287V1288E1289S1290T1291I1292


 


D1293T1294V1295F1296T1297E1298S1299H1300Q1301D1302V1303R1304V1305K1306L1307H1308P1309Q1310I1311E1312


 


S1313H1314Q1315D1316F1317R1318A1319K1320N1321P1322I1323V1324E1325S1326R1327K1328P1329D1330Y1331Q1332


 


V1333E1334W1335T1336D1337L1338R1339T1340E1341S1342S1343N1344D1345R1346N1347A1348Q1349D1350I1351S1352


 


N1353R1354I1355L1356S1357R1358N1359F1360V1361R1362L1363Y1364V1365P1366G1367S1368S1369L1370Y1371T1372


 


H1373G1374L1375F1376A1377Y1378G1379R1380M1381L1382L1383M1384P1385K1386


 


H1387M1388F1389D1390M1391L1392N1393G1394S1395V1396E1397I1398V1399S1400I1401A1402D1403K1404G1405N1406


 


T1407R1408


 


V1409H1410V1411K1412I1413Q1414S1415H1416K1417T1418V1419T1420R1421G1422G1423Y1424E1425V1426D1427I1428


 


V1429I1430C1431E1432M1433G1434N1435S1436I1437S1438A1439R1440K1441D1442I1443T1444S1445Y1446F1447P1448


 


T1449V1450K1451E1452L1453P1454


 


G1455L1456T1457G1458M1459M1460S1461S1462G1463R1464M1465R1466V1467F1468S1469T1470A1471K1472F1473K1474


 


A1475S1476


 


D1477S1478C1479S1480Y1481L1482M1483P1484Q1485D1486F1487V1488A1489K1490Y1491I1492A1493A1494V1495D1496


 


H1497I1498T1499S1500K1501S1502P1503E1504K1505K1506S1507Y1508F1509I1510R1511Q1512G1513F1514E1515A1516


 


E1517S1518D1519S1520M1521Q1522G1523D1524C1525C1526S1527P1528Y1529V1530L1531F1532N1533S1534A1535S1536


 


R1537A1538K1539I1540V1541G1542L1543H1544C1545A1546G1547F1548D1549G1550T1551A1552R1553V1554F1555A1556


 


Q1557I1558I1559T1560Q1561E1562D1563I1564M1565A1566A1567T1568P1569T1570T1571H1572A1573G1574R1575V1576


 


T1577T1578E1579F1580P1581H1582T1583S1584L1585R1586D1587S1588P1589L1590P1591


 


N1592S1593M1594A1595I1596G1597S1598V1599K1600T1601A1602P1603N1604P1605T1606K1607S1608E1609I1610T1611


 


R1612S1613P1614


 


I1615H1616G1617C1618F1619P1620V1621R1622T1623A1624P1625A1626T1627L1628Y1629S1630P1631T1632E1633N1634


 


L1635L1636I1637


 


K1637N1639A1640M1641K1642V1643T1644K1645N1646V1647E1648L1649L1650E1651E1652D1653L1654I1655D1656A1657


 


C1658V1659


 


H1660D1661V1662K1663R1664I1665L1666N1667A1668P1669G1670V1671S1672D1673V1674E1675K1676R1677V1678L1679


 


T1680H1681E1682E1683S1684I1685T1686G1687I1688E1689N1690R1691Q1692Y1693M1694N1695A1696L1697N1698R1699


 


S1700T1701S1702A1703G1704F1705


 


P1706Y1707S1708S1709R1710K1711A1712K1713G1714K1715S1716G1717K1718Q1719T1720W1721L1722G1723S1724E1725


 


E1726F1727I1728V1729D1730N1731P1732D1733L1734K1735E1736H1737V1738E1739K1740I1741V1742D1743K1744A1745


 


K1746D1747G1748I1749V1750


 


D1751V1752S1753L1754G1755I1756F1757A1758A1759T1760L1761K1762D1763E1764R1765R1766P1767L1768E1769K1770


 


V1771Q1772A1773N1774K1775T1776R1777V1778F1779A1780A1781S1782N1783Q1784G1785L1786A1787L1788A1789L1790


 


R1791R1792Y1793Y1794L1795


 


S1796F1797L1798D1799H1800V1801M1802T1803N1804R1805I1806D1807N1808E1809I1810G1811L1812G1813V1814N1815


 


V1816Y1817S1818Y1819D1820W1821T1822R1823I1824V1825N1826K1827L1828K1829R1830V1831G1832D1833K1834V1835


 


I1836A1837G1838D1839F1840


 


S1841N1842F1843D1844G1845S1846L1847N1848S1849Q1850I1851L1852S1853R1854V1855S1856E1857I1858V1859T1860


 


D1861W1862Y1863G1864D1865D1866A1867E1868N1869G1870L1871I1872R1873H1874T1875L1876L1877E1878Y1879L1880


 


F1881N1882A1883T1884W1885L1886M1887N1888G1889K1890V1891F1892Q1893L1894N1895H1896S1897Q1898P1899S1900


 


G1901N1902P1903L1904T1905T1906L1907I1908N1909C1910V1911Y1912N1913M1914I1915I1916F1917R1918Y1919V1920


 


Y1921L1922L1923A1924Q1925R1926E1927N1928G1929F1930P1931M1932T1933L1934S1935G1936F1937T1938T1939N1940


 


V1941A1942C1943I1944F1945Y1946G1947D1948D1949S1950L1951C1952S1953V1954S1955D1956K1957V1958S1959E1960


 


W1961F1962N1963Q1964H1965V1966I1967T1968R1969L1970M1971A1972A1973T1974G1975H1976E1977


 


Y1978T1979D1980E1981T1982K1983S1984G1985S1986P1987P1988P1989Y1990R1991S1992L1993N1994E1995V1996T1997


 


F1998L1999K2000R2001E2002F2003V2004L2005R2006D2007H2008F2009W2010I2011A2012P2013L2014S2015R2016N2017


 


T2018I2019E2020D2021M2022C2023M2024W2025S2026R2027K2028N2029I2030D2031A2032Q2033D2034A2035L2036L2037


 


Q2038T2039T2040R2041I2042A2043S2044F2045E2046A2047S2048L2049H2050E2051K2052N2053Y2054F2055L2056M2057


 


F2058C2059D2060V2061I2062K2063K2064A2065C2066R2067N2068


 


A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082S2083F2084L2085L2086A2087Q2088


 


Q2089G2090R2091A2092G2093A2094H2095D2096S2097E2098F2099L2100S2101Q2102L2103L2104D2105L2106N2107


 


Replikin Sequences in Amino-Terminal Portion of Peptide


(SEQ ID NO: 114)










 (1)
H20R21A22F23Y24V25M26N27D28D29G30E31N32R33I34Y35S36L37I38G39T40L41R42R43A44P45A46F47K48V49



 



G50S51R52R53Y54K55S56H57I58P59Y60R61R62K63


 








(SEQ ID NO: 115)










 (2)
K48V49G50S51R52R53Y54K55S56H57



 








(SEQ ID NO: 116)










 (3)
K48V49G50S51R52R53Y54K55S56H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74



 








(SEQ ID NO: 117)










 (4)
K55S56H57I58P59Y60R61R62K63



 








(SEQ ID NO: 118)










 (5)
K55S56H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74



 








(SEQ ID NO: 119)










 (6)
H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88



 



V89I90S91E92N93K94


 








(SEQ ID NO: 120)










 (7)
H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88



 



V89I90S91E92N93K94V95Q96P97D98S99M100L101K102


 








(SEQ ID NO: 121)










 (8)
H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88V89I90S91E92N93K94V95Q96P97D98S99M100L101K102



 








(SEQ ID NO: 122)










 (9)
H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88V89I90S91E92N93K94



 








(SEQ ID NO: 123)










(10)
K145C146N147D148C149I150L151K152M153N154R155N156V157E158Y159P160Y161H162



 








(SEQ ID NO: 124)










(11)
K469I470I471D472L473I474K475E476T477F478V479S480L481F482F483A484I485L486T487K488S489L490Y491P492



 



I493I494Q495G496I497S498S499Y500A501L502R503N504N505L506M507A508N509H510


 








(SEQ ID NO: 125)










(12)
K634S635R636K637N638L639D640L641L642K643E644Y645P646S647L648D649S650L651L652S653I654F655N656Y657



 



F658H659


 








(SEQ ID NO: 126)










(13)
K637N638L639D640L641L642K643E644Y645P646S647L648D649S650L651L652S653I654F655N656Y657F658H659



 








(SEQ ID NO: 127)










(14)
H697R698E699I700S701S702R703L704K705E706A707R708N709S710V711K712



 








Replikin Sequences in Mid-Molecule Portion of Peptide



(SEQ ID NO: 128)










(15)
K705E706A707R708N709S710V711K712D713L714I715A716K717A718Q719V720Y721L722T723C724G725D726G727S728



 



R729V730P731P732V733V734V735Y736M737Y738G739D740A741G742C743G744K745T746E747L748S749M750A751L752


 



Q753D754H755


 








(SEQ ID NO: 129)










(16)
H755F756A757T758K759Y760F761G762E763V764P765K766



 








(SEQ ID NO: 130)










(17)
H755F756A757T758K759Y760F761G762E763V764P765K766K767



 








(SEQ ID NO: 131)










(18)
H755F756A757T758K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774



 








(SEQ ID NO: 132)










(19)
H755F756A757T758K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774A775E776N777E778



 



F779W780D781G782V783K784


 








(SEQ ID NO: 133)










(20)
K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774A775E776N777E778F779W780D781G782



 



V783K784Q785S786H787


 








(SEQ ID NO: 134)










(21)
K766K767D768V769I770Y771S772R773K774A775E776N777E778F779W780D781G782V783K784Q785S786H787



 








(SEQ ID NO: 135)










(22)
K767D768V769I770Y771S772R773K774A775E776N777E778F779V780D781G782V783K784Q785S786H787



 








(SEQ ID NO: 136)










(23)
K774A775E776N777E778F779W780D781G782V783K784Q785S786H787



 








(SEQ ID NO: 137)










(24)
K774A775E776N777E778F779W780D781G782V783K784Q785S786H787K788I789I790A791Y792D793D794V795L796Q797



 



I798V799D800S801A802Q803K804P805N806P807E808L809F810E811F812I813R814L815N816N817S818D819P820Y821


 



Q822V823H824


 








(SEQ ID NO: 138)










(25)
H858S859A860D861A862F863R864R865R866L867D868L869C870V871Y872V873D874V875K876D877E878F879A880R881



 



I882V883A884G885S886K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906


 








(SEQ ID NO: 139)










(26)
H858S859A860D861A862F863R864R865R866L867D868L869C870V871Y872V873D874V875K876D877E878F879A880R881



 



I882V883A884G885S886K887G888H889R890K891V892P893C894E895Q896K897


 








(SEQ ID NO: 140)










(27)
K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910



 



M911K912H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929E930L931H932


 








(SEQ ID NO: 141)










(28)
K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901



 








(SEQ ID NO: 142)










(29)
K887G888H889R890K891V892P893C894E895Q896K897



 








(SEQ ID NO: 143)










(30)
K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909



 








(SEQ ID NO: 144)










(31)
K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910



 



M911K912H913


 








(SEQ ID NO: 145)










(32)
H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912



 



H913E914I915V916A917G918T919Y920K921


 








(SEQ ID NO: 146)










(33)
H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906



 








(SEQ ID NO: 147)










(34)
H889R890K891V892P893C894E895Q896K897



 








(SEQ ID NO: 148)










(35)
H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912



 








(SEQ ID NO: 149)










(36)
K891V892P893C894E895Q896K897I898W899L900H901



 








(SEQ ID NO: 150)










(37)
K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909



 








(SEQ ID NO: 151)










(38)
K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913



 








(SEQ ID NO: 152)










(39)
H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912



 



H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929E930L931H932


 








(SEQ ID NO: 153)










(40)
K897I898W899L900H901Q902N903P904G905K906



 








(SEQ ID NO: 154)










(41)
K897I898W899L900H901Q902N903P904G905K906T907Q908H909



 








(SEQ ID NO: 155)










(42)
K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913



 








(SEQ ID NO: 156)










(43)
K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913E914I915V916A917G918T919Y920



 



K921I922T923P924E925T926A927V928Y929E930L931H932


 








(SEQ ID NO: 157)










(44)
H901Q902N903P904G905K906T907Q908H909D910M911K912



 








(SEQ ID NO: 158)










(45)
H901Q902N903P904G905K906T907Q908H909D910M911K912H913E914I915V916A917G918T919Y920K921



 








(SEQ ID NO: 159)










(46)
K906T907Q908H909D910M911K912



 








(SEQ ID NO: 160)










(47)
K906T907Q908H909D910M911K912H913



 








(SEQ ID NO: 161)










(48)
K906T907Q908H909D910M911K912H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929



 








(SEQ ID NO: 162)










(49)
H909D910M911K912H913E914I915V916A917G918T919Y920K921



 








(SEQ ID NO: 163)










(50)
K912H913E914I915V916A917G918T919Y920K921



 








(SEQ ID NO: 164)










(51)
K912H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929E930L931H932



 








(SEQ ID NO: 165)










(52)
H1119W1120I1121S1122I1123S1124A1125V1126I1127G1128S1129A1130L1131L1132I1133G1134G1135V1136S1137



 



S1138A1139V1140K1141C1142A1143I1144K1145C1145R1147V1148R1149K1150


 








(SEQ ID NO: 166)










(53)
H1278Q1279D1280F1281K1282P1283K1284D1285A1286I1287V1288E1289S1290I1291I1292D1293I1294V1295F1296



 



T1297E1298


 



S1299H1300Q1301D1302V1303R1304V1305K1306L1307H1308P1309Q1310I1311E1312S1313H1314Q1315D1316F1317


 



R1318A1319K1320N1321P1322I1323V1324E1325S1326R1327K1328


 








(SEQ ID NO: 167)










(54)
H1300Q1301D1302V1303R1304V1305K1306L1307H1308P1309Q1310I1311E1312S1313H1314Q1315D1316F1317R1318



 



A1319K1320N1321P1322I1323V1324E1325S1326R1327K1328


 








(SEQ ID NO: 168)










(55)
H1308P1309Q1310I1311E1312S1313H1314Q1315D1316F1317R1318A1319K1320N1321P1322I1323V1324E1325S1326



 



R1327K1328


 








(SEQ ID NO: 169)










(56)
H1314Q1315D1316F1317R1318A1319K1320N1321P1322I1323V1324E1325S1326R1327K1328



 








(SEQ ID NO: 170)










(57)
H1373G1374L1375F1376A1377Y1378G1379R1380M1381L1382L1383M1384P1385K1386H1387M1388F1389D1390M1391



 



L1392


 



N1393G1394S1395V1396E1397I1398V1399S1400I1401A1402D1403K1404G1405N1406T1407R1408V1409H1410V1411


 



K1412


 








(SEQ ID NO: 171)










(58)
H1387M1388F1389D1390M1391L1392N1393G1394S1395V1396E1397I1398V1399S1400I1401A1402D1403K1404G1405



 



N1406T1407R1408V1409H1410V1411K1412


 








(SEQ ID NO: 172)










(59)
K1404G1405N1406T1407R1408V1409H1410V1411K1412



 








(SEQ ID NO: 173)










(60)
K1404G1405N1406T1407R1408V1409H1410V1411K1412I1413Q1414S1415H1416



 








Replikin Sequences in Carboxy-Terminal Portion of Peptide










(61)
H1410V1411K1412I1413Q1414S1415H1416K1417T1418V1419T1420R1421G1422G1423Y1424E1425V1426D1427I1428



 



V1429I1430


 



C1431E1432M1433G1434N1435S1436I1437S1438A1439R1440K1441D1442I1443T1444S1445Y1446F1447P1448T1449


 



V1450K1451


 








(SEQ ID N0 175)










(62)
H1416K1417T1418V1419T1420R1421G1422G1423Y1424E1425V1426D1427I1428V1429I1430C1431E1432M1433G1434



 



N1435S1436I1437S1438A1439R1440K1441D1442I1443T1444S1445Y1446F1447P1448T1449V1450K1451


 








(SEQ ID NO: 176)










(63)
H1582T1583S1584L1585R1586D1587S1588P1589L1590P1591N1592S1593M1594A1595I1596G1597S1598V1599K1600



 



T1601A1602P1603N1604P1605T1606K1607


 








(SEQ ID NO: 177)










(64)
K1600T1601A1602P1603N1604P1605T1606K1607S1608E1609I1610T1611R1612S1613P1614I1615H1616



 








(SEQ ID NO: 178)










(65)
H1616G1617C1618F1619P1620V1621R1622T1623A1624P1625A1626T1627L1628Y1629S1630P1631T1632E1633N1634



 



L1635L1636I1637K1638N1639A1640M1641K1642V1643T1644K1645


 








(SEQ ID NO: 179)










(66)
K1638N1639A1640M1641K1642V1643T1644K1645N1646V1647E1648L1649L1650E1651E1652D1653L1654I1655D1656



 



A1657C1658V1659H1660


 








(SEQ ID NO: 180)










(67)
K1638N1639A1640M1641K1642V1643T1644K1645N1646V1647E1648L1649L1650E1651E1652D1653L1654I1655D1656



 



A1657C1658V1659H1660D1661V1662K1663R1664I1665L1666N1667A1668P1669G1670V1671S1672D1673V1674E1675


 



K1676R1677V1678L1679T1680H1681


 








(SEQ ID NO: 181)










(68)
H1681E1682E1683S1684I1685T1686G1687I1688E1689N1690R1691Q1692Y1693M1694N1695A1696L1697N1698R1699



 



S1700T1701S1702A1703G1704F1705P1706Y1707S1708S1709R1710K1711A1712K1713G1714K1715S1716G1717K1718


 








(SEQ ID NO: 182)










(69)
K1711A1712K1713G1714K1715S1716G1717K1718Q1719T1720W1721L1722G1723S1724E1725E1726F1727I1728V1729



 



D1730N1731P1732D1733L1734K1735E1736H1737


 








(SEQ ID NO: 183)










(70)
K1735E1736H1737V1738E1739K1740I1741V1742D1743K1744



 








(SEQ ID NO: 184)










(71)
H1737V1738E1739K1740I1741V1742D1743K1744A1745K1746D1747G1748I1749V1750D1751V1752S1753L1754G1755



 



I1756F1757A1758A1759T1760L1761K1762D1763E1764R1765R1766P1767L1768E1769K1770


 








(SEQ ID NO: 185)










(72)
H1737V1738E1739K1740I1741V1742D1743K1744A1745K1746



 








(SEQ ID NO: 186)










(73)
K1762D1763E1764R1765R1766P1767L1768E1769K1770V1771Q1772A1773N1774K1775T1776R1777V1778F1779A1780



 



A1781S1782N1783Q1784G1785L1786A1787L1788A1789L1790R1791R1792Y1793Y1794L1795S1796F1797L1798D1799


 



H1800


 








(SEQ ID NO: 187)










(74)
H1800V1801M1802T1803N1804R1805I1806D1807N1808E1809I1810G1811L1812G1813V1814N1815V1816Y1817S1818



 



Y1819D1820W1821T1822R1823I1824V1825N1826K1827L1828K1829R1830V1831G1832D1833K1834


 








(SEQ ID NO: 188)










(75)
K1827L1828K1829R1830V1831G1832D1833K1834V1835I1836A1837G1838D1839F1840S1841N1842F1843D1844G1845



 



S1846L1847N1848S1849Q1850I1851L1852S1853R1854V1855S1856E1857I1858V1859T1860D1861W1862Y1863G1864


 



D1865D1866A1867E1868N1869G1870L1871I1872R1873H1874


 








(SEQ ID NO: 189)










(76)
H2050E2051K2052N2053Y2054F2055L2056M2057F2058C2059D2060V2061I2062K2063K2064A2065C2066R2067N2068



 



A2069G2070Y2071K2072


 








(SEQ ID NO: 190)










(77)
H2050E2051K2052N2053Y2054F2055L2056M2057F2058C2059D2060V2061I2062K2063K2064A2065C2066R2067N2068



 



A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082


 








(SEQ ID NO: 191)










(78)
K2063K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077



 








(SEQ ID NO: 192)










(79)
K2063K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081



 



K2082S2083F2084L2085L2086A2087Q2088Q2089G2090R2091A2092G2093A2094H2095


 








(SEQ ID NO: 193)










(80)
K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082



 



S2083F2084L2085L2086A2087Q2088Q2089G2090R2091A2092G2093A2094H2095


 








(SEQ ID NO: 194)










(81)
K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077



 








(SEQ ID NO: 195)










(82)
K2072E2073A2074C2075L2076H2077E2078L20792080C2081K2082



 








(SEQ ID NO: 196)










(83)
K2072E2073A2074C2075L2076H2077E2078L20792080C2081K2082S2083F2084L2085L2086A2087Q2088Q2089G2090



 



R2091A2092G2093A2094H2095






Replikin Count=Number of Replikins per 100 amino acids=83/2107=3.9

EXAMPLE 6
Cyclic Production of West Nile Virus Replikins and Annual Human Morbidity and Methods of Prediction
In a further aspect of the invention, correlation between virus biochemical cycles and virus morbidity cycles are identified and used to predict increases in morbidity 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 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 at least four successive time points and identifying at least two cycles of increasing mean Replikin Counts over the at least four time points; and (4) predicting an increase in morbidity 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 embodiment, specific conserved Replikin sequences are identified within the step-wise cycles for example, KIIQKAHK (SEQ ID NO: 199), HLKCRVKMEK (SEQ ID NO: 200), KLTSGHLK (SEQ ID NO: 201), and HNDKRADPAFVCK (SEQ ID NO: 202).
The following data in West Nile virus provides an example of cycling in mean Replikin Count in a virus wherein the cycle predicts morbidity. The data additionally further support vaccines in aquaculture in invertebrates because they support the principles upon which such Replikin vaccines and other therapies are based including, in particular, the role Replikin sequences play in virulence and morbidity in viral diseases, the correlation of Replikin Count in diseases generally with pathogenicity, and the targeting of the Replikin structure in controlling rapid replication and disease.
Cycles are detectable because of repeating conserved virus structures and continuity of the Replikin phenomenon through time. The identified cycles provide a novel method of (1) determining the growth, spread, and path of an emerging disease, (2) predicting and tracking the occurrence and intensity of viral and other organism outbreaks by tracking changes in Replikin Count manually or using computer programs such as ReplikinsForecastTM (Replikins LLC) (see, e.g., U.S. application Ser. No. 11/116,203, filed Apr. 28, 2005, which is incorporated herein in its entirety by reference), (3) 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, (4) 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 (5) 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.
FIG. 7 provides the data for cycling of Replikin Count in West Nile Virus in correlation with cycling of West Nile Virus morbidity. The annual Replikin Count of the Envelope Protein of WNV (black), mean and standard deviation, is compared to the annual number of human cases in the U.S. per CDC reports (gray).
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 as defined by Replikin Count, preceding 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 virus. Since the relationship has already been demonstrated in several species, including crustacea, horses, and humans, it appears to be a broadly distributed general principle. 2004 to 2008: 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 FIG. 7, two cycles of Replikin concentration (count) and two cycles of WNV human morbidity may be observed. Two ‘Virus Replikins Cycles’, 2000-2003, and 2004-2008, as reflected by the Replikin Count of the virus, and two ‘human-infectivity cycles’, of the same dates, as reflected by the number of human WNV cases per year, are shown to correlate in the Figure. That Replikin Count data cycle in this manner was suggested from previous influenza virus data in previous applications, with H1N1 and H3N2, but not as clearly shown because the actual number of cases due to the H1N1 or H3N2 particular strain were not recorded (rather than just “outbreak”, “epidemic” or “pandemic”) as they are here for West Nile Virus.
The rising numbers for both the Replikin Count and the number of cases in the second cycle, 2004-2008, when compared to the first cycle, suggests an increased or ‘improved’ infective efficiency accompanying an increased Replikin Count in the second cycle, 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 cycle is related to the appearance of new Replikins 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, where 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.
FIG. 7 illustrates that early detection of changes in Replikin Count may be directly translated in a rapid response with vaccines to the emerging Replikin structures that may be synthesized in seven days or fewer after identification of the emerging Replikin sequences using, for example, ReplikinForecastTM software (Replikins LLC).
The following data provides 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. The data is reflected in FIG. 7.

















No. of
Mean






Isolates
Replikin



PubMed Accession Number
per
Count per


Year
Replikin Count West Nile Virus Envelope Protein
year
year
S.D.
Significance




















2000
ABR19638 102 AAK06624 97 AAG02039 98 AAG02038 97
4
2.9
0.1
low p < .001, prev







p < .001


2001
AAM70028 28 AAL07765 6 AAL07764 6 AAL07763 6 AAL07762 6
130
3.6
2.0
low p < .02, prev p < .001



AAL07761 6 AAL14222 30 AAL14221 30 AAL14220 30 AAL14219 30



AAL14218 30 AAL14217 30 AAL14216 30 AAL14215 30 AAK58104 30



AAK58103 31 AAK58102 30 AAK58101 30 AAK58100 30 AAK58099 31



AAK58098 30 AAK58097 30 AAK58096 30 AAK52303 30 AAK52302 30



AAK52301 30 AAK52300 30 AAK62766 32 AAK62765 32 AAK62764 32



AAK62763 32 AAK62762 32 AAK62761 32 AAK62760 32 AAK62759 32



AAK62758 32 AAK62757 32 AAK62756 32 AAK91592 20 ABR19637 111



AAM81753 97 AAM81752 97 AAM81751 97 AAM81750 97 AAM81749 97



AAK67141 7 AAK67140 7 AAK67139 7 AAK67138 7 AAK67137 7



AAK67136 7 AAK67135 7 AAK67134 7 AAK67133 7 AAK67132 7



AAK67131 7 AAK67130 7 AAK67129 7 AAK67128 7 AAK67127 7



AAK67126 7 AAK67125 7 AAK67124 3 AAK67123 7 AAK67122 7



AAK67121 7 AAK67120 7 AAK67119 7 AAK67118 7 AAK67117 7



AAK67116 7 AAK67115 7 AAK67114 7 AAK67113 7 AAK67112 7



AAK67111 7 AAK67110 7 AAK67109 7 AAK67108 7 AAK67107 7



AAK67106 7 AAK67105 7 AAK67104 7 AAK67103 7 AAK67102 7



AAK67101 7 AAK67100 7 AAK67099 7 AAK67098 7 AAK67097 7



AAK67096 7 AAK67095 7 AAK67094 7 AAK67093 7 AAK67092 7



AAK67091 7 AAK67090 7 AAK67089 7 AAK67088 7 AAK67087 7



AAK67086 7 AAK67085 7 AAK67084 7 AAK67083 7 AAK67082 7



AAK67081 7 AAK67080 7 AAK67079 7 AAK67078 7 AAK67077 7



AAK67076 7 AAK67075 7 AAK67074 7 AAK67073 7 AAK67072 7



AAK67071 7 AAK67070 5 AAK67069 7 AAK67068 7 AAK67067 7



AAK67066 7 AAK67065 7 AAK67064 7 AAL87748 19 AAL87747 18



AAL87746 19 AAL87745 18 AAL37596 18 AAM21944 24 AAM21941 32


2002
AAM09856 6 AAM09855 6 AAM09854 6 AAO26579 30 AAO26578 30
18
4.7
1.5
low p < .001, prev



AAN77484 3 AAN85090 97 AAO73303 36 AAO73302 36 AAO73301 36



p < .005



AAO73300 36 AAO73299 36 AAO73298 36 AAO73297 36 AAO73296 36



AAO73295 36 AAL87234 96 CAD60131 96


2003
AAP20887 96 AAR10793 6 AAR10784 6 AAR17575 32 AAR17574 32
94
5.3
1.5
low p < .001, prev p < .05



AAR17573 32 AAR17572 32 AAR17571 32 AAR17570 32 AAR17569 32



AAR17568 32 AAR17567 32 AAR17566 32 AAR17565 32 AAR17564 32



AAR17563 32 AAR17562 32 AAR17561 32 AAR17560 32 AAR17559 32



AAR17558 32 AAR17557 32 AAR17556 32 AAR17555 32 AAR17554 32



AAR17553 32 AAR17552 32 AAR17551 32 AAR17550 32 AAR17549 32



AAR17548 32 AAR17547 32 AAR17546 32 AAR17545 32 AAR17544 32



AAR17543 32 AAR17542 32 AAQ87608 16 AAQ87607 16 AAQ87606 14



AAR10804 6 AAR10803 6 AAR10802 6 AAR10801 6 AAR10800 6



AAR10799 6 AAR10798 6 AAR10797 6 AAR10796 6 AAR10795 6



AAR10794 6 AAR10792 6 AAR10791 6 AAR10790 6 AAR10789 6



AAR10788 6 AAR10787 6 AAR10786 6 AAR10785 6 AAR10783 6



AAR10782 6 AAR10781 6 AAR10780 6 AAQ88403 10 AAQ88402 10



AAX99361 97 AAR84198 36 AAQ55854 97 AAR14153 36 AAR84614 95



AAR06948 36 AAR06947 36 AAR06946 36 AAR06945 36 AAR06944 36



AAR06943 36 AAR06942 36 AAR06941 36 AAR06940 36 AAR06939 36



AAR06938 36 AAR06937 36 AAR06936 35 AAR06935 36 AAR06934 36



AAR06933 36 AAR06932 36 AAR06931 36 AAQ00999 100 AAQ00998 97



AAP22087 97 AAP22086 97 AAP22089 97 AAP22088 96


2004
AAT11553 32 AAT11552 32 AAT11551 32 AAT11550 32 AAT11549 32
55
4.2
1.7
low p < .001,



AAT11548 32 AAT11547 32 AAT11546 32 AAT11545 32 AAT11544 32



prev p < .001



AAT11543 32 AAT11542 32 AAT11541 32 AAT11540 32 AAT11539 32



AAT11538 32 AAT11537 32 AAT11536 32 AAT11535 32 AAT11534 28



AAS75296 6 AAS75295 6 AAS75294 6 AAS75293 6 AAS75292 6 AAS75291



6 AAT95390 108 AAU00153 96 AAV54504 97 AAT02759 111 ABG67747 99



ABG67746 99 BAD34491 97 BAD34490 97 BAD34489 97 BAD34488 97



ABV82765 97 AAZ91684 106 AAW56064 97 AAW56066 97 AAW56065 97



AAW28871 97 AAV49728 6 AAV49727 6 AAV49726 6 AAV49725 6



AAV49724 6 AAT92099 97 AAT92098 97 AAV52690 96 AAV52689 97



AAV52688 97 AAV52687 97 AAV68177 97 AAX09982 97


2005
YP_001527880 32 ABC18309 8 ABC18308 9 ABC02196 3 AAY67877 9
125
4.3
1.8
low p < .001, prev p > .50



AAY67876 11 AAY67875 11 AAY67874 8 AAY67873 8 AAY67872 8



AAY67871 8 AAY67870 8 AAY67869 8 AAY67868 8 AAY67867 8



AAY67866 8 AAY57985 8 ABB01532 97 ABC40712 100 YP_001527877 97



ABB01533 101 ABA62343 97 AAY32590 36 AAY32589 36 YP_001527879



4 AAY55949 97 AAY29684 6 AAY29685 6 AAY29683 6 AAY29682 6



AAY29681 6 AAY29680 6 AAY29679 6 AAY29678 6 AAY29677 7



AAY29676 7 AAZ32750 97 AAZ32749 97 AAZ32748 94 AAZ32747 94



AAZ32746 94 AAZ32745 94 AAZ32744 94 AAZ32743 94 AAZ32742 94



AAZ32741 95 AAZ32740 96 AAZ32739 97 AAZ32738 97 AAZ32737 97



AAZ32736 97 AAZ32735 97 AAZ32734 96 AAZ32733 96 AAZ32732 97



AAZ32731 97 AAZ32730 97 AAZ32729 97 ABC49716 111 ABA43046 36



ABA43045 36 ABA43044 36 ABA43043 36 ABA43042 36 ABA43041 36



ABA43040 36 ABA43039 36 ABA43038 36 ABA43037 36 ABA43036 36



ABA43035 36 ABA43034 36 ABA43033 37 ABA43032 37 ABA43031 36



ABA43030 37 ABA43029 37 ABA43028 36 ABA43027 36 ABA43026 36



ABA43025 36 ABA43024 36 ABA43023 36 ABA43022 36 ABA43021 36



ABA43020 36 ABA43019 36 ABA43018 36 ABA43017 36 ABA43016 36



ABA43015 36 ABA43014 36 ABA43013 36 ABA43012 36 ABA43011 36



ABA43010 36 ABA43009 34 ABA43008 36 ABA43007 36 ABA43006 36



ABA43005 36 ABA43004 36 ABA43003 36 ABA54595 97 ABA54594 97



ABA54593 97 ABA54592 97 ABA54591 97 ABA54590 97 ABA54589 97



ABA54588 97 ABA54587 97 ABA54586 97 ABA54585 98 ABA54584 97



ABA54583 105 ABA54582 97 ABA54581 93 ABA54580 97 ABA54579 97



ABA54578 97 ABA54577 97 ABA54576 97 ABA54575 97 AAY54162 97


2006
ABI81406 32 ABI81405 32 ABI81404 32 ABI81403 32 ABI81402 32
312
6.0
1.3
low p < .001,



ABI81401 32 ABI81400 32 ABI81399 32 ABI81398 32 ABI81397 32



prev p < .001



ABI81396 32 ABI81395 32 ABI81394 32 ABI81393 32 ABI81392 32



ABI81391 32 ABI81390 32 ABI81389 32 ABI81388 32 ABI81387 32



ABI81386 32 ABI81385 32 ABI81384 32 ABI81383 32 ABI81382 32



ABI81381 32 ABI81380 32 ABI81379 32 ABI81378 32 ABI81377 32



ABI81376 32 ABI81375 32 ABI81374 32 ABI81373 32 ABI81372 32



ABI81371 32 ABI81370 32 ABI81369 32 ABI81368 32 ABI81367 32



ABI81366 32 ABI81365 32 ABI81364 32 ABI81363 32 ABI81362 32



ABI81361 32 ABI81360 32 ABI81359 32 ABI81358 32 ABI81357 32



ABI81356 32 ABI81355 32 ABI81354 32 ABI81353 32 ABI81351 32



ABI81350 32 ABI81349 32 ABI81348 32 ABI81347 32 ABI81346 32



ABI81345 32 ABI81344 32 ABI81343 32 ABI81342 32 ABI81341 32



ABI81340 32 ABI81339 32 ABI81338 32 ABI81337 32 ABI81336 32



ABI81335 32 ABI81334 32 ABI81333 32 ABI81332 32 ABI81331 32



ABI81330 32 ABI81329 32 ABI81328 32 ABI81327 32 ABI81326 32



ABI81325 32 ABI81324 32 ABI81323 32 ABI81322 32 ABI81321 34



ABI81320 32 ABI81319 32 ABI81318 32 ABI81317 32 ABI81316 32



ABI81315 32 ABI81314 32 ABI81313 32 ABI81312 32 ABI81311 32



ABI81310 32 ABI81309 32 ABI81308 32 ABI81307 32 ABI81306 32



ABI81305 32 ABI81304 32 ABI81303 32 ABI81302 32 ABI81301 32



ABI81300 32 ABI81299 32 ABI81298 32 ABI81297 32 ABI81296 32



ABI81295 32 ABI81294 32 ABI81293 32 ABI81292 32 ABI81291 32



ABI81290 32 ABI81289 32 ABI81288 32 ABI81287 32 ABI81286 32



ABI81285 32 ABI81284 32 ABI81283 32 ABI81282 32 ABI81281 32



ABI81280 32 ABI81279 32 ABI81278 32 ABI81277 32 ABI81276 32



ABI81275 32 ABI81274 32 ABI81273 32 ABI81272 32 ABI81271 32



ABI81270 32 ABI81269 32 ABI81268 32 ABI81267 32 ABI81266 32



ABI81265 32 ABI81264 32 ABI81263 32 ABI81262 32 ABI81261 32



ABI81260 32 ABI81259 32 ABI81258 32 ABI81257 32 ABI81256 32



ABI81255 32 ABI81254 32 ABI81253 32 ABI81252 32 ABI81251 32



ABI81250 32 ABI81249 32 ABI81248 32 ABI81247 32 ABI81246 32



ABI81245 32 ABI81244 32 ABI81243 32 ABI81242 32 ABI81241 32



ABI81240 32 ABI81239 32 ABI81238 32 ABI81237 32 ABI81236 32



ABI81235 32 ABI81234 32 ABI81233 32 ABI81232 32 ABI81231 32



ABI81230 32 ABI81229 32 ABI81228 32 ABJ90133 32 ABJ90132 32



ABJ90131 32 ABJ90130 32 ABJ90129 32 ABJ90128 32 ABJ90127 32



ABJ90126 32 ABJ90125 32 ABJ90124 32 ABJ90123 32 ABJ90122 32



ABJ90121 32 ABJ90120 32 ABJ90119 32 ABJ90118 32 ABJ90117 32



ABJ90116 32 ABJ90115 32 ABJ90114 32 ABJ90113 32 ABJ90112 32



ABJ90111 32 ABJ90110 32 ABJ90109 32 ABJ90108 32 ABJ90107 32



ABJ90106 32 ABJ90105 32 ABJ90104 32 ABJ90103 32 ABJ90102 32



ABJ90101 32 ABJ90100 32 ABJ90099 32 ABJ90098 32 ABJ90097 32



ABJ90096 32 ABJ90095 32 ABJ90094 32 ABJ90093 32 ABJ90092 32



ABJ90091 32 ABJ90090 32 ABJ90089 32 ABJ90088 32 ABJ90087 32



ABJ90086 32 ABJ90085 32 ABJ90084 32 ABJ90083 32 ABJ90082 32



ABJ90081 32 ABJ90080 32 ABJ90079 32 ABJ90078 32 ABJ90077 32



ABJ90076 32 ABJ90075 32 ABJ90074 32 ABJ90073 32 ABJ90072 32



ABJ90071 32 ABJ90070 32 ABJ90069 32 ABJ90068 32 ABJ90067 32



ABJ90066 32 CAL49454 98 ABI97486 99 ABG36517 36 ABG81344 92



ABG81343 97 ABG81342 97 ABG81341 97 ABG81340 99 ABG76816 41



ABG76815 43 ABG76814 43 ABG76813 43 ABG76812 43 ABG76811 43



ABG76810 43 ABG76809 43 ABG76808 43 ABG76807 43 ABG76806 43



ABG76805 43 ABG76804 43 ABG76803 43 ABG76802 43 ABG76801 43



ABG76800 43 ABG76799 43 ABG76798 43 ABG76797 43 ABG76796 43



ABG76795 43 ABI26622 40 ABI26621 40 ABD19642 97 ABD19641 97



ABD19640 97 ABD19513 97 ABD19512 96 ABD19511 97 ABD19510 97



ABD85083 98 ABD85082 93 ABD85081 97 ABD85080 97 ABD85078 97



ABD85077 97 ABD85076 97 ABD85075 97 ABD85074 99 ABD85073 97



ABD85072 99 ABD85070 97 ABD85069 96 ABD85068 97 ABD85067 97



ABD85066 95 ABD85065 97 ABD85064 97 ABD67762 97 ABD67761 97



ABD67760 97 ABD67759 97 ABD67758 97 ABD67757 97


2007
ABR19639 111 ABV22897 97 ABU54838 97 ABU52997 98 ABQ52692 97
(Incom-
(Incom-
(Incomplete)
(Incomplete) low



ABO69610 36 ABO69609 36 ABO69608 36 ABO69607 36 ABO69606 36
plete)
plete)
1.2
p < .001, prev p < .001



ABO69605 36 ABO69604 36 ABO69603 36 ABO69602 36 ABO69601 36
27
4.6



ABO69600 36 ABO69599 36 ABO69598 36 ABO69597 36 ABO69596 36



ABO69595 36 ABO69594 36 ABO69593 36 ABO69592 36 ABU41789 114



CAM91200 97 ABR10608 56


2008
ABZ10682 21 ABZ10681 29 ABZ10680 29 ABZ10679 29 ABZ10678 29
(Incom-
(Incom-
(Incomplete)
(Incomplete) low




plete)
plete)
0.7
p < .002, prev p < .04




5
5.5









Examples of Conserved WNV Replikins
Sequence History by Year: Note that entries for years before WNV appeared in the U.S., approx. 2000, are from non-U.S. specimens, as from the Middle East and Africa.








KIIQKAHK (SEQ ID NO: 199)



All occurrences of the sequence by year:


1988


P14335 position 835, BAA00176 position 835.


 


1999


AAF20205 position 835, AAF20092 position 835,


 


AAG02040 position 835, AAF18443 position 835.


 


2000


AAK06624 position 835, AAG02039 position 835,


 


AAG02038 position 835.


 


2001


AAM81753 position 835, AAM81752 position 835,


 


AAM81751 position 835, AAM81750 position 835,


 


AAM81749 position 835.


 


2002


AAN85090 position 835, AAL87234 position 835,


 


CAD60131, position 835.


 


2003


AAP20882 position 835, AAX99361 position 835,


 


AAQ55854 position 835, AAR84614 position 835,


 


AAQ00999 position 835, XAP22087 position 835,


 


AAP22086 position 835, AAP22089 position 835.


 


2004


AAU00153 position 835, AAV54504 position 835,


 


ABG67747 position 835, ABG67746 position 835,


 


BAD34491 position 835, BAD34490 position 835,


 


BAD34489 position 835, BAD34488 position 835,


 


ABV82765 position 835, AAW56064 position 835,


 


AAW56066 position 835, AAW56065 position 835,


 


AAW28871 position 835, AA197099 position 835,


 


AAT92098 position 835, AAV52690 position 835,


 


AAV52689 position 835, AAV52688 position 835,


 


AAV52687 position 835, AAV68177 position 835,


 


AAX09982 position 835.


 


2005


YP_001527877 position 835, ABB01533 position 835,


 


ABA62343 position 835, AAY55949 position 835,


 


AAZ32750 position 835, AAZ32749 position 835,


 


AAZ32748 position 835, AAZ32747 position 835,


 


AAZ32746 position 835, AAZ32745 position 835,


 


AAZ32744 position 835, AAZ32743 position 835,


 


AAZ32747 position 835, AAZ32741 position 835,


 


AAZ32740 position 835, AAZ32739 position 835,


 


AAZ32738 position 835, AAZ32737 position 835,


 


AAZ32736 position 835, AAZ32735 position 835,


 


AAZ32734 position 835, AAZ37733 position 835,


 


AAZ32732 position 835, AAZ32731 position 835,


 


AAZ32730 position 835, AAZ32729 position 835,


 


ABA54595 position 835, ABA54594 position 835,


 


ABA54593 position 835, ABA54592 position 835,


 


ABA54591 position 835, ABA54590 position 835,


 


ABA54589 position 835, ABAA5488 position 835,


 


ABA54587 position 835, ABA54586 position 835,


 


ABA54585 position 835, ABA54584 position 835,


 


ABA54583 position 835, ABA54582 position 835,


 


ABA54581 position 835, ABA54580 position 835,


 


ABA54579 position 835, ABA54578 position 835,


 


ABA54577 position 835, ABA54576 position 835,


 


ABA54575 position 835, AAY54162 position 835.


 


2006


CAL49454 position 835, ABI97486 position 835,


 


ABG81344 position 835, ABG81343 position 835,


 


ABG81342 position 835, ABG81341 position 835,


 


ABG81340 position 835, ABG76816 position 835,


 


ABG76815 position 835, ABG76814 position 835,


 


ABG76813 position 835, ABG76812 position 835,


 


ABG76811 position 835, ABG76810 position 835,


 


ABG76809 position 835, ABG76808 position 835,


 


ABG76807 position 835, ABG76806 position 835,


 


ABG76805 position 835, ABG76804 position 835,


 


ABG76803 position 835, ABG76802 position 835,


 


ABG76801 position 835, ABG76800 position 835,


 


ABG76799 position 835, ABG76798 position 835,


 


ABG76797 position 835, ABG76796 position 835,


 


ABG76795 position 835, ABD19642 position 835,


 


ABD19641 position 835, ABD19640 position 835,


 


ABD19513 position 835, ABD19512 position 835,


 


ABD19511 position 835, ABD19510 position 835,


 


ABD85083 position 835, ABD85082 position 835,


 


ABD85081 position 835, ABD85080 position 835,


 


ABD85078 position 835, ABD85077 position 835,


 


ABD85076 position 835, ABD85075 position 835,


 


ABD85074 position 835, ABD85073 position 835,


 


ABD85072 position 835, ABD85070 position 835,


 


ABD85069 position 835, ABD85068 position 835,


 


ABD85067 position 835, ABD85066 position 835,


 


ABD85065 position 835, ABD85064 position 835,


 


ABD67762 position 835, ABD67761 position 835,


 


ABD67760 position 835, ABD67759 position 835,


 


ABD67758 position 835, ABD67757 position 835,


 


ABD67756 position 835.


 


2007


ABV22897 position 835, ABU54838 position 835,


 


ABU52997 position 835, ABQ52692 position 835,


 


CAM91200 position 835, ABR10608 position 377.


 


Sequence History by Year


HLKCRVKMEK (SEQ ID NO: 200)


All occurences of the sequence by year:


1982


ABC497717 position 575.


 


1985


P06935 position 571, AAA48498 position 571.


 


1988


P14335 position 575, BAA00176 position 575.


 


1989


ABR19636 position 575.


 


1993


NP_776014 position 281, NP_041724 position 571.


 


1998


AAD28624 position 550.


 


1999


AAD31720 position 285, AAF20205 position 575,


 


AAF26360 position 575, AAD28623 position 550,


 


AAF20092 position 575, AAG02040 position 575,


 


AAF18443 position 575.


 


2000


ABR19638 position 575, AAK06624 position 575,


 


AAG02039 position 575, AAG02038 position 575.


 


2001


AAM70028 position 285, AAL14222 position 285,


 


AAL14221 position 285, AAL14220 position 285,


 


AAL14219 position 285, AAL14218 position 285,


 


AAL14217 position 285, AAL14216 position 285,


 


AAL14215 position 285, AAK58104 position 285,


 


AAK58103 position 285, AAK58102 position 285,


 


AAK58101 position 285, AAK58100 position 285,


 


AAK58099 position 285, AAK58098 position 285,


 


AAK58097 position 285, AAK58096 position 285,


 


AAK52303 position 285, AAK52302 position 285,


 


AAK52301 position 285, AAK52300 position 285,


 


AAK62766 position 285, AAK62765 position 285,


 


AAK62764 position 285, AAK62763 position 285,


 


AAK62762 position 285, AAK62761 position 285,


 


AAK67760 position 285, AAK62759 position 285,


 


AAK62758 position 285, AAK62757 position 285,


 


AAK62756 position 285, AAK91592 position 176,


 


ABR19637 position 575, AAM81753 position 575,


 


AAM81752 position 575, AAM81751 position 575,


 


AAM81750 position 575, AAM81749 position 575,


 


AAM21944 position 198, AAM21941 position 264.


 


2002


AAO26579 position 285, AA026578 position 285,


 


AAN85090 position 575, AAO73303 position 452,


 


AAO73302 position 285, AA073301 position 452,


 


AAO73300 position 452, AAO73299 position 452,


 


AAO73298 position 285, AA073297 position 452,


 


AAO73296 position 452, AAO73295 position 452,


 


AAL87234 position 285, CAD60131 position 575.


 


2003


AAP20887 position 575, AAR10793 position 128,


 


AAR10784 position 128, AAR17575 position 285,


 


AAR17574 position 285, AAR17573 position 285,


 


AAR17572 position 285, AAR17571 position 285,


 


AAR17570 position 285, AAR17569 position 285,


 


AAR17568 position 285, AAR17567 position 285,


 


AAR17566 position 285, AAR17565 position 285,


 


AAR17564 position 285, AAR17563 position 285,


 


AAR17562 position 285, AAR17561 position 285,


 


AAR17560 position 285, AAR17559 position 285,


 


AAR17558 position 285, AAR17557 position 285,


 


AAR17556 position 285, AAR17545 position 285,


 


AAR17554 position 285, AAR17553 position 285,


 


AAR17552 position 285, AAR17555 position 285,


 


AAR17550 position 285, AAR17549 position 285,


 


AAR17548 position 285, AAR17547 position 285,


 


AAR17546 position 285, AAR17545 position 285,


 


AAR17544 position 285, AAR17543 position 285,


 


AAR17542 position 285, AAO87608 position 194,


 


AAO87607 position 194, AAO87606 position 58,


 


AAR10804 position 128, AAR10803 position 128,


 


AAR10802 position 128, AAR10801 position 128,


 


AAR10800 position 128, AAR10799 position 128,


 


AAR10798 position 128, AAR10797 position 128,


 


AAR10796 position 128, AAR10795 position 128,


 


AAR10794 position 128, AAR10792 position 128,


 


AAR10791 position 128, AAR10790 position 128,


 


AAR10789 position 128, AAR10788 position 128,


 


AAR10787 position 128, AAR10786 position 128,


 


AAR10785 position 128, AAR10783 position 128,


 


AAR10782 position 128, AAR10781 position 128,


 


AAR10780 position 128, AAX99361 position 575,


 


AAR84198 position 452, AAO55854 position 575 


 


AAR14153 position 452, AAR84614 position 575,


 


AAR06948 position 452, AAR06947 position 452,


 


AAR06946 position 452, AAR06945 position 452,


 


AAR06944 position 452, AAR06943 position 452,


 


AAR06942 position 452, AAR06941 position 452,


 


AAR06940 position 452, AAR06939 position 452,


 


AAR06938 position 452, AAR06937 position 452,


 


AAR06936 position 452, AAR06935 position 452,


 


AAR06934 position 452, AAR06933 position 452,


 


AAR06932 position 452, AAR06931 position 452,


 


AAO00999 position 575, AAO00998 position 575,


 


AAP22087 position 575, AAP22086 position 575,


 


AAP22089 position 575, AAP22088 position 572,


 


2004


AAT11553 position 285, AAT11552 position 285,


 


AAT11551 position 285, AAT11550 position 285,


 


AAT11549 position 285, AAT11548 position 285,


 


AAT11547 position 285, AAT11546 position 285,


 


AAT11545 position 285, AAT11544 position 285,


 


AAT11543 position 285, AAT11542 position 285,


 


AAT11541 position 285, AAT11540 position 285,


 


AAT11539 position 285, AAT11538 position 285,


 


AAT11537 position 285, AAT11536 position 285,


 


AAT11535 position 285, AAT11534 position 285,


 


AAS75296 position 128, AAS75295 position 128,


 


AAS75294 position 128, AAS75293 position 128,


 


AAS75292 position 128, AAS75291 position 128,


 


AAT95390 position 575, AAU00153 position 575,


 


AAV54504 position 575, AAT02759 position 571,


 


ABG67747 position 575, ABG67746 position 575,


 


BAD34491 position 575, BAD34490 position 575,


 


BAD34489 position 575, BAD34488 position 575,


 


ABV82765 position 575, AAZ91684 position 575,


 


AAW56064 position 575, AAW56066 position 575,


 


AAW56065 position 575, AAW28871 position 575,


 


AAT92099 position 575, AAT92098 position 575,


 


AAV52690 position 575, AAV52689 position 575,


 


AAV52688 position 575, AAV52687 position 575,


 


AAV68177 position 575, AAX09982 position 575.


 


2005


YP_001527880 position 285, ABC40712 position 575,


 


YP_001527877 position 575, ABB01533 position 575,


 


ABA62343 position 575, AAY32590 position 452,


 


AAY32589 position 452, AAY55949 position 575,


 


AAZ32750 position 575, AAZ32749 position 575,


 


AAZ32740 position 575, AAZ32739 position 575,


 


AAZ32738 position 575, AAZ32737 position 575,


 


AAZ32736 position 575, AAZ32735 position 575,


 


AAZ32734 position 575, AAZ32733 position 575,


 


AAZ32732 position 575, AAZ32731 position 575,


 


AAZ32730 position 575, AAZ32729 position 575,


 


ABC49716 position 571, ABA43046 position 452,


 


ABA43045 position 452, ABA43044 position 452,


 


ABA43043 position 452, ABA43042 position 452,


 


ABA43041 position 452, ABA43040 position 452,


 


ABA43039 position 452, ABA43038 position 452,


 


ABA43037 position 452, ABA43036 position 452,


 


ABA43035 position 452, ABA43034 position 452,


 


ABA43033 position 452, ABA43032 position 452,


 


ABA43031 position 452, ABA43030 position 452,


 


ABA43029 position 452, ABA43028 position 452,


 


ABA43027 position 452, ABA43026 position 452,


 


ABA43025 position 452, ABA43024 position 452,


 


ABA43023 position 452, ABA43022 position 452,


 


ABA43021 position 452, ABA43020 position 452,


 


ABA43019 position 452, ABA43018 position 452,


 


ABA43017 position 452, ABA43016 position 452,


 


ABA43015 position 452, ABA43014 position 452,


 


ABA43013 position 452, ABA43012 position 452,


 


ABA43011 position 452, ABA43010 position 452,


 


ABA43009 position 452, ABA43008 position 452,


 


ABA43007 position 452, ABA43006 position 452,


 


ABA43005 position 452, ABA43004 position 452,


 


ABA43003 position 452, ABA54595 position 575,


 


ABA54594 position 575, ABA54593 position 575,


 


ABA54592 position 575, ABA54591 position 575,


 


ABA54590 position 575, ABA54589 position 575,


 


ABA54588 position 575, ABA54587 position 575,


 


ABA54586 position 575, ABA54585 position 575,


 


ABA54584 position 575, ABA54583 position 575,


 


ABA54582 position 575, ABA54581 position 575,


 


ABA54580 position 575, ABA54579 position 575,


 


ABA54578 position 575, ABA54577 position 575,


 


ABA54576 position 575, ABA54575 position 575,


 


AAY54162 position 575.


 


2006


ABI81406 position 275, ABI81405 position 275,


 


ABI81404 position 275, ABI81403 position 275,


 


ABI81402 position 275, ABI81401 position 275,


 


ABI81400 position 275, ABI81399 position 275,


 


ABI81398 position 275, ABI81397 position 275,


 


ABI81396 position 275, ABI81395 position 275,


 


ABI81394 position 275, ABI81393 position 275,


 


ABI81392 position 275, ABI81391 position 275,


 


ABI81390 position 275, ABI81389 position 275,


 


ABI81388 position 275, ABI81387 position 275,


 


ABI81386 position 275, ABI81385 position 275,


 


ABI81384 position 275, ABI81383 position 275,


 


ABI81382 position 275, ABI81381 position 275,


 


ABI81380 position 275, ABI81379 position 275,


 


ABI81378 position 275, ABI81377 position 275,


 


ABI81376 position 275, ABI81375 position 275,


 


ABI81374 position 275, ABI81373 position 275,


 


ABI81372 position 275, ABI81371 position 275,


 


ABI81370 position 275, ABI81369 position 275,


 


ABI81368 position 275, ABI81367 position 275,


 


ABI81366 position 275, ABI81365 position 275,


 


ABI81364 position 275, ABI81363 position 275,


 


ABI81362 position 275, ABI81361 position 275,


 


ABI81360 position 275, ABI81359 position 275,


 


ABI81358 position 275, ABI81357 position 275,


 


ABI81356 position 275, ABI81355 position 275,


 


ABI81354 position 275, ABI81353 position 275,


 


ABI81351 position 275, ABI81350 position 275,


 


ABI81349 position 275, ABI81348 position 275,


 


ABI81347 position 275, ABI81346 position 275,


 


ABI81345 position 275, ABI81344 position 275,


 


ABI81343 position 275, ABI81342 position 275,


 


ABI81341 position 275, ABI81340 position 275,


 


ABI81339 position 275, ABI81338 position 275,


 


ABI81337 position 275, ABI81336 position 275,


 


ABI81335 position 275, ABI81334 position 275,


 


ABI81333 position 275, ABI81332 position 275,


 


ABI81331 position 275, ABI81330 position 275,


 


ABI81329 position 275, ABI81328 position 275,


 


ABI81327 position 275, ABI81326 position 275,


 


ABI81325 position 275, ABI81324 position 275,


 


ABI81323 position 275, ABI81322 position 275,


 


ABI81321 position 275, ABI81320 position 275,


 


ABI81319 position 275, ABI81318 position 275,


 


ABI81317 position 275, ABI81316 position 275,


 


ABI81315 position 275, ABI81314 position 275,


 


ABI81313 position 275, ABI81312 position 275,


 


ABI81311 position 275, ABI81310 position 275,


 


ABI81309 position 275, ABI81308 position 275,


 


ABI81307 position 275, ABI81306 position 275,


 


ABI81305 position 275, ABI81304 position 275,


 


ABI81303 position 275, ABI81302 position 275,


 


ABI81301 position 275, ABI81300 position 275,


 


ABI81299 position 275, ABI81298 position 275,


 


ABI81297 position 275, ABI81296 position 275,


 


ABI81295 position 275, ABI81294 position 275,


 


ABI81293 position 275, ABI81292 position 275,


 


ABI81291 position 275, ABI81290 position 275,


 


ABI81289 position 275, ABI81288 position 275,


 


ABI81287 position 275, ABI81286 position 275,


 


ABI81285 position 275, ABI81284 position 275,


 


ABI81283 position 275, ABI81282 position 275,


 


ABI81281 position 275, ABI81280 position 275,


 


ABI81279 position 275, ABI81278 position 275,


 


ABI81277 position 275, ABI81276 position 275,


 


ABI81275 position 275, ABI81274 position 275,


 


ABI81273 position 275, ABI81272 position 275,


 


ABI81271 position 275, ABI81270 position 275,


 


ABI81269 position 275, ABI81268 position 275,


 


ABI81267 position 275, ABI81266 position 275,


 


ABI81265 position 275, ABI81264 position 275,


 


ABI81263 position 275, ABI81262 position 275,


 


ABI81261 position 275, ABI81260 position 275,


 


ABI81259 position 275, ABI81258 position 275,


 


ABI81257 position 275, ABI81256 position 275,


 


ABI81255 position 275, ABI81254 position 275,


 


ABI81253 position 275, ABI81252 position 275,


 


ABI81251 position 275, ABI81250 position 275,


 


ABI81249 position 275, ABI81248 position 275,


 


ABI81247 position 275, ABI81246 position 275,


 


ABI81245 position 275, ABI81244 position 275,


 


ABI81243 position 275, ABI81242 position 275,


 


ABI81241 position 275, ABI81240 position 275,


 


ABI81239 position 275, ABI81238 position 275,


 


ABI81237 position 275, ABI81236 position 275,


 


ABI81235 position 275, ABI81234 position 275,


 


ABI81233 position 275, ABI81232 position 275,


 


ABI81231 position 275, ABI81230 position 275,


 


ABI81229 position 275, ABI81228 position 275,


 


ABI81133 position 275, ABI81901 position 275,


 


ABJ90131 position 309, ABJ90130 position 309,


 


ABJ90129 position 309, ABJ90128 position 309,


 


ABJ90127 position 309, ABJ90126 position 309,


 


ABJ90125 position 309, ABJ90124 position 309,


 


ABJ90123 position 309, ABJ90122 position 309,


 


ABJ90121 position 309, ABJ90120 position 309,


 


ABJ90119 position 309, ABJ90l18 position 309,


 


ABJ90117 position 309, ABJ90116 position 309,


 


ABJ90115 position 309, ABJ90114 position 309,


 


ABJ90113 position 309, ABJ90112 position 309,


 


ABJ90111 position 309, ABJ90110 position 309,


 


ABJ90109 position 309, ABJ90108 position 309,


 


ABJ90107 position 309, ABJ90106 position 309,


 


ABJ90105 position 309, ABJ90104 position 309,


 


ABJ90103 position 309, ABJ90102 position 309,


 


ABJ90101 position 309, ABJ90100 position 309,


 


ABJ90099 position 309, ABJ90098 position 309,


 


ABJ90097 position 309, ABJ90096 position 309,


 


ABJ90095 position 309, ABJ90094 position 309,


 


ABJ90093 position 309, ABJ90092 position 309,


 


ABJ90091 position 309, ABJ90090 position 309,


 


ABJ90088 position 309, ABJ90087 position 309,


 


ABJ90086 position 309, ABJ90085 position 309,


 


ABJ90084 position 309, ABJ90083 position 309,


 


ABJ90082 position 309, ABJ90081 position 309,


 


ABJ90080 position 309, ABJ90079 position 309,


 


ABJ90078 position 309, ABJ90077 position 309,


 


ABJ90076 position 309, ABJ90075 position 309,


 


ABJ90074 position 309, ABJ90073 position 309,


 


ABJ90072 position 309, ABJ90071 position 309,


 


ABJ90070 position 309, ABJ90069 position 309,


 


ABJ90068 position 309, ABJ90067 position 309,


 


ABJ90066 position 309, CAL49454 position 575,


 


ABI97486 position 575, ABG36517 position 452,


 


ABG81344 position 575, ABG81343 position 575,


 


ABG81342 position 575, ABG81341 position 575,


 


ABG81340 position 575, ABG76816 position 575,


 


ABG76815 position 575, ABG76814 position 575,


 


ABG76813 position 575, ABG76812 position 575,


 


ABG76811 position 575, ABG76810 position 575,


 


ABG76809 position 575, ABG76808 position 575,


 


ABG76807 position 575, ABG76806 position 575,


 


ABG76805 position 575, ABG76804 position 575,


 


ABG76803 position 575, ABG76802 position 575,


 


ABG76801 position 575, ABG76800 position 575,


 


ABG76799 position 575, ABG76798 position 575,


 


ABG76797 position 575, ABG76796 position 575,


 


ABG76795 position 575, ABI26622 position 575,


 


ABI26621 position 575, ABD19642 position 575,


 


ABD19641 position 575, ABD19640 position 575,


 


ABD19513 position 575, ABD19512 position 575,


 


ABD19511 position 575, ABD19510 position 575,


 


ABD85083 position 575, ABD85082 position 575,


 


ABD85081 position 575, ABD85080 position 575,


 


ABD85078 position 575, ABD85077 position 575,


 


ABD85076 position 575, ABD85075 position 575,


 


ABD85074 position 575, ABD85073 position 575,


 


ABD85072 position 575, ABD85070 position 575,


 


ABD85069 position 575, ABD85068 position 575,


 


ABD85067 position 575, ABD85066 position 575,


 


ABD85065 position 575, ABD85064 position 575,


 


ABD67762 position 575, ABD67761 position 575,


 


ABD67760 position 575, ABD67759 position 575,


 


ABD67758 position 575, ABD67757 position 575,


 


ABD67756 position 575, CAM90885 position 285,


 


CAM90884 position 285.


 


2007


ABR19639 position 575, ABV22897 position 575,


 


ABU54838 position 575, ABU52997 position 575,


 


ABO52692 position 575, ABO69610 position 452,


 


ABO69609 position 452, ABO69608 position 452,


 


ABU69607 position 452, ABO69606 position 452,


 


ABO69605 position 452, ABO69604 position 452,


 


ABO69603 position 452, ABO69602 position 452,


 


ABO69601 position 452, ABO69600 position 452,


 


ABO69599 position 452, ABO69598 position 452,


 


ABO69597 position 452, ABO69596 position 452,


 


ABO69595 position 452, ABO69594 position 452,


 


ABO69593 position 452, ABO69592 position 452,


 


ABU41789 position 575, CAMP1200 position 575.


 


2008


ABZ10682 position 285, A13Z10681 position 285,


 


ABZ10680 position 285, ABZ10679 position 285,


 


ABZ10678 position 285.


 


Sequence History by Year


KLTSGHLK (SEQ ID NO: 201)


All occurences of the sequence by year:


1982


ABC49717 position 570.


 


1985


P06935 position 566, AAA48498 position 566.


 


1988


P14335 position 570, BAA00176 position 570.


 


1989


ABR19636 position 570.


 


1993


NP_776014 position 276,NP_041724 position 566.


 


1998


AAD28624 position 545, AAW81711 position 570.


 


1999


AAD21720 position 280, AAF20205 position 570,


 


AAF26360 position 570, AAD28623 position 545,


 


AAF20092 position 570, AAG02040 position 570,


 


AAF18443 position 570.


 


2000


ABR19638 position 570, AAK06624 position 570,


 


AA602039 position 570, AAG02038 position 570.


 


2001


AAM70028 position 280, AAL14222 position 280,


 


AAL14221 position 280, AAL14220 position 280,


 


AAL14219 position 280, AAL14218 position 280,


 


AAL14217 position 280, AAL14216 position 280,


 


AAL14215 position 280, AAK58104 position 280,


 


AAK58103 position 280, AAK58102 position 280,


 


AAK58101 position 280, AAK58100 position 280,


 


AAK58099 position 280, AAK58098 position 280,


 


AAK58097 position 280, AAK58096 position 280,


 


AAK52303 position 280, AAK52302 position 280,


 


AAK52301 position 280, AAK52300 position 280,


 


AAK62766 position 280, AAK62765 position 280,


 


AAK62764 position 280, AAK62763 position 280,


 


AAK62766 position 280, AAK62761 position 280,


 


AAK62760 position 280, AAK62759 position 280,


 


AAK62758 position 280, AAK62757 position 280,


 


AAK62756 position 280, AAK91592 position 171,


 


ABR19637 position 570, AAM81753 position 570,


 


AAM81752 position 570, AAM81751 position 570,


 


AAM81750 position 570, AAM81749 position 570,


 


AAM21944 position 193, AAM21941 position 259.


 


2002


AAO26579 position 280, AAO26578 position 280,


 


AAN77484 position 129, AAN85090 position 570,


 


AAO73303 position 447, AAO73302 position 447,


 


AAO73301 position 447, AAO73300 position 447,


 


AAO73299 position 447, XAO73298 position 447,


 


AAO73297 position 447, AAO73296 position 447,


 


AAO73295 position 447, AAL87234 position 570,


 


CAD60131 position 570.


 


2003


AAP20887 position 570, AAR10793 position 123,


 


AAR10784 position 123, AAR17575 position 280,


 


AAR17574 position 280, AAR17573 position 280,


 


AAR17572 position 280, AAR17571 position 280,


 


AAR17570 position 280, AAR17569 position 280,


 


AAR17568 position 280, AARI7567 position 280,


 


AAR17566 position 280, AAR17565 position 280,


 


AAR17564 position 280, AAR17563 position 280,


 


AAR17562 position 280, AAR17561 position 280,


 


AAR17560 position 280, AAR17559 position 280,


 


AAR17558 position 280, AAR17557 position 280,


 


AAR17556 position 280, AAR17555 position 280,


 


AAR17554 position 280, AAR17553 position 280,


 


AAR17552 position 280, AAR17551 position 280,


 


AAR17550 position 280, AAR17549 position 280,


 


AAR17548 position 280, AAR17547 position 280,


 


AAR17546 position 280, AAR17545 position 280,


 


AAR17544 position 280, AAR17543 position 280,


 


AAR17542 position 280, AAO87608 position 189,


 


AAQ87607 position 189, AAO87606 position 53,


 


AAR10804 position 123, AAR10803 position 123,


 


AAR10802 position 123, AAR10801 position 123


 


AAR10800 position 123, AAR10799 position 123,


 


AAR10798 position 123, AAR10797 position 123


 


AAR10796 position 123, AAR10795 position 123,


 


AAR10794 position 123, AAR10792 position 123


 


AAR10791 position 123, AAR10790 position 123,


 


AAR10789 position 123, AAR10788 position 123


 


AAR10787 position 123, AAR10786 position 123,


 


AAR10785 position 123, AAR10783 position 123


 


AAR10782 position 123, AAR10781 position 123,


 


AAR10780 position 123, AAX99361 position 570,


 


AAR84198 position 447, AAO55854 position 570,


 


AAR14153 position 447, AAR84614 position 570,


 


AAR06948 position 447, AAR06947 position 447,


 


AAR06946 position 447, AAR06945 position 447,


 


AAR06944 position 447, AAR06943 position 447,


 


AAR06942 position 447, AAR06941 position 447,


 


AAR06940 position 447, AAR06939 position 447,


 


AAR06938 position 447, AAR06937 position 447,


 


AAR06936 position 447, AAR06935 position 447,


 


AAR06934 position 447, AAR06933 position 447,


 


AAR06932 position 447, AAR06931 position 447,


 


AAO00999 position 570, AAO00998 position 570,


 


AAP22087 position 570, AAP22086 position 570,


 


AAP22089 position 570.


 


2004


AAT11593 position 280, AAT11552 position 280,


 


AAT11551 position 280, AAT11550 position 280,


 


AAT11549 position 280, AAT11548 position 280,


 


AAT11547 position 280, AAT11546 position 280,


 


AAT11545 position 280, AAT11544 position 280,


 


AAT11543 position 280, AAT11542 position 280,


 


AAT11541 position 280, AAT11540 position 280,


 


AAT11539 position 280, AAT11538 position 280,


 


AAT11537 position 280, AAT11536 position 280,


 


AAT115S5 position 280, AAT11534 position 280,


 


AAS75296 position 123, AAS75295 position 123,


 


AAS75294 position 123, AAS75293 position 123,


 


AAS75292 position 123, AAS75291 position 123,


 


AAT95390 position 570, AAU00153 position 570,


 


AAV54504 position 570, AAT02759 position 566,


 


ABG67747 position 570, ABG67746 position 570,


 


BAD34491 position 570, BAD34490 position 570,


 


BAD34489 position 570, BAD34488 position 570,


 


ABV82765 position 570, AAZ91684 position 570,


 


AAW56064 position 570, AAW56066 position 570,


 


AAW56065 position 570, AAW28871 position 570,


 


AXT92099 position 570, AAT92098 position 570,


 


AAV52690 position 570, AAV52689 position 570,


 


AAV52688 position 570, AAV52687 position 570,


 


AAV68177 position 570, AAX09982 position 570.


 


2005


YP_001527880 position 280, ABC40712 position 570,


 


YP_001527877 position 570, ABB01533 position 570,


 


ABA62343 position 570, AAY32590 position 447,


 


AAY32589 position 447, AAY55949 position 570,


 


AAZ32750 position 570, AAZ32749 position 570,


 


AAZ32748 position 570, AAZ32747 position 570,


 


AAZ32746 position 570, AAZ32745 position 570,


 


AAZ32744 position 570, AAZ32743 position 570,


 


AAZ32742 position 570, AAZ32741 position 570,


 


AAZ32740 position 570, AAZ32739 position 570,


 


AAZ32738 position 570, AAZ32737 position 570,


 


AAZ32736 position 570, AAZ32735 position 570,


 


AAZ32734 position 570, AAZ32733 position 570,


 


AAZ32732 position 570, AAZ32731 position 570,


 


AAZ32730 position 570, AAZ32729 position 570,


 


ABC49716 position 566, ABA43046 position 570,


 


ABA43045 position 447, ABA43044 position 447,


 


ABA43043 position 447, ABA43042 position 570,


 


ABA43041 position 447, ABA43040 position 447,


 


ABA43039 position 447, ABA43038 position 570,


 


ABA43037 position 447, ABA43036 position 447,


 


ABA43035 position 447, ABA43034 position 570,


 


ABA43033 position 447, ABA43032 position 447,


 


ABA43031 position 447, ABA43030 position 570,


 


ABA43029 position 447, ABA43028 position 447,


 


ABA43027 position 447, ABA43026 position 570,


 


ABA43025 position 447, ABA43024 position 447,


 


ABA43023 position 447, ABA43022 position 570,


 


ABA43021 position 447, ABA43020 position 447,


 


ABA43019 position 447, ABA43018 position 570,


 


ABA43017 position 447, ABA43016 position 447,


 


ABA43015 position 447, ABA43014 position 570,


 


ABA43013 position 447, ABA43012 position 447,


 


ABA43011 position 447, ABA43010 position 570,


 


ABA43009 position 447, ABA43008 position 447,


 


ABA43007 position 447, ABA43006 position 570,


 


ABA43005 position 447, ABA43004 position 447,


 


ABA43003 position 447, ABA54595 position 570,


 


ABA54594 position 570, ABA54593 position 570,


 


ABA54592 position 570, ABA54591 position 570,


 


ABA54590 position 570, ABA54589 position 570,


 


ABA54588 position 570. ABA54587 position 570,


 


ABA54586 position 570, ABA54585 position 570,


 


ABA54584 position 570, ABA54583 position 570,


 


ABA54582 position 570, ABA54581 position 570,


 


ABA54580 position 570, ABA54579 position 570,


 


ABA54578 position 570, ABA54547 position 570,


 


ABA54576 position 570, ABA54575 position 570,


 


AAY54162 position 570.


 


2006


ABI81406 position 270, ABI81405 position 270,


 


ABI81404 position 270, ABI81403 position 270,


 


ABI81402 position 270, ABI81401 position 270,


 


ABI81400 position 270, ABI81399 position 270,


 


ABI81398 position 270, ABI81397 position 270,


 


ABI81396 position 270, ABI81395 position 270,


 


ABI81394 position 270, ABI81393 position 270,


 


ABI81392 position 270, ABI81391 position 270,


 


ABI81390 position 270, ABI81389 position 270,


 


ABI81388 position 270, ABI81387 position 270,


 


ABI81386 position 270, ABI81385 position 270,


 


ABI81384 position 270, ABI81383 position 270,


 


ABI81382 position 270, ABI81381 position 270,


 


ABI81380 position 270, ABI81379 position 270,


 


ABI81378 position 270, ABI81377 position 270,


 


ABI81376 position 270, ABI81375 position 270,


 


ABI81374 position 270, ABI81373 position 270,


 


ABI81372 position 270, ABI81371 position 270,


 


ABI81370 position 270, ABI81369 position 270,


 


ABI81368 position 270, ABI81367 position 270,


 


ABI81366 position 270, ABI81365 position 270,


 


ABI81364 position 270, ABI81363 position 270,


 


ABI81362 position 270, ABI81361 position 270,


 


ABI81360 position 270, ABI81359 position 270,


 


ABI81358 position 270, ABI81357 position 270,


 


ABI81356 position 270, ABI81355 position 270,


 


ABI81354 position 270, ABI81353 position 270,


 


ABI81351 position 270, ABI81350 position 270,


 


ABI81349 position 270, ABI81348 position 270,


 


ABI81347 position 270, ABI81346 position 270,


 


ABI81345 position 270, ABI81344 position 270,


 


ABI81343 position 270, ABI81342 position 270,


 


ABI81341 position 270, ABI81340 position 270,


 


ABI81339 position 270, ABI81338 position 270,


 


ABI81337 position 270, ABI81336 position 270,


 


ABI81335 position 270, ABI81334 position 270,


 


ABI81333 position 270, ABI81332 position 270,


 


ABI81331 position 270, ABI81330 position 270,


 


ABI81329 position 270, ABI81328 position 270,


 


ABI81327 position 270, ABI81326 position 270,


 


ABI81325 position 270, ABI81324 position 270,


 


ABI81323 position 270, ABI81322 position 270,


 


ABI81321 position 270, ABI81320 position 270,


 


ABI81319 position 270, ABI81318 position 270,


 


ABI81317 position 270, ABI81316 position 270,


 


ABI81315 position 270, ABI81314 position 270,


 


ABI81313 position 270, ABI81312 position 270,


 


ABI81311 position 270, ABI81310 position 270,


 


ABI81309 position 270, ABI81308 position 270,


 


ABI81307 position 270, ABI81306 position 270,


 


ABI81305 position 270, ABI81304 position 270,


 


ABI81303 position 270, ABI81302 position 270,


 


ABI81301 position 270, ABI81300 position 270,


 


ABI81299 position 270, ABI81298 position 270,


 


ABI81297 position 270, ABI81296 position 270,


 


ABI81295 position 270, ABI81294 position 270,


 


ABI81293 position 270, ABI81292 position 270,


 


ABI81291 position 270, ABI81290 position 270,


 


ABI81289 position 270, ABI81288 position 270,


 


ABI81287 position 270, ABI81286 position 270,


 


ABI81285 position 270, ABI81284 position 270,


 


ABI81283 position 270, ABI81282 position 270,


 


ABI81281 position 270, ABI81280 position 270,


 


ABI81279 position 270, ABI81278 position 270,


 


ABI81277 position 270, ABI81276 position 270,


 


ABI81275 position 270, ABI81274 position 270,


 


ABI81273 position 270, ABI81272 position 270,


 


ABI81271 position 270, ABI81270 position 270,


 


ABI81269 position 270, ABI81268 position 270,


 


ABI81267 position 270, ABI81266 position 270,


 


ABI81265 position 270, ABI81264 position 270,


 


ABI81263 position 270, ABI81262 position 270,


 


ABI81261 position 270, ABI81260 position 270,


 


ABI81259 position 270, ABI81258 position 270,


 


ABI81257 position 270, ABI81256 position 270,


 


ABI81255 position 270, ABI81254 position 270,


 


ABI81253 position 270, ABI81252 position 270,


 


ABI81251 position 270, ABI81250 position 270,


 


ABI81249 position 270, ABI81248 position 270,


 


ABI81247 position 270, ABI81246 position 270,


 


ABI81245 position 270, ABI81244 position 270,


 


ABI81243 position 270, ABI81242 position 270,


 


ABI81241 position 270, ABI81240 position 270,


 


ABI81239 position 270, ABI81238 position 270,


 


ABI81237 position 270, ABI81236 position 270,


 


ABI81235 position 270, ABI81234 position 270,


 


ABI81233 position 270, ABI81232 position 270,


 


ABI81231 position 270, ABI81230 position 270,


 


ABI81229 position 270, ABI81228 position 270,


 


ABJ90133 position 304, ABJ90132 position 304,


 


ABJ90131 position 304, ABJ90130 position 304,


 


ABJ90129 position 304, ABJ90128 position 304,


 


ABJ90127 position 304, ABJ90126 position 304,


 


ABJ90125 position 304, ABJ90124 position 304,


 


ABJ90123 position 304, ABJ90122 position 304,


 


ABJ90121 position 304, ABJ90120 position 304,


 


ABJ90119 position 304, ABJ90118 position 304,


 


ABJ90117 position 304, ABJ90116 position 304,


 


ABJ90115 position 304, ABJ90114 position 304,


 


ABJ90113 position 304, ABJ90112 position 304,


 


ABJ90111 position 304, ABJ90110 position 304,


 


ABJ90109 position 304, ABJ90108 position 304,


 


ABJ90107 position 304, ABJ90106 position 304,


 


ABJ90105 position 304, ABJ90104 position 304,


 


ABJ90103 position 304, ABJ90102 position 304,


 


ABJ90101 position 304, ABJ90100 position 304,


 


ABJ90099 position 304, ABJ90098 position 304,


 


ABJ90097 position 304, ABJ90096 position 304,


 


ABJ90095 position 304, ABJ90094 position 304,


 


ABJ90093 position 304, ABJ90092 position 304,


 


ABJ90091 position 304, ABJ90090 position 304,


 


ABJ90089 position 304, ABJ90088 position 304,


 


ABJ90087 position 304, ABJ90086 position 304,


 


ABJ90085 position 304, ABJ90084 position 304,


 


ABJ90083 position 304, ABJ90082 position 304,


 


ABJ90081 position 304, ABJ90080 position 304,


 


ABJ90079 position 304, ABJ90078 position 304,


 


ABJ90077 position 304, ABJ90076 position 304,


 


ABJ90075 position 304, ABJ90074 position 304,


 


ABJ90073 position 304, ABJ90072 position 304,


 


ABJ90071 position 304, ABJ90070 position 304,


 


ABJ90069 position 304, ABJ90068 position 304,


 


ABJ90067 position 304, ABJ90066 position 304,


 


CAL49454 position 570, ABI97486 position 570,


 


ABG36517 position 447, ABG81344 position 570,


 


ABG81343 position 570, ABG81342 position 570,


 


ABG81341 position 570, ABG81340 position 570,


 


ABG76816 position 570, ABG76815 position 570,


 


ABG76814 position 570, ABG76813 position 570,


 


ABG76812 position 570, ABG76811 position 570,


 


ABG76810 position 570, ABG76809 position 570,


 


ABG76808 position 570, ABG76807 position 570,


 


ABG76806 position 570, ABG76805 position 570,


 


ABG76804 position 570, ABG76803 position 570,


 


ABG76802 position 570, ABG76801 position 570,


 


ABG76800 position 570, ABG76799 position 570,


 


ABG76798 position 570, ABG76797 position 570,


 


ABG76796 position 570, ABG76795 position 570,


 


ABI26622 position 570, ABI26621 position 570,


 


ABD19642 position 570, ABD19641 position 570,


 


ABD19640 position 570, ABD19513 position 570,


 


ABD19512 position 570, ABD19511 position 570,


 


ABD85010 position 570, ABD85083 position 570,


 


ABD85082 position 570, ABD85081 position 570,


 


ABD85080 position 570, ABD85078 position 570,


 


ABD85077 position 570, ABD85076 position 570,


 


ABD85075 position 570, ABD85074 position 570,


 


ABD85073 position 570, ABD85072 position 570,


 


ABD85070 position 570, ABD85069 position 570,


 


ABD85068 position 570, ABD85067 position 570,


 


ABD85066 position 570, ABD85065 position 570,


 


ABD67764 position 570, ABD67762 position 570,


 


ABD67761 position 570, ABD67760 position 570,


 


ABD67759 position 570, ABD67758 position 570,


 


ABD67757 position 570, ABD67756 position 570,


 


CAM90885 position 280, CAM90884 position 280.


 


2007


ABR19639 position 570, ABV22897 position 570,


 


ABU54838 position 570, ABU52997 position 570,


 


ABQ52692 position 570, ABO69610 position 447,


 


ABO69609 position 447, ABO69608 position 447,


 


ABO69607 position 447, ABO69606 position 447,


 


ABO69605 position 447, ABO69604 position 447,


 


ABO69603 position 447, ABO69602 position 447,


 


ABO69601 position 447, ABO69600 position 447,


 


ABO69599 position 447, ABO69598 position 447,


 


ABO69597 position 447, ABO69596 position 447,


 


ABO69595 position 447, ABO69594 position 447,


 


ABO69593 position 447, ABO69592 position 447,


 


ABU41789 position 570, CAM91200 position 570.


 


2008


ABZ10682 position 280, ABZl0681 position 280,


 


ABZ10680 position 280, ABZ10679 position 280,


 


AB710678 position 280.


 


A “more recent arrival’- a WNV replikin which


appeared in 1998


Sequence Histony by Year


HNDKRADPAFVCK (SEQ ID NO: 202)


All occurrences of the sequence by year:


1998


AAD28624 position 346.


 


2000


AAG2039 position 371.


 


2001


AAL87748 position 346, AAL87746 position 346.


 


2003


AAR84614 position 371.






The sequence listing, saved as file named 47504-seqlisting.txt, created on Apr. 23, 2008, and totaling 87000 bytes, is hereby incorporated by reference in its entirety.

## Claims

1. A method of increasing survival in shrimp exposed to taura syndrome virus comprising administering to at least one shrimp a substance comprising a mixture of isolated or synthetic peptides, wherein each peptide of the mixture consists of 7 to 50 amino acids and wherein the mixture includes peptides comprising at least each of the following sequences of KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), and HVEKIVDKAK (SEQ ID NO: 11).

2. The method of claim 1, wherein said isolated or synthetic peptides are synthetic.

3. The method of claim 1, wherein said substance comprises a mixture consisting of the isolated or synthetic peptides KVGSRRYKSH (SEQ ID NO: 1), HFATKCFGEVPKK (SEQ ID NO: 2), KAENEFWDGVKQSH (SEQ ID NO: 3), KGHRKVPCEQK (SEQ ID NO: 4), HRKVPCEQK (SEQ ID NO: 5), KVPCEQKIWLH (SEQ ID NO: 6), KIWLHQNPGK (SEQ ID NO: 7), HQNPGKTQQDMK (SEQ ID NO: 8), KGNTRVHVK (SEQ ID NO: 9), KEHVEKIVDK (SEQ ID NO: 10), and HVEKIVDKAK (SEQ ID NO: 11).

4. The method of claim 1, wherein said isolated or synthetic peptides consist of up to 15 amino acids.

5. The method of claim 1, wherein said substance further comprises a pharmaceutically acceptable carrier.

6. The method of claim 1, wherein said substance comprises feed.

7. The method of claim 6, wherein said feed comprises a daily ration of feed for shrimp.

8. The method of claim 3, wherein said substance comprises an equal mixture by weight of each of said isolated or synthetic peptides.

9. The method of claim 3, wherein said substance is administered to shrimp at a dose of about 0.01 mg to about 2 mg per gram of body weight of each shrimp per day.

10. The method of claim 9, wherein said dose is about 0.2 mg to 1.0 mg per gram of body weight of each shrimp per day.

11. The method of claim 10, wherein said dose is about 0.5 mg per gram of body weight of each shrimp per day.

12. The method of claim 1, wherein said substance is administered to shrimp as a prophylactic therapy prior to the onset of symptoms of a taura syndrome virus.

13. The method of claim 1, wherein said substance is administered to shrimp as a therapy after the onset of symptoms of taura syndrome virus.

14. The method of claim 1, wherein said substance is administered to shrimp as a prophylactic therapy at sub-therapeutic concentrations.

15. The method of claim 14, wherein said substance is administered over substantially the entire life cycle of at least one shrimp.

16. The method of claim 1, wherein said substance is administered orally, via submersion of the invertebrate in an aqueous medium containing said substance, or via injection.

17. The method of claim 1, wherein said substance is administered orally.