Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOUNDS FOR MITIGATING
PATHOGENIC OUTBREAKS USING REPLIKIN COUNT CYCLES
[0001] This application claims priority to U.S. Provisional Appln. Ser. No.
61/143,618, filed
January 9, 2009, U.S. Provisional Appln. Ser. No. 61/087,354, filed August 8,
2008, U.S.
Provisional Appln. Ser. No. 61/054,010, filed May 16, 2008, U.S. Appln. Ser.
No. 12/108,458,
filed April 23, 2008, and PCT/US2008/61336, filed April 23, 2008, each of
which is
incorporated herein by reference in its entirety. This application
additionally incorporates herein
by reference: U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008, U.S.
Provisional Appln.
Ser. No. 60/991,676, filed November 30, 2007, U.S. Appln. Ser. No. 11/923,559,
filed October
24, 2007, U.S. Provisional Appln. Ser. No. 60/982,336, filed October 24, 2007,
U.S. Provisional
Appln. Ser. No. 60/982,333, filed October 24, 2007, U.S. Provisional Appln.
Ser. No.
60/982,338, filed October 24, 2007, U.S. Provisional Appln. Ser. No.
60/935,816, filed August
31, 2007, U.S. Provisional Appln. Ser. No. 60/935,499 filed August 16, 2007,
U.S. Provisional
Appln. Ser. No. 60/954,743, filed August 8, 2007, U.S. Appln. Ser. No.
11/755,597, filed May
30, 2007, U.S. Provisional Appln. Ser. No. 60/898,097, filed January 30, 2007,
U.S. Provisional
Appln. Ser. No. 60/880,966, filed January 18, 2007, U.S. Provisional Appln.
Ser. No.
60/853,744, filed October 24, 2006, U.S. Appln. Ser. No. 11/355,120, filed
February 16, 2006,
U.S. Appln. Ser. No. 11/116,203, filed April 28, 2005, U.S. Appln. Ser. No.
10/860,050, filed
June 4, 2004, now U.S. Patent No. 7,442,761, U.S. Appln. Ser. No. 10/189,437,
filed July 8,
2002, now U.S. Patent No. 7,452,963, U.S. Appln. Ser. No. 10/105,232, filed
March 26, 2002,
now U.S. Patent No. 7,189,800, U.S. Appln. Ser. No. 09/984,057, filed October
26, 2001, now
U.S. Patent No. 7,420,028, and U.S. Appln. Ser. No. 09/984,056, filed October
26, 2001, now
U. S. Patent No. 7,176,275, each in its entirety.
BACKGROUND OF THE INVENTION
[0002] In surveys of global health, infectious disease often accounts for as
many as five of
the top ten causes of death in lower- and middle-income countries and
respiratory infections are
often assigned as the fourth leading cause of death in higher-income
countries. Further,
pathogenic outbreaks and pandemics continue to threaten human populations from
previously
unknown or otherwise mutated pathogenic diseases. Previously unknown or
otherwise mutated
pathogenic diseases often occur when a pathogen diverges from an established
host, such as pigs
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or chickens, into a new host, such as humans. In view of this phenomenon, new
strategies are
continually needed for mitigating pathogenic outbreaks from previously-known
or previously-
unknown pathogens. Several such threatening pathogenic diseases include
malaria, influenza,
West Nile virus, foot-and-mouth disease, and other threats to global health in
both humans and
animals. Therapies or methods of treatment that are useful across different
pathogenic strains or
even across pathogenic groups are especially helpful in improving the fight
against mutable
pathogenic disease and outbreaks of previously-unknown pathogens.
[0003] Among the most threatening of global infectious diseases is malaria.
Malaria kills a
million or more people each year in tropical and sub-tropical environments.
Malaria is most
commonly and seriously caused by the trypanosome Plasmodiumfalciparum, which
is
reportedly responsible for ninety percent of malarial deaths. The majority of
death from malarial
infection is recorded in young children. P. falciparum is vectored by female
Anopheles
mosquitoes. Once in the blood stream of a human, the trypanosome multiplies
rapidly within red
blood cells causing anemia, flu-like symptoms, and sometimes coma and death.
Partly effective
vaccines are only now beginning to be marketed for malaria and no wholly-
effective vaccine has
yet been registered for sale in industrialized countries. As such, there
continues to be a need in
the art for improved methods of predicting and identifying increases in
virulence, morbidity, and
mortality in and from malaria.
[0004] Another threat to public health is West Nile virus (WNV), which causes
encephalitis
and other serious neuroinvasive diseases in a small percentage of human
infections. In about
four percent of reported cases, the resulting neuroinvasive disease results in
death. WNV is
flaviviridae virus, first observed in North America in 1999 and now considered
endemic in the
United States. The virus is spread to humans through mosquito (and related
insect) bites. WNV
is a single-stranded sense RNA virus and is a member of the Japanese
encephalitis virus
antigenic complex, which includes several medically important viruses
associated with human
encephalitis: Japanese encephalitis, St. Louis encephalitis, Murray Valley
encephalitis, and
Kunjin encephalitis, an Australian subtype of WNV.
[0005] Since introduction of the disease to the United States in 1999, there
have been more
than 16,000 reported cases of WNV in humans and more than 650 reported deaths.
In addition,
more than 21,000 cases have been reported in horses. Currently, the only
available approved
strategies to combat WNV in humans are nationwide active surveillance in
conjunction with
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mosquito control efforts and individual protection with insect repellents.
There is a need in the
art, therefore, for methods of predicting increases in virulence of WNV prior
to epidemics and
for therapies for preventing, mitigating, and treating WNV infections.
[0006] Influenza is an acute respiratory illness of global importance.
Virulent and lethal
outbreaks of influenza continue to threaten world health. Researchers,
government officials, and
medical practitioners are increasingly aware of the continuing threat of a
pandemic of virulent
and lethal influenza requiring new methods of treatment and novel therapeutic
compounds.
Researchers, government officials, and medical practitioners likewise
recognize that the
continuing threat of pandemic influenza requires new and more effective
methods of predicting
and tracking lethal outbreaks of influenza.
[0007] Influenza vaccines remain the most effective defense against influenza
virus, but
because of the ability of the virus to mutate, and the availability of non-
human host reservoirs, it
is expected that influenza will remain an emergent or re-emergent infectious
threat. Global
influenza surveillance indicates that influenza viruses may vary within a
country and between
countries and continents during an influenza season. Virologic surveillance is
of importance in
monitoring antigenic shift and drift. Disease surveillance is also important
in assessing the
impact of epidemics. Both types of information have provided the basis of
vaccine composition
and use of antivirals. However, traditionally there has been only annual post
hoc hematological
classification of the increasing number of emerging influenza virus strains,
and no specific
chemical structure of the viruses was identified as an indicator of
approaching influenza
epidemic or pandemic. Until the discovery of Replikin chemistry in the virus
genome structure,
the only basis for annual classification of influenza virus as present or
absent in a given year was
identification by serological testing of the hemagglutinin and neuraminidase
proteins in an
isolate of virus. The activity of a strain of influenza was, therefore, only
recorded after the fact
of the occurrence of the outbreak, never in advance.
[0008] There is a continuing need in the art for quantitative methods of
tracking and
predicting increases in virulence and lethality of influenza prior to
outbreaks. There is likewise a
need in the art for quantitative methods of preventing and treating outbreaks
caused by virulent
strains of influenza. Because of the annual administration of influenza
vaccines and the short
period of time when a vaccine can be administered, strategies directed at
improving vaccine
coverage are of critical importance.
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[0009] Replikin peptides are a family of small peptides that have been
correlated with the
phenomenon of rapid replication in malaria, influenza, West Nile virus, foot
and mouth disease,
and many other pathogens. Replikin peptides have likewise been correlated with
the
phenomenon of rapid replication in viruses and organisms generally.
[00010] Identification of Replikin peptides has provided targets for detection
and treatment of
pathogens, including vaccine development against virulent pathogens such as
malaria, influenza
virus, West Nile virus, and foot and mouth disease virus. In general,
knowledge of and
identification of this family of peptides enables development of effective
therapies and vaccines
for any pathogen that harbors Replikins. The phenomenon of the association of
Replikins with
rapid replication and virulence has been fully described in U.S. Patent No.
7,189,800, U.S. Patent
No. 7,176,275, U.S. Patent No. 7,442,761, and U.S. Appln. Ser. No. 11/355,120.
Both Replikin
concentration (number of Replikins per 100 amino acids) and Replikin
composition have been
correlated with the functional phenomenon of rapid replication.
[00011] There continues to be a need in the art, however, for improved methods
of predicting
and identifying increases in virulence, morbidity, and lethality and
expansions of and outbreaks
of virulent pathogens. There is likewise a need in the art for improved
methods of preventing
and treating outbreaks and expansions of virulent pathogens using Replikin
sequences identified
with increases in virulence, morbidity, and lethality of expanding pathogenic
populations.
SUMMARY OF THE INVENTION
[00012] The present invention provides a quantitative cyclic structure
comprising Replikin
peptide concentrations identified in a strain of microorganism through time,
wherein said cyclic
structure correlates in time with the expansion and/or contraction of a
population of said strain of
microorganism, the infectivity of said strain of microorganism, and/or the
lethality of said strain
of microorganism.
[00013] Further, the present invention provides methods of preventing,
mitigating, and
treating outbreaks of a pathogen comprising predicting an expansion of a
population of a strain
of pathogen or an increase in the virulence, morbidity, and/or lethality of a
strain of pathogen as
compared to another strain of the same or a related pathogen and administering
to an animal or
patient a compound comprising an isolated or synthesized portion of the
structure or genome of
the pathogen to mitigate, prevent, or treat the predicted outbreak of the
pathogen.
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[00014] The present invention further provides methods of predicting an
expansion of a strain
of pathogen or an increase in the virulence, morbidity, and/or mortality of a
pathogen comprising
identifying a cycle in the Replikin Count in a protein fragment, protein,
genome fragment, or
genome of a pathogen and predicting an increase in the virulence, morbidity,
and/or mortality of
said pathogen within the identified cycle in Replikin Count. The present
invention further
provides Replikin peptides identified within a pathogen predicted to be
expanding or to have an
increase in virulence, morbidity, and/or mortality as diagnostic, therapeutic,
or preventive agents
against an outbreak of the pathogen.
[00015] A first non-limiting aspect of the invention provides a method of
preventing,
mitigating, or treating an outbreak of a pathogen predicted to have an
expansion of population
comprising predicting an expansion of the population of a first pathogen
comprising
identifying at least one cycle of Replikin concentration in isolates of the
pathogen
and predicting that an expansion of the population of the first pathogen will
take place after the
occurrence of a rising portion of the at least one cycle of Replikin
concentration, and
administering to an animal or patient a compound comprising an isolated or
synthesized portion of the structure or genome of the pathogen to mitigate,
prevent, or treat the
predicted outbreak of the pathogen.
[00016] A further embodiment of the first aspect of the invention provides a
method of
preventing, mitigating, or treating an outbreak of pathogen comprising
predicting an expansion of the population or an increase in virulence,
morbidity,
and/or mortality of an isolate or plurality of isolates of a first strain of
pathogen as compared to
another isolate or plurality of isolates of the same or a related strain of
pathogen comprising: (1)
identifying a first cycle in the Replikin concentration of a plurality of
isolates of said first strain
of pathogen, (2) identifying a first peak in the Replikin concentration within
the identified first
cycle at a first time point or time period, and (3) predicting an increase in
the virulence of an
isolate of the same or related strain of pathogen isolated at a second time
point or time period
subsequent to the first time point or time period; and
administering to an animal or a patient a compound comprising an isolated or
synthesized portion of the structure or genome of the at least one isolate of
the pathogen to
prevent, mitigate, or treat the outbreak of the pathogen.
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[00017] In a non-limiting embodiment of the first aspect of the invention, the
pathogen is an
influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth
disease virus, taura
syndrome virus, white spot syndrome virus, porcine reproductive and
respiratory syndrome
virus, porcine circovirus, Helicobacter pylori, Entamoeba invadens, L.
legionella, S. aureus,
maize streak virus, bovine herpes virus, feline immunodeficiency virus, human
immunodeficiency virus, rous sarcoma virus, avian sarcoma virus, sindbis
virus, hepatitis virus,
b. anthracis, or any other infectious agent. In a non-limiting embodiment, the
influenza virus is
an H1N1, H2N2, H3N2, H5N1, H3N8, or H9N2 strain of influenza virus.
[00018] In a further non-limiting embodiment of the first aspect of the
invention, said
expansion of a strain of pathogen or increase in virulence, morbidity, and/or
mortality of an
isolate or plurality of isolates of a strain of pathogen comprises identifying
a second cycle in the
Replikin concentration of a plurality of isolates of a second strain of
pathogen that shares
synchrony with said first cycle in the Replikin concentration of said
plurality of isolates of said
first strain of pathogen and identifying a first peak in the Replikin
concentration within the
identified first cycle at a first time point or time period and identifying a
first peak in the
Replikin concentration within the identified second cycle of said second
strain of pathogen at a
second time point or time period that is similar to said first time point or
time period and
predicting an increase in the virulence of said first strain of pathogen
following the first time
point or time period. In a non-limiting embodiment, the pathogen is an
influenza virus, a
malarial trypanosome, a West Nile virus, a foot and mouth disease virus, or
any other infectious
agent.
[00019] In a non-limiting embodiment of the first aspect of the invention,
said pathogen is an
influenza virus. In a further non-limiting embodiment, said first strain of
influenza is any strain
different from said second strain of influenza. In another non-limiting
embodiment, said first
strain of influenza is H5N1 and said second strain of influenza is H9N2, or
vice versa.
[00020] In a further non-limiting embodiment of the first aspect of the
invention, said isolated
or synthesized portion of the structure or genome of the at least one isolate
of a pathogen is a
protein or protein fragment comprising a Replikin peptide and/or a Replikin
Peak Gene, a
Replikin peptide identified within a Replikin Peak Gene, or any structure or
portion of the
structure of said pathogen. In another embodiment, said isolated or
synthesized portion of the
structure or genome is a nucleic acid encoding a Replikin Peak Gene, a
Replikin peptide or a
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plurality of Replikin peptides within a Replikin Peak Gene, or a Replikin
peptide or plurality of
Replikin peptides.
[00021] In another non-limiting embodiment of the first aspect of the present
invention, the
second time point or time period is up to three years after the first time
point or time period. In a
further non-limiting embodiment, the second time point or time period is about
one year after the
first time point or time period. In a further non-limiting embodiment, the
second time point or
time period is about six months after the first time point or time period. In
a further non-limiting
embodiment, the second time point or time period is the next season of a
pathogen following the
first time point or time period. In a further non-limiting embodiment, the
second time point or
time period is the next season of influenza following the first time point or
first time period. In a
further non-limiting embodiment, the next influenza season is the next winter
season in a
geographic region following the first time point or time period. In another
non-limiting
embodiment, the second time point or time period is the next season of malaria
following the
first time point or first time period. In a further non-limiting embodiment,
the next season of
malaria is the next rainy season. In another non-limiting embodiment, the
second time point or
time period is the next season of West Nile virus. In a further non-limiting
embodiment, the next
season of West Nile virus is a summer season.
[00022] In another non-limiting embodiment of the first aspect of the present
invention, the
identified peak in the cycle of Replikin concentration has a higher Replikin
concentration than a
chronologically earlier peak in the cycle of Replikin concentration. In a
further non-limiting
embodiment of the invention, the identified peak in the cycle of Replikin
concentration is
significantly higher than the earlier peak. In a further non-limiting
embodiment the identified
peak is significantly higher than the earlier peak with a p value less than
0.01. In a further non-
limiting embodiment the identified peak is significantly higher than the
earlier peak with a p
value less than 0.001.
[00023] A second non-limiting aspect of the invention provides a method of
predicting an
expansion of the population of a first pathogen comprising identifying at
least one cycle of
Replikin concentration in isolates of the pathogen and predicting that an
expansion of the
population of the first pathogen will take place after the occurrence of a
rising portion of the at
least one cycle of Replikin concentration, wherein the at least one cycle is
cycle A.
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[00024] In a further embodiment of the second aspect of the invention, the
rising portion
comprises a peak wherein said expansion of the population of the first
pathogen is predicted after
the occurrence of the peak. In a further embodiment, the cycle comprises at
least a first rising
portion and a second rising portion, wherein said first rising portion occurs
prior in time to said
second rising portion. In a further embodiment, the cycle comprises at least
three rising portions,
wherein the at least three rising portions are at least rising portion A',
rising portion B' and rising
portion U. In a further embodiment, the rising portion B' comprises a peak and
the rising
portion A' comprises a peak, and the peak of rising portion B' has a greater
Replikin
concentration than the peak of rising portion A'. In a further non-limiting
embodiment, the
method of prediction further comprises processing the method on a computer. In
a further non-
limiting embodiment, the cycle comprises more than one cycle including, for
example, from
peak to trough to peak to trough or from trough to peak to trough to peak. In
a further non-
limiting embodiment, the cycle comprises three peaks or three troughs or more.
[00025] In a further embodiment of the second aspect of the present invention,
the method of
prediction comprises identifying at least one other cycle of Replikin
concentration in isolates of
at least one other strain of pathogen, wherein the at least one other cycle is
cycle B, and wherein
cycle B shares synchrony with cycle A; and predicting that an expansion of the
population of the
first pathogen will occur after the occurrence of a rising portion in cycle A
that corresponds to a
rising portion in cycle B. In a further embodiment, the first pathogen is a
first strain of influenza
virus and the one other pathogen is a different strain of influenza virus. In
a further embodiment,
the first pathogen is an H5N1 strain of influenza virus and the one other
strain of pathogen is an
H9N2 strain of influenza virus. In a further embodiment, the expansion of the
population of the
first pathogen is predicted within three years after the peak. In a further
embodiment, the
expansion of the population of the first pathogen is predicted within one year
after said peak. In
a further embodiment, the expansion of the population of the first pathogen is
predicted after the
next virulence season of the pathogen.
[00026] A further embodiment of the second aspect of the invention provides a
method of
predicting an expansion of a population of a pathogen or an increase in the
virulence, morbidity,
and/or mortality of a pathogen relative to the population or the virulence,
morbidity, and/or
mortality of another pathogen of the same species or of another pathogen of a
related species
comprising: (1) identifying a cycle in the Replikin concentration of isolates
of a plurality of the
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pathogen, (2) identifying a first peak in the Replikin concentration of
isolates of a plurality of
said pathogen within the identified cycle at a first time point or time
period, and (3) predicting an
expansion of the population of a pathogen of the same or a related species or
an increase in the
virulence, morbidity, and/or mortality of a pathogen of the same or a related
species isolated at a
second time point or time period subsequent to the first time point or time
period.
[00027] In a non-limiting embodiment of the second aspect of the invention,
the pathogen
may be, but is not limited to, a malarial trypanosome, West Nile virus,
influenza virus, equine
influenza virus, coronavirus, foot and mouth disease virus, taura syndrome
virus, white spot
syndrome virus, or other pathogen or infectious agent.
[00028] A non-limiting embodiment of the second aspect of the present
invention, the
pathogen is a malarial trypanosome. In another non-limiting embodiment, the
trypanosome is P.
falciparum, P. vivax, P. ovale, or P. malariae. In a further non-limiting
embodiment, the
trypanosome is P. falciparum. In a further non-limiting embodiment, the method
predicts an
increase in mortality from malarial infection.
[00029] In another embodiment of the second aspect of the present invention,
the identified
Replikin cycle represents Replikin concentrations identified in a histidine
rich protein of P.
falciparum. In another non-limiting embodiment of the present invention, the
identified Replikin
cycle represents Replikin concentrations identified in the histidine-rich
protein of P. falciparum.
[00030] In another non-limiting embodiment of the second aspect of the present
invention, the
pathogen is a West Nile virus. In a further embodiment, the identified
Replikin cycle represents
concentration identified in the envelope protein of West Nile virus. In
another non-limiting
embodiment, the pathogen is a foot and mouth disease virus. In a further
embodiment, the
identified Replikin cycle represents concentrations identified in the VP 1
protein of foot and
mouth disease virus. In another non-limiting embodiment, the pathogen is an
influenza virus. In
a further embodiment, the identified Replikin cycle represents concentrations
identified in the
pB1 gene area of influenza virus. In another non-limiting embodiment, the
influenza virus is an
H1N1, H2N2, H3N2, H3N8, H5N1, or H9N2 strain of influenza virus.
[00031] In another non-limiting embodiment of the second aspect of the present
invention, the
second time point or time period is up to three years after the first time
point or time period. In a
further non-limiting embodiment, the second time point or time period is about
one year after the
first time point or time period. In a further non-limiting embodiment, the
second time point or
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time period is about six months after the first time point or time period. In
a further non-limiting
embodiment, the second time point or time period is the next season of a
pathogen following the
first time point or time period. In a further non-limiting embodiment, the
second time point or
time period is the next season of influenza following the first time point or
first time period. In a
further non-limiting embodiment, the next influenza season is the next winter
season in a
geographic region following the first time point or time period. In a further
non-limiting
embodiment, the second time point or time period is following the next dry
season after the first
time point or time period. In a further non-limiting embodiment, the second
time point or time
period is the next season of malaria following the first time point or first
time period. In a further
non-limiting embodiment, the next season is the next rainy season.
[00032] In another non-limiting embodiment of the second aspect of the present
invention, the
identified peak in the cycle of Replikin concentration has a higher Replikin
concentration than a
chronologically earlier peak in the cycle of Replikin concentration. In a
further non-limiting
embodiment of the invention, the identified peak in the cycle of Replikin
concentration is
significantly higher than the earlier peak. In a further non-limiting
embodiment the identified
peak is significantly higher than the earlier peak with a p value less than
0.01. In a further non-
limiting embodiment the identified peak is significantly higher than the
earlier peak with a p
value less than 0.001.
[00033] In a further non-limiting embodiment of the second aspect of the
invention, predicting
said expansion of population or said increase in virulence, morbidity, and/or
mortality of an
isolate of a pathogen comprises identifying a second cycle in the Replikin
concentration of a
plurality of isolates of a second strain or related strain of pathogen that
shares synchrony with
said first cycle in the Replikin concentration of said plurality of isolates
of said first strain of
pathogen and identifying a first peak in the Replikin concentration within the
identified first
cycle at a first time point or time period and identifying a first peak in the
Replikin concentration
within the identified second cycle of said second strain of pathogen or
related strain of pathogen
at a second time point or time period that is similar to said first time point
or time period and
predicting an expansion of the population or an increase in the virulence,
morbidity, and/or
mortality of said first strain of pathogen following the first time point or
time period. In a non-
limiting embodiment, the pathogen is a malarial trypanosome, a West Nile
virus, a foot and
mouth disease virus, or any other infectious agent.
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[00034] In a non-limiting embodiment, said pathogen is an influenza virus. In
a further non-
limiting embodiment, said first strain of influenza is any strain different
from said second strain
of influenza. In another non-limiting embodiment, said first strain of
influenza is H5N1 and said
second strain of influenza is H9N2, or vice versa. In another embodiment, the
strain is any
influenza strain and the related strain is any other strain wherein a
relationship with said first
strain is determined by comparing the Replikin cycles of said strain and said
related strain. In
another embodiment, the strains are related because the Replikin cycles share
synchrony.
[00035] A further non-limiting embodiment of the second aspect of the
invention provides a
method of predicting an expanding population of a pathogen or an increase in
virulence,
morbidity, and/or mortality in a pathogen comprising: (1) determining the mean
Replikin Count
in a plurality of isolates of at least two strains of pathogen at a plurality
of successive time
points; (2) comparing the mean Replikin Count at at least four successive time
points for each
strain and identifying at least one cycle of increasing mean Replikin Counts
over the at least four
time points for each of the at least two strains; (3) identifying at least
partial synchrony between
the at least one cycle of increasing mean Replikin Counts for each of the at
least two strains; and
(4) predicting an increase in virulence following in time the increase in mean
Replikin Count in
the at least one cycle in said at least two strains wherein said at least one
cycle in said at least
two strains occurs at a corresponding time period. In a further non-limiting
embodiment, step-
wise cycles are identified between successive time points. In a further non-
limiting embodiment,
specific conserved Replikin sequences are identified within the step-wise
cycles. In a further
non-limiting embodiment, Replikin sequences are identified at the peak of a
stepwise cycle. The
Replikin sequences identified at the peak of a stepwise cycle are useful for
developing a vaccine
or therapeutic composition of an isolated or synthesized Replikin peptide for
use in preventing or
treating outbreaks of malaria with relatively higher mortality. In a further
embodiment, the
pathogen is influenza. In a further embodiment, the at least two strains of
influenza are H9N2
and H5N1.
[00036] Another non-limiting embodiment of the second aspect of the invention
provides a
method of predicting a contraction or failure of a population of a strain of
pathogen, wherein an
isolate of said pathogen is isolated at a time point or time period subsequent
to a decreasing
portion of a Replikin cycle.
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[00037] A further non-limiting embodiment of the second aspect of the
invention provides a
method for making a vaccine comprising predicting an expanding population of a
pathogen or
related strain of pathogen or an increase in virulence, morbidity, and/or
mortality of a pathogen
or a related strain of pathogen and identifying a portion of the structure or
genome of said
isolated influenza virus to be comprised in a vaccine.
[00038] A further non-limiting embodiment of the second aspect of the present
invention
provides an isolated or synthesized portion of the structure or genome of a
pathogen wherein said
pathogen is predicted to have an expansion of the population of the pathogen.
In a further
embodiment, the isolated or synthesized portion is a protein, protein
fragment, or peptide
comprising a Replikin peptide or a Replikin Peak Gene. In a further non-
limiting embodiment,
the isolated or synthesized portion of the structure or genome of a pathogen
consists of one or
more Replikin peptides and/or one or more Replikin Peak Genes. In a further
non-limiting
embodiment, the one or more Replikin peptides are conserved during a cycle in
Replikin
concentration at at least two successive time points or time periods in the
cycle.
[00039] Another non-limiting embodiment of the second aspect of the present
invention
provides Replikin peptides for diagnostic, therapeutic, and/or preventive
purposes identified as
conserved in an isolate of said pathogen from among a plurality of isolates of
said pathogen,
wherein said isolates are isolated during a cycle in Replikin concentration at
at least two
successive time points or time periods, and the cycle preferably includes at
least two peaks or
two troughs.
[00040] In a further non-limiting embodiment of the second aspect of the
invention, the
pathogen is an influenza virus. In a further non-limiting embodiment, the
Replikin peptide is at
least one of HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1),
KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH
(SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4),
HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH
(SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ
ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID
NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or
HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12).
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[00041] In a further non-limiting embodiment of the second aspect of the
invention, the
pathogen is a West Nile virus. In a further non-limiting embodiment, the
Replikin peptide is at
least one of KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK
(SEQ ID NO: 15), or HNDKRADPAFVCK (SEQ ID NO: 16).
[00042] In a further non-limiting embodiment, the pathogen is a foot and mouth
disease virus.
In a further non-limiting embodiment, the Replikin peptide is at least one of
HKQKIIAPAK
(SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18).
[00043] In a further non-limiting embodiment, the pathogen is malaria. In a
further non-
limiting embodiment, the Replikin peptide is at least one of a Replikin
peptide identified from at
least one of the following accession numbers: ABU43157, CAD49281, CAD49281, or
XP001349534.
[00044] Any of the above-listed or herein identified Replikin peptides may be
comprised in an
immunogenic compound of the invention.
[00045] A further non-limiting embodiment of the second aspect of the
invention provides a
computer readable medium having stored thereon instructions which, when
executed, cause a
processor to perform a method of predicting an expansion of a strain of
pathogen or an increase
in virulence, morbidity, and/or mortality of a pathogen. In a further
embodiment, the processor
reports a prediction to a display, user, researcher, or other machine or
person. In a further
embodiment, the processor identifies to a display, user, researcher, or other
machine or person, a
portion of a pathogen predicted to be an expanding pathogen or predicted to
increase in
virulence, morbidity, and/or mortality, wherein said portion may be employed
as a therapeutic or
diagnostic compound. Said portion may be a Replikin peptide or plurality of
Replikin peptides
or any other structure or portion of said genome of said pathogen including a
Replikin Peak
Gene.
[00046] A third non-limiting aspect of the present invention provides Replikin
peptides for
diagnostic, therapeutic, and/or preventive purposes identified in an isolate
of a pathogen, wherein
said isolate is isolated during a rising portion of a cycle in Replikin
concentration from among a
plurality of isolates of the pathogen, or is isolated at a peak in a cycle in
Replikin concentration
from among a plurality of isolates of a pathogen, or isolated subsequent to a
peak in a cycle in
Replikin concentration from among a plurality of isolates of a pathogen.
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[00047] Another non-limiting embodiment of the third aspect of the present
invention
provides Replikin peptides for diagnostic, therapeutic, and/or preventive
purposes identified as
conserved in an isolate of a pathogen from among a plurality of isolates said
pathogen, wherein
said isolates are isolated during a cycle in Replikin concentration at at
least two successive time
points or time periods, and the cycle includes at least two peaks or two
troughs.
[00048] In a non-limiting embodiment of the third aspect of the present
invention, the
pathogen is a malarial trypanosome. In a further non-limiting embodiment, the
identified cycle
is in the histidine rich protein of P. falciparum. In another non-limiting
embodiment, the
identified cycle is in the ATP-ase protein of P. falciparum. In another non-
limiting embodiment,
the identified cycle is in a Replikin Peak Gene of a trypanosome that causes
malaria.
[00049] In another non-limiting embodiment of the third aspect of the present
invention, the
pathogen is a West Nile virus. In a further non-limiting embodiment, the
identified cycle is in
the envelope protein of West Nile virus. In another non-limiting embodiment,
the pathogen is a
foot and mouth disease virus. In a further non-limiting embodiment, the
identified cycle is in a
VP1 protein of a foot and mouth disease virus.
[00050] In another non-limiting embodiment of the third aspect of the present
invention, the
pathogen is an influenza virus. In another non-limiting embodiment, the
influenza virus is an
H1N1, H2N2, H3N2, H3N8, H5N1, or H9N2 influenza virus. In a further non-
limiting
embodiment, the identified cycle is in the neuraminidase or hemagglutinin
protein of an
influenza virus.
[00051] A fourth non-limiting aspect of the present invention provides an
immunogenic
composition comprising a Replikin peptide identified in an isolate of a
pathogen, wherein said
isolate is isolated during a rising portion of a cycle in Replikin
concentration from among a
plurality of isolates of said pathogen, or is isolated at a peak in the
identified cycle in Replikin
concentration from among a plurality of isolates of the pathogen, or is
isolated subsequent to a
peak in the identified cycle in Replikin concentration from among a plurality
of isolates of the
pathogen.
[00052] In another non-limiting embodiment of the fourth aspect of the present
invention, the
immunogenic composition is a vaccine for prevention or treatment of an
infection of a pathogen.
Another non-limiting embodiment of the present invention provides an antibody
to a Replikin
peptide identified in an isolate of the pathogen, wherein said isolate is
identified during a rising
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portion of a cycle in Replikin concentration, or is identified at a peak in a
cycle in Replikin
concentration, or is identified subsequent to a peak in a cycle in Replikin
concentration. In
another non-limiting embodiment, the pathogen is a West Nile virus. In another
non-limiting
embodiment, the pathogen is a foot and mouth disease virus.
[00053] A fifth non-limiting aspect of the invention provides a method of
preventing,
mitigating, or treating an outbreak of a pathogen comprising
predicting an expansion of a strain of pathogen comprising (1) determining a
mean
Replikin Count and a standard deviation of said mean Replikin Count for a
plurality of isolates
of a strain of pathogen for a first time period in a first geographic region,
(2) determining a
Replikin Count of at least one isolate of the same or a related strain of
pathogen from a second
time period and/or second geographic region wherein said second time period is
different from
said first time period and/or said second geographic region is different from
said first geographic
region, and (3) predicting an expansion of said strain of pathogen isolated in
said second time
period and/or second geographic region if the Replikin Count of said at least
one isolate is
greater than one standard deviation of the mean of the Replikin Count of the
plurality of isolates
isolated in said first time period and in said first geographic region; and
administering to an animal or a patient a compound comprising an isolated or
synthesized portion of the structure or genome of the at least one isolate of
influenza virus to
prevent or treat the outbreak of influenza virus.
[00054] In a non-limiting embodiment of the fifth aspect of the invention,
said pathogen is an
influenza virus, a malarial trypanosome, a West Nile virus, a foot and mouth
disease virus, or
any other kind of infectious agent.
[00055] In a non-limiting embodiment of the fifth aspect of the invention,
said first time
period is one year and said first geographic region is a country. In a further
embodiment, said
second time period is one year. In a further embodiment, said second
geographic region is a
country. In a further embodiment, where the pathogen is influenza virus, said
first geographic
region is China. In a further embodiment, where the pathogen is a malarial
trypanosome, said
first geographic region is India. In a further embodiment, where the pathogen
is West Nile virus,
said first geographic region is a state within the United States.
[00056] In another non-limiting embodiment of the fifth aspect of the
invention, said plurality
of isolates of a strain of pathogen for a first time period in a first
geographic region is a plurality
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of isolates from all publicly available sequences in said first time period in
said first geographic
region. In another non-limiting embodiment, said plurality of isolates is all
isolates from a
species of animal. In another non-limiting embodiment, said plurality of
isolates is all isolates
from a particular species of bird such as swans, chickens, falcons, turkeys,
ducks, or other
domestic or wild birds.
[00057] In a further non-limiting embodiment of the fifth aspect of the
invention, said isolated
or synthesized portion of the structure or genome of the at least one isolate
of pathogen is a
protein or protein fragment comprising a Replikin peptide. In a further
embodiment, said protein
or protein fragment is a Replikin peptide. In another embodiment, said protein
or protein
fragment comprises a Replikin Peak Gene. In a further embodiment, said protein
or protein
fragment is a Replikin Peak Gene. In a further embodiment, said protein or
protein fragment is a
Replikin peptide identified within a Replikin Peak Gene. In another
embodiment, said isolated
or synthesized portion of the structure or genome is a nucleic acid encoding a
Replikin Peak
Gene, a nucleic acid encoding a Replikin peptide or plurality of Replikin
peptides within a
Replikin Peak Gene, or a nucleic acid encoding a Replikin peptide.
[00058] In another non-limiting embodiment of the fifth aspect of the
invention, the at least
one isolate of the same strain of pathogen from a second time period and/or
second geographic
region is a plurality of isolates from said second time period and/or second
geographic region
and the Replikin Count of each isolate of the plurality of isolates from said
second time period
and/or second geographic region is compared separately to said one standard
deviation of said
mean Replikin Count.
[00059] In a further non-limiting embodiment of the fifth aspect of the
present invention, an
expansion of said strain of pathogen isolated in said second time period
and/or second
geographic region is predicted if the number of Replikin Counts of said
plurality of isolates from
said second period and/or said second geographic region that is greater than
one standard
deviation of the mean of the Replikin Count of the plurality of isolates
isolated in said first time
period in said first geographic region, is greater than the number of Replikin
Counts of said
plurality of isolates from said second time period and/or said second
geographic region that is
less than said one standard deviation of the mean.
[00060] In a further non-limiting embodiment of the fifth aspect of the
invention, the Replikin
Count is the concentration of Replikin peptides identified encoded in the
genome of an isolate of
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the pathogen. In a further embodiment, the Replikin Count is the concentration
of Replikin
peptides identified in the expressed proteins of an isolate of the pathogen.
In a further
embodiment, the Replikin Count is the concentration of Replikin peptides
identified in at least
one protein or gene area of an isolate of the pathogen. In a further
embodiment, the gene area is
the pB 1 gene area of the genome of influenza virus, the histidine-rich
protein gene area of a
malarial trypanosome, the VP1 gene area of foot and mouth disease virus, or
the envelope
protein gene area of West Nile virus. In another embodiment, the Replikin
Count is the
concentration of Replikin peptides identified in at least one protein fragment
of an isolate of the
pathogen. In a further embodiment, the Replikin Count is the concentration of
Replikin peptides
identified in a Replikin Peak Gene of an isolate of the pathogen. In a further
embodiment, the
Replikin Peak Gene is identified in the polymerase area of an influenza virus
genome. In a
further embodiment, the Replikin Peak Gene is identified in the pB 1 area of
an influenza virus
genome. In a further embodiment, the Replikin Peak Gene is identified in the
histidine-rich
protein area of a malarial trypanosome, the VP1 area of a foot and mouth
disease virus, or the
envelope protein of a West Nile virus.
[00061] A sixth non-limiting aspect of the present invention provides a method
of predicting
an expansion of a strain of pathogen comprising
(1) determining a mean Replikin Count and a standard deviation of said mean
Replikin Count for a plurality of isolates of said strain of pathogen for a
first time period in a
first geographic region;
(2) determining a Replikin Count of at least one isolate of the same or a
related
strain of pathogen from a second time period and/or second geographic region
wherein said
second time period is different from said first time period and/or said second
geographic region
is different from said first geographic region; and
(3) predicting an expansion of said strain of pathogen isolated in said second
time
period and/or second geographic region if the Replikin Count of said at least
one isolate from a
second time period and/or second geographic region is greater than one
standard deviation of the
mean of the Replikin Count of the plurality of isolates isolated in said first
time period and in
said first geographic region.
[00062] In a non-limiting embodiment the method of predicting further
comprises processing
the method on a computer.
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[00063] A non-limiting embodiment of the sixth aspect of the invention
contemplates that the
pathogen is an influenza virus, a malarial trypanosome, a West Nile virus, a
foot and mouth
disease virus, or any other kind of infectious agent.
[00064] A further non-limiting embodiment of the sixth aspect of the invention
provides a
method for making a vaccine comprising predicting an expansion of said strain
of pathogen
isolated in said second time period and/or second geographic region and
identifying a portion of
the structure or genome of said isolated influenza virus to comprise a
vaccine.
[00065] In a further non-limiting embodiment of the sixth aspect of the
invention, the at least
one isolate of the same strain of pathogen from a second time period and/or
second geographic
region is a plurality of isolates from said second time period and/or second
geographic region. In
a further non-limiting embodiment, the Replikin Count of each isolate of the
plurality of isolates
from said second time period and/or second geographic region is compared
separately to said one
standard deviation of the mean.
[00066] In another non-limiting embodiment of the sixth aspect of the
invention, an expansion
of a strain of pathogen isolated in said second time period and/or said second
geographic region
is predicted if the number of Replikin Counts of said plurality of isolates
from said second time
period and/or said second geographic region that is greater than one standard
deviation of the
mean of the Replikin Count of the plurality of isolates isolated in said first
time period in said
first geographic region, is greater than the number of Replikin Counts of said
plurality of isolates
from said second time period and/or said second geographic region that is less
than said one
standard deviation of the mean. In a further non-limiting embodiment, an
expansion of a strain
of influenza virus isolated in said second time period and/or second
geographic region is
predicted if the ratio of the number of Replikin Counts of said plurality of
isolates from said
second time period and/or said second geographic region that is greater than
said one standard
deviation of the mean, divided by the number of Replikin Counts of said
plurality of isolates
from said second time period and/or said second geographic region that is less
than said one
standard deviation of the mean, is greater than one.
[00067] A further non-limiting embodiment of the sixth aspect of the present
invention
provides Replikin peptides for diagnostic, therapeutic, and/or preventive
purposes identified in
an isolate of a pathogen predicted to have an expanding population. In another
non-limiting
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embodiment, the Replikin peptides for diagnostic, therapeutic, and/or
preventive purposes are
conserved over time or across geographic regions.
[00068] Another non-limiting embodiment of the sixth aspect of the invention
provides a
method of predicting a contraction or failure of a population of a strain of
pathogen, wherein a
Replikin Count of at least one isolate of a strain of pathogen from a first
time period and/or first
geographic region is less than one standard deviation of the mean of the
Replikin Count of a
plurality of isolates of influenza from a second time period and second
geographic region.
Another non-limiting embodiment provides a method of predicting a contraction
or failure of a
population of a strain of pathogen, wherein the number of Replikin Counts of a
plurality of
isolates from a first time period and/or a first geographic region greater
than one standard
deviation of the mean of the Replikin Count of a plurality of isolates from a
second time period
in a second geographic region, is less than the number of Replikin Counts of
the plurality of
isolates from the first time period and/or the first geographic region that is
less than said one
standard deviation of the mean. In a further non-limiting embodiment, said
contraction or failure
is predicted if the ratio of the number of Replikin Counts of said plurality
of isolates from said
first time period and/or said first geographic region that are greater than
said standard deviation
of the mean, divided by the number of Replikin Counts of said plurality of
isolates from said first
time period and/or said first geographic region that are less than said
standard deviation of the
mean, is less than one.
[00069] A further non-limiting embodiment of the sixth aspect of the invention
provides a
computer readable medium having stored thereon instructions which, when
executed, cause a
processor to perform a method of predicting an expansion of a strain of
pathogen or the
expansion of a virus or organism. In a further embodiment, the processor
reports a prediction to
a display, user, researcher, or other machine or person. In a further
embodiment, the processor
identifies to a display, user, researcher, or other machine or person, a
portion of a pathogen
predicted to be an expanding pathogen, wherein said portion may be employed as
a therapeutic
or diagnostic compound. Said portion may be a Replikin peptide or plurality of
Replikin
peptides or any other structure or portion of said genome of said pathogen
including a Replikin
Peak Gene.
[00070] A seventh non-limiting aspect of the present invention provides an
immunogenic
composition comprising a portion of the structure or genome of an isolate of a
pathogen, wherein
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said isolate of said pathogen is (1) an isolate having a Replikin Count
greater than one standard
deviation of a mean Replikin Count of a plurality of isolates of pathogen
isolated in a different
time period and/or in a different geographical region, (2) an isolate from a
first time period
and/or geographical region wherein the number of a plurality of isolates from
the first time
period and/or geographical region having a Replikin Count greater than said
one standard
deviation of the mean is greater than the number of isolates having a Replikin
Count less than
said one standard deviation of the mean, (3) isolated during a rising portion
of a cycle or a set of
two or more synchronous cycles in Replikin concentration from among a
plurality of isolates of
influenza, and/or (4) isolated at a peak in the identified cycle or set of
synchronous cycles in
Replikin concentration from among a plurality of isolates of influenza.
[00071] In another non-limiting embodiment the seventh aspect of the present
invention, the
immunogenic composition is a vaccine for prevention or treatment of an
infection of a pathogen.
Another non-limiting embodiment provides an antibody to a Replikin peptide
identified in an
isolate of pathogen predicted to have an increase in virulence, morbidity,
and/or lethality or
expansion of its population.
BRIEF DESCRIPTION OF THE DRAWINGS
[00072] Figure 1 illustrates cycling between 1986 and 2007 of mean annual
Replikin
concentration in the histidine rich protein of Plasmodium falciparum for
sequences available at
www.pubmed.com for isolates from 1986 through 2007. In Figure 1, three rising
portions of
cycles of Replikin concentration and two decreasing portions of cycles of
Replikin concentration
are observable with peaks at 1987 and 1999. A first rising portion and
decreasing portion of a
cycle is observed from 1986 to 1995. A second rising portion and decreasing
portion of a cycle
is observed from 1996 to 2005. A new cycle appears to have begun between 2005
and 2007.
The peak of the first rising portion was identified in 1987 with a mean annual
Replikin Count of
38.2 and standard deviation of 23.5. The peak of the second rising portion
was identified in
1999 with a step-wise even higher mean annual Replikin Count of 62.9 and
standard deviation of
63. Both the 1987 peak and the 1999 peak were observed to be related to higher
human
mortality. Following the 1999 peak, mean annual Replikin Counts were observed
to fall to a low
of 7.4 in 2005 with a standard deviation of 6.5. Mortality rates likewise
fell between 2000 and
2005. A new malaria Replikin cycle appears to have begun in 2005 with the
observed mean
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annual Replikin Count increasing from 7.4 6.5 in 2005 to 17.2 19 in 2007. The
beginning of
the new cycle provides a prediction that Replikin Count may continue to
increase along with an
increase in malaria mortality rate.
[00073] Figure 2 illustrates that mortality rates per 1000 clinical cases of
malaria in humans
generally correlate with mean annual Replikin Count in sequences of the P.
falciparum ATP-ase
enzyme publicly available at www.pubmed.com. Mean annual Replikin Counts of P.
falciparum
ATP-ase increased from 1997 to 1998 along with an increase in mortality per
malaria case from
1997 and 1998 to 1999. The mean annual Replikin Count of P. falciparum ATP-ase
decreased
from 1998 to 2006 along with the mortality rates from 1999 to 2005 (consistent
mortality data is
considered presently available only through 2005). The data for Figure 2 may
be seen in Table 6
below. Mortality rates in Figure 2 and Table 6 are recorded as declared by the
World Health
Organization. See www.who.int.
[00074] Figure 3 illustrates cycling of mean annual Replikin Count in West
Nile virus in
correlation with cycling of West Nile virus morbidity. The mean annual
Replikin Count of the
Envelope Protein of WNV (black) and standard deviation (capped line) is
compared to the
annual number of human cases in the United States as reported by the Centers
for Disease
Control (CDC) (gray). Mean annual Replikin Count was analyzed in envelope
protein sequences
from isolates isolated between 2000 and 2006 and publicly available at
www.pubmed.com. In
Figure 3, the standard deviation of the mean of the Replikin Count of the
envelope protein is
observed to increase markedly from 2000 to 2001 (p<O.001). This change has
been observed to
signal rapid replication and expansion of the range of the Replikin Count
preceding virus
outbreak in all common strains of influenza virus (not the same virus genus as
WNV) as standard
deviation within a virus population increases. The increase in mean Replikin
Count in WNV
from 2000 to 2003 appears to accompany, or precede, the increase in the number
of human
WNV cases recorded independently and published by the CDC. A decrease in mean
annual
Replikin Count and recorded human cases of WNV is observed following 2003. In
2006, an
increase is observed in the Replikin Count followed by an increase in 2007 of
the number of
human cases. As a result, Figure 3 illustrates two rising portions and one
decreasing portion in a
cycle of Replikin concentration and two rising portions and one decreasing
portion in a cycle of
WNV human morbidity, the first rising portion from 2000 to 2003 and the second
rising portion
from 2004 to 2006/2007. Conserved viral Replikin structures within the
envelope protein are
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observed throughout the illustrated cycles and the relationship between
Replikin structure and
rapid replication and virulence are observed through time.
[00075] Figure 4 illustrates cycling in Replikin concentration in the whole
genome of foot and
mouth disease virus (FMDV) type 0 isolated between 1999 and 2008 and reported
at
www.pubmed.com. The data demonstrate that annual Replikin Counts (Mean and
Standard
Deviation (SD)) for isolates of FMDV type 0 occurred with two rising portions
and one
decreasing portion. A first rising portion and a first decreasing portion are
observed between
1999 and 2005. A second rising portion is observed beginning in 2005 through
2008. The cycle
is presently incomplete since a second trough is not yet observable. In Figure
4, mean annual
Replikin Count is observed to provide advance warning signals (with p<O.001)
prior to severe
FMDV outbreaks in the U.K. and the Netherlands in 2001-2002, Mean annual
Replikin Count is
further observed to provide advance warning signals (with p<0.001) prior to
severe FMDV
outbreaks in the Middle East, Africa, India, and Asia (including China) in
2008-2009. Replikin
cycles are detectable because of repeating conserved virus structures and
continuity of the
Replikin phenomenon through time. The data in Figure 4 demonstrate that the
highest mean
annual Replikin Counts over the ten year period reflected in Figure 4 were
observed in 2007 and
2008.
[00076] Figure 5 illustrates cycles of mean annual Replikin Count in influenza
sequences
from the pB l gene area for isolates isolated between 1993 and 2008 and
reported at
www.pubmed.com. In Figure 5, the mean annual Replikin Count of the pBl gene
area of
isolates of H9N2 is shown in light gray columns with standard deviation shown
above in dark
gray columns. The number of poultry flocks reported in Israel with H9N2
infection is provided
in white columns. The data illustrate an increase in mean annual Replikin
Count that
corresponds to an increase in influenza outbreaks in flocks of poultry in
Israel between 2000 and
2004. The standard deviation data further emphasize the extent of expanding
Replikin Counts
within the annual H9N2 influenza population.
[00077] Figure 6 illustrates synchronous cycles of mean annual Replikin Counts
in the pB 1
gene area of H9N2 and H5N1 influenza isolates. The data represent analysis of
sequences of
isolates isolated between 1993 and 2008 and reported at www.pubmed.com. In
Figure 6, annual
mean Replikin Count for H9N2 is reported in light gray columns with standard
deviation
reported above in dark gray columns. Annual mean Replikin Count for H5N1 is
reported in
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black columns with standard deviation reported above in white columns. Figure
6 visibly
illustrates synchrony between the H9N2 and H5N1 Replikin Cycles. The
synchronous cycles
individually and together predict H5N1 outbreaks in Hong Kong in 1997, 2002,
2004, 2007, and
a present outbreak of H5N1 and H9N2 in 2008-2009. Because the cycles of
different strains
correspond with a level of synchrony, the predictive capacity of the
individual cycles is increased
by the correspondence. Further an interrelationship between H5N1 and H9N2 is
demonstrated
suggesting that H9N2 may be a candidate for a future influenza pandemic just
as H5N1 has been
known to be a candidate for such a pandemic.
[00078] Figure 7 illustrates cycling in mean annual Replikin Counts in the pB
1 gene area 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, a
first peak is
followed by a decline (likely due to immunity in the hosts), then by a second
recovery peak 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. The data
supports a prediction of an increase in virulence and morbidity following a
peak in a cycle of
mean annual Replikin Count in influenza virus. For influenza strains that
result in mortality, an
increase in virulence and morbidity was accompanied by increased mortality in
the pandemics of
the 20th Century.
[00079] Figure 8 illustrates the same data as Figure 7 but is expanded in size
for better
viewing of the data for individual years. Figure 8(A-C) illustrates cycles in
Replikin Count in
strains of influenza related to outbreaks of influenza between 1917 and 2007.
The data illustrate
an increase in Replikin Count before and accompanying each influenza A
pandemic and
outbreak since 1918 and low Replikin Counts during quiescent periods of
influenza A infection
and continually in non-lethal Influenza B. The graph provides annual Replikin
Counts from
1917-2007 for all Replikin Peak Genes isolated in silico in the pB 1 gene area
of influenza strains
having amino acid or nucleic acid sequences publicly available at PubMed. Data
is provided (1)
for non-lethal human Influenza B between 1940 and 2007 (thick solid line) and
(2) for both the
lethal and non-lethal periods of human Influenza A viruses between 1917 and
2007. Human
Influenza A strains are (1) HiN1 (thin solid line), (2) H2N2 (long-short-long
dashed line), (3)
H3N2 (medium dashed line), and (4) H5N1 (long dashed line). H5N1 strains
isolated from
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chicken are illustrated by a short dashed line. The total number of sequences
analyzed for the
data (N) is 14,227. Listed pandemics, epidemics and outbreaks are the 1918
HINT pandemic,
the 1930's HINT epidemic, the 1957 H2N2 pandemic, the 1968 H3Nl pandemic, the
1977-78
H3N2 outbreaks and the H5N1 outbreaks of 2001-2004 and 2007. A 1997 outbreak
of H5N1 is
not shown in Figures 7 and 8. Over a ninety year period, pandemics, epidemics
and outbreaks
are associated with Replikin Counts of four or above in the RPG of influenza
strains. Over the
same period, constant low Replikin Counts of less than four may be observed
during quiescent
non-lethal periods of influenza A infections and low Replikin Counts of less
than four may be
observed in non-lethal Influenza B.
[00080] Figure 9 illustrates an immune response with protective effect
following
administration of a vaccine comprising a mixture of peptides of SEQ ID NO(s):
1-12 to chickens
later challenged with Low-Path H5N1 virus. Eighty chickens were divided into
four groups of
twenty chickens each on a first day after hatch. Group 1 was a negative
control subjected to
neither vaccination nor infection with the Low-Path H5N1 virus. Group 2 was a
vaccine control
subjected to vaccination intranasally on day 1 after hatch, intraocularly on
day 7 after hatch, and
via spray inhalation on day 14 after hatch. Group 2 was not subjected to
infection with the Low-
Path H5N1 virus. Group 3 was subjected to vaccination on the same schedule as
Group 2 and
Low-Path H5N1 was introduced to the cleft palate of the chickens on day 28.
Group 4 was a
challenged control that was not vaccinated but was infected with H5N1 on day
28 via the cleft
palate. On days 7, 14, and 21, between six and nine chickens from each group
were tested for
serum production of antibodies against H5N1 virus. The data from the serum
antibody tests are
contained in Table 15 and illustrated in Figure 9. Figure 9 illustrates that
only one of seven
(14%) chickens tested in Group 3 (vaccinated and challenged with virus) was
observed to
produce antibody in serum seven days after challenge while four of seven
chickens (57%) tested
in Group 4 (not vaccinated but challenged) were observed to produce antibody
in serum seven
days after challenge. Figure 9 further illustrates that only three of six
chickens (50%) tested in
Group 3 (vaccinated and challenged) were observed to produce antibody in serum
fourteen days
after challenge while seven of nine (78%) chickens tested in Group 4 (not
vaccinated but
challenged) were observed to produce antibody in serum fourteen days after
challenge. Figure 9
further illustrates that two of seven (29%) chickens tested in Group 3 were
observed to produce
antibody in serum twenty-one days after challenge while three of nine (33%)
chickens tested in
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Group 4 were observed to produce antibody in serum twenty-one days after
challenge. In the
vaccine control (Group 2) six of six (100%) chickens tested were observed to
produce antibody
in serum fourteen days after challenge while no chickens tested on days 7 or
21 were observed to
produce antibody in serum. In the negative control (Group 1), no chickens were
observed to
produce antibody in serum on any day of testing. In combination with data
provided in Example
demonstrating that no H5N1 virus was observed by PCR detection excreted in
feces or saliva
from chickens in Groups 1, 2, and 3 (negative control, vaccine control, a
vaccine/challenge
groups, respectively) and that H5N1 virus was observed by PCR detection
excreted in feces and
saliva for all chickens in Group 4 (challenge control), one of ordinary skill
in the art concludes
that chickens in the vaccinated groups (Groups 2 and 3) produced an immune
response to the
vaccine and that chickens in the vaccinated and challenged group (Group 3)
were provided a
measure of protection from the Low-Path H5N1 challenge on day 28 following
hatch.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[00081] As used herein, a "Replikin cycle" or "a cycle of Replikin
concentration" or "a cycle
of Replikin Count" means Replikin concentrations of a plurality of isolates of
a species of virus
or organism wherein at least four of said plurality of isolates are isolated
at successive time
points or in successive time periods, wherein a Replikin concentration of a
second individual
isolate or second mean of a plurality of isolates at a second time point or
time period is higher
than a Replikin concentration of a first individual isolate or a first mean of
a plurality of isolates
at a first time point or time period, a Replikin concentration of a third
individual isolate or a third
mean of a plurality of isolates at a third time point or time period is lower
than the Replikin
concentration at a second time point or time period, and a Replikin
concentration of a fourth
individual isolate or fourth mean of a plurality of isolates at a fourth time
point or time period is
higher than the Replikin concentration at a third time point or time period;
or wherein a Replikin
concentration of a second individual isolate or second mean of a plurality of
isolates at a second
time point or time period is lower than a Replikin concentration of a first
individual isolate or a
first mean of a plurality of isolates at a first time point or time period, a
Replikin concentration of
a third individual isolate or a third mean of a plurality of isolates at a
third time point or time
period is higher than the Replikin concentration at a second time point or
time period, and a
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Replikin concentration of a fourth individual isolate or fourth mean of a
plurality of isolates at a
fourth time point or time period is lower than the Replikin concentration at a
third time point or
time period. Within the Replikin cycle, cycle of Replikin concentration, or
cycle of Replikin
Count, the second time point or time period must be later in time than the
first time point or time
period, the third time point or time period must be later in time than the
second time point or
time period, and the fourth time point or time period must be later in time
than the third time
point or time period. Within a Replikin cycle, any rising portion is
predictive of an expansion in
population or an increase in virulence, morbidity, and/or mortality of a
pathogen in hosts and any
decreasing portion is predictive of a contracting population or a decrease in
virulence, morbidity,
and/or mortality of pathogen in hosts. A cycle need not be complete to be
predictive, a
decreasing portion followed by a rising portion is predictive of an expanding
population or an
increase in virulence, morbidity, and/or mortality. Likewise, a rising portion
followed by a
decreasing portion followed by a rising portion is predictive of an expanding
population or an
increase in virulence, morbidity, and/or mortality. As such, cycles need not
be complete cycles
to provide predictive capacity concerning an expansion or contraction (or
change in virulence,
morbidity, and/or mortality) of a pathogen in hosts.
[00082] As used herein, a "step-wise" cycle is any set of cycles wherein a
first Replikin cycle
peak in time is lower than a second Replikin cycle peak in time or a first
Replikin cycle peak in
time is higher than a second Replikin cycle peak in time. A step-wise cycle
also occurs when
successive peaks are observed to move lower. A step-wise cycle may also be
observed if
successive troughs move higher or lower. Step-wise cycles provide additional
predictive
capacity for predictions of expansion or contraction of a population.
[00083] As used herein a Replikin cycle that is "synchronous," shares
"synchrony," or any
other related word, with another Replikin cycle means a cycle having a period
or phase or any
portion of the cycle that is similar to some period, phase, or portion of the
cycle wherein said
similarity may be determined visually, mathematically, statistically, or by
any other method
known or hereinafter known by one of skill in the art. Synchronous cycles do
not necessarily
share portions that arise or occur at exactly the same time. Synchronous
cycles in related
pathogens will at times be shifted by some measure of time from one another
and may shift in
time from one another in any portion of either cycle. A portion of a Replikin
cycle
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"corresponds" in time with another Replikin cycle if there is a similarity
between the portions of
the cycle. Any correspondence need not be exact.
[00084] As used herein, a "rising portion" of a Replikin cycle means the
Replikin
concentration of an isolate or the mean Replikin concentration of a plurality
of isolates, wherein
the isolate or isolates were isolated at a time point or time period of the
Replikin cycle where the
trend of Replikin concentration in the Replikin cycle is increasing from at
least a first time point
or time period to at least a second time point or time period. Additionally,
the rising portion may
include a peak.
[00085] As used herein, a "decreasing portion" of a Replikin cycles means the
opposite of a
rising portion, wherein a decreasing portion may include a trough.
[00086] As used herein, a "peak" in a Replikin cycle means a second time point
or time
period within a Replikin cycle, wherein the Replikin concentration at a first
time point or time
period sequentially preceding the second time point or time period is lower
than the Replikin
concentration at the second time point or time period, and the Replikin
concentration at a third
time point or time period sequentially following the second time point or time
period is lower
than the Replikin concentration at the second time point or time period. One
of skill in the art
will understand that because of the variability of biological systems, a peak
may include a
general region of a cycle that is generally higher than a sequentially
preceding region and
generally higher than a sequentially following region rather than an exact
time point or time
period.
[00087] As used herein, a "trough" in a Replikin cycle means the opposite of a
peak in a
Replikin cycle.
[00088] As used herein, a "Replikin Count Virus Expansion Index" or "RCVE
Index" or a
"Replikin Count Expansion Index" or "RCE Index" is the number of Replikin
Counts of a
plurality of isolates from a first time period and/or first geographic region
that are greater than
one standard deviation of the mean of the Replikin Count of a plurality of
isolates isolated in a
second time period and in a second geographic region, divided by the number of
Replikin Counts
of said plurality of isolates from said first time period and/or said first
geographic region that are
less than one standard deviation of the mean of the Replikin Count of the
plurality of isolates
isolated in said second time period in said second geographic region. An RCE
or RCVE Index
predicts the expansion of a pathogen in a particular region and/or time period
if the ratio of the
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RCE or RCVE Index is greater than one. An RCE or RCVE Index predicts the
contraction,
retraction, reduction, or failure of a pathogen in a particular region and/or
time period if the ratio
of the RCE or RCVE Index is less than one. An RCE or RCVE Index predicts
equilibrium
between expansion and contraction in the pathogen population if the ratio of
the RCVE Index is
equal to one.
[00089] As used herein, a "related pathogen" means a first pathogen that is of
the same
species, genus, or family as a second pathogen for which a relationship is
known now or
hereafter by one of skill in the art. A related pathogen may be a first
pathogen that is of the same
species but a different strain from a second pathogen. A related pathogen may
be a first
pathogen that is the same or different species from a second pathogen and
shares a host,
reservoir, or vector with the second pathogen. Even if a first pathogen is not
of the same species,
genus, or family as a second pathogen, the first pathogen is related to the
second pathogen if the
first pathogen has a Replikin cycle that is synchronous with the Replikin
cycle of the second
pathogen. One of skill in the art will recognize the many ways that a first
pathogen may be
related to a second pathogen. A related pathogen may be within the same family
as a first
pathogen. A related pathogen may be within the same genus as a first pathogen.
A related
pathogen may be within the same species as a first pathogen. A related
pathogen may be within
the same strain as a first pathogen.
[00090] As used herein, different "time periods" or different "time points"
are any two time
periods or time points that may be differentiated from each other. For
example, an isolate of an
organism or virus isolated during the year 2004 may be considered to be
isolated in a different
time period than an isolate of the same organism or virus isolated during the
year 2005.
Likewise, an isolate of an organism or virus isolated in May 2004 may be
considered to be
isolated in a different time period than an isolate of the same organism or
virus isolated in June
2004. When comparing Replikin concentrations of different isolates, one may
use comparable
time periods. For example, an isolate from 2004 may be compared to at least
one other isolate
from some other year such as 2002 or 2005. Likewise, an isolate from May 2004
may be
compared to at least one isolate from some other month of some year, for
example, an isolate
from December 2003 or from June 2004.
[00091] As used herein, an "isolate" is any virus or organism isolated from a
natural source
wherein a natural source includes, but is not limited to, a reservoir of an
organism or virus, a
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vector of an organism or virus, or a host of an organism or virus.
"Obtaining," "isolating," or
"identifying" an isolate is any action by which an amino acid or nucleic acid
sequence within an
isolate is obtained including, but not limited to, isolating an isolate and
sequencing any portion
of the genome or protein sequences of the isolate, obtaining any nucleic acid
sequence or amino
acid sequence of an isolate from any medium, including from a database such as
PubMed,
wherein the nucleic acid sequence or amino acid sequence may be analyzed for
Replikin
concentration, or any other means of obtaining the Replikin concentration of a
virus isolated
from a natural source at a time point or within a time period.
[00092] As used herein, "an earlier-arising" virus or organism or a virus or
organism isolated
at "an earlier time point" or during "an earlier time period" is a specimen of
a virus or organism
collected from a natural source of the virus or organism on a date prior to
the date on which
another specimen of the virus or organism was collected from a natural source.
A "later-arising"
virus or organism or a virus or organism isolated at a "later time point" or
during a "later time
period" is a specimen of a virus or organism collected from a natural source
of the virus
(including, but not limited to, a reservoir, a vector, or a host) or a natural
source of the organism
on a date subsequent to the date on which another specimen of the virus or
organism was
collected from a natural source.
[00093] As used herein, the "next virulence season" of a pathogen is a time
period in which an
increase in morbidity of a pathogen is expected based on seasonal changes,
such as a change
from summer to winter or a change from a wet season to a dry season, wherein
the pathogen was
experiencing less morbidity in a previous sequential time period prior to the
time period in which
the increase in morbidity is expected to occur.
[00094] As used herein, the term "dry season" or "winter season" with respect
to malaria
describes a season in any geographical region wherein mosquito activity
(including feeding and
reproduction) is significantly less than during other times of the year. A
peak in a Replikin cycle
before a dry season or winter season predicts an increase in virulence,
morbidity, and/or
mortality in malaria in the following rainy season or summer season when
mosquito activity is
greatest.
[00095] As used herein "trypanosome that causes malaria," "malarial
trypanosome" or
"trypanosome" in singular or plural means any Plasmodium species or other
species known now
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or hereafter to cause malaria. Malarial trypanosomes include but are not
limited to Plasmodium
falciparum, Plasmodium vivax, Plasmodium ovate, and Plasmodium malariae.
[00096] As used herein, a "Replikin Peak Gene (RPG)" (or sometimes a Replikin
Peak
Gene Area-RPGA) means a segment of a genome, protein, segment of protein, or
protein
fragment in which an expressed gene or gene segment has a highest
concentration of continuous,
non-interrupted and overlapping Replikin sequences (number of Replikin
sequences per 100
amino acids) when compared to other segments or named genes of the genome.
Generally, a
whole protein or gene or gene segment that contains the amino acid portion
having the highest
concentration of continuous Replikin sequences is also referred to as the
Replikin Peak Gene.
More than one RPG may be identified within a gene, gene segment, protein, or
protein fragment.
An RPG may have a terminal lysine or a terminal histidine, two terminal
lysines, or a terminal
lysine and a terminal histidine. For diagnostic, therapeutic and preventive
purposes, an RPG
may have a terminal lysine or a terminal histidine, two terminal lysines, or a
terminal lysine and
a terminal histidine or may likewise have neither a terminal lysine nor a
terminal histidine so
long as the terminal portion of the RPG contains a Replikin sequence or
Replikin sequences
defined by the definition of a Replikin sequence, namely, an amino acid
sequence having about 7
to about 50 amino acids comprising:
(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.
Further, for diagnostic, therapeutic, preventive and predictive purposes, an
RPG may include the
protein or protein fragment that contains an identified RPG. For predictive
purposes, a Replikin
Count in the RPG may be used to track changes in virulence and lethality.
Likewise the RPG
may be used as an immunogenic compound or as a vaccine. Whole proteins or
protein fragments
containing RPGs are likewise useful for diagnostic, therapeutic and preventive
purposes, such as,
for example, to be included in immunogenic compounds, vaccines and for
production of
therapeutic or diagnostic antibodies.
[00097] As used herein, a "Replikin sequence" is an amino acid sequence of 7
to about 50
amino acids comprising or consisting of a Replikin motif wherein the Replikin
motif comprises:
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(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 or a histidine residue at the other
terminus. For diagnostic,
therapeutic, and preventive purposes, a Replikin sequence may or may not have
defined termini.
[00098] The term "Replikin sequence" can also refer to a nucleic acid sequence
encoding an
amino acid sequence having about 7 to about 50 amino acids comprising:
(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.
[00099] As used herein, the term "peptide" or "protein" refers to a compound
of two or
more amino acids in which the carboxyl group of one amino acid is attached to
an amino group
of another amino acid via a peptide bond. As used herein, "isolated" or
"synthesized" peptide or
biologically active portion thereof refers to a peptide that is, after
purification, substantially free
of cellular material or other contaminating proteins or peptides from the cell
or tissue source
from which the peptide is derived, or substantially free from chemical
precursors or other
chemicals when chemically synthesized by any method, or substantially free
from contaminating
peptides when synthesized by recombinant gene techniques or a protein or
peptide that has been
isolated in silico from nucleic acid or amino acid sequences that are
available through public or
private databases or sequence collections. An "encoded" or "expressed"
protein, protein
sequence, protein fragment sequence, or peptide sequence is a sequence encoded
by a nucleic
acid sequence that encodes the amino acids of the protein or peptide sequence
with any codon
known to one of ordinary skill in the art now or hereafter. It should be noted
that it is well-
known in the art that, due to redundancy in the genetic code, individual
nucleotides can be
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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.
[000100] As used herein, "outbreak" is an increase in virulence, morbidity,
and/or mortality
in a pathogenic disease or an expansion in the population of pathogen as
compared to a baseline
of an earlier occurring epidemiological pattern of infection in the same
disease. One of ordinary
skill in the art will know how to determine an epidemiological baseline.
[000101] As used herein, "morbidity," is the number of cases of a disease
caused by the
virus, either in excess of zero cases in the past or in excess of a baseline
of endemic cases in the
past. Therefore the baseline of endemic cases, in epidemiological terms, may,
for example,
relate to whether none or some cases were present in a geographic region in
the immediate past.
The past, in epidemiological terms, may mean more than one year and can mean
several years or
more as understood by one of ordinary skill in the art. The past may also mean
less than one
year as determined by one of ordinary skill in the art. In the case of
annually-recurrent common
influenza and seasonal malaria and West Nile virus, for example, the baseline
often reflects an
annual recurrence or expansion and contraction of these diseases.
[000102] As used herein, "expansion" of a pathogen or a population of pathogen
and
"expanding" pathogen or population of pathogen means an increase in virulence,
morbidity,
and/or lethality of a pathogen (e.g., strain of P. falciparum, a strain of
influenza virus, etc.)
and/or an expansion of the population of a pathogen (e.g., strain of P.
falciparum, a strain of
influenza virus, etc.) wherein said expansion includes an increase in the
occurrence of the
pathogen in a given geographic region or in a given time period or both, or a
spreading of the
occurrence of the pathogen to another geographic region.
[000103] As used herein, an increase or decrease in "virulence" includes an
increase or
decrease in virulence, morbidity, lethality, host mortality, and/or expansion
of a pathogen, such
as an influenza virus.
[000104] As used herein, "geographic region" or similar term is an area
differentiated from
another area by space. For example, China is a geographic region that may be
differentiated
from the geographic region of India. Likewise a geographic region may be a
town, or city, or
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continent or any area differentiable from another area. A geographic region
may encompass the
entire earth if an isolate or plurality of isolates from a given time period
is compared to isolates
from another time period over the entire earth and no geographic
differentiation is undertaken for
the comparison.
[000105] As used herein, "conserved" or "conservation" refers to conservation
of particular
amino acids due to lack of substitution.
[000106] As used herein, "Replikin Count" or "Replikin Concentration" refers
to the number
of Replikin sequences per 100 amino acids in a protein, protein fragment,
virus, or organism. A
higher Replikin concentration in a first strain of a virus or organism has
been found to correlate
with more rapid replication of the first virus or organism as compared to a
second, earlier-arising
or later-arising strain of the virus or organism having a lower Replikin
concentration. Replikin
concentration is determined by counting the number of Replikin sequences in a
given sequence
wherein a Replikin sequence is a peptide of 7 to about 50 amino acid residues
with a lysine
residue on one end and a lysine residue or a histidine residue on the other
end wherein the
peptide comprises (1) a lysine residue six to ten residues from another lysine
residue, (2) a
histidine residue, (3) and 6% or more lysine residues, or wherein a Replikin
sequence is a nucleic
acid that encodes a Replikin peptide sequence.
[000107] As used herein, the term "continuous Replikin sequences" means a
series of two or
more Replikin sequences that are overlapped and/or are directly covalently
linked.
Replikin Cycles in Pathogens
[000108] The present invention provides methods of preventing, mitigating, and
treating
outbreaks of a pathogen by predicting an expansion of a strain of pathogen or
an increase in the
virulence, morbidity, and/or lethality of a strain of pathogen as compared to
another strain of the
same pathogen and administering to an animal or patient a compound comprising
an isolated or
synthesized portion of the structure or genome of the pathogen to mitigate,
prevent, or treat the
predicted outbreak of the pathogen. The present invention further provides
methods of
predicting an expanding population of a pathogen or an increase in the
virulence, morbidity,
and/or mortality of a pathogen comprising identifying a cycle in the Replikin
Count in a protein
fragment, protein, genome fragment, or genome of a pathogen and predicting an
expansion of the
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population of the pathogen or an increase in the virulence, morbidity, and/or
mortality of the
pathogen within the identified cycle in Replikin Count.
[000109] An increase in the virulence, morbidity, or mortality of a pathogen
relative to the
virulence, morbidity, and/or mortality of another pathogen of the same species
may be predicted
by identifying a peak in a cycle or cycles in the concentration of Replikin
sequences in the
pathogen and predicting an expansion of the population of the pathogen or an
increase in the
virulence, morbidity, and/or mortality of a pathogen of the same or a related
species isolated
subsequent to the peak. A Replikin cycle is a cycle in the concentration of
Replikin sequences
identified in at least four isolates of a species of virus or organism
isolated at successive times
where (1) the concentration in the first isolate-in-time is higher than the
concentration in the
second isolate-in-time, the concentration in the third isolate-in-time is
higher than the
concentration in the second isolate-in-time, and the concentration of the
fourth isolate-in-time is
lower than the concentration in the third isolate-in-time, or (2) the
concentration in the first
isolate-in-time is lower than the concentration in the second isolate-in-time,
the concentration in
the third isolate-in-time is lower than the concentration in the second
isolate-in-time, and the
concentration of the fourth isolate-in-time is higher than the concentration
in the third isolate-in-
time. Within a Replikin cycle, an increase in virulence, morbidity, and/or
mortality of a
pathogen may be predicted for a pathogen arising during a rising portion of
the cycle or
subsequent to the peak of a cycle. An expanding population may represent an
increase in
population in a region or expansion from one region into another region. In
determining a
Replikin cycle, Replikin Counts may represent individual isolates, or mean
Replikin Counts of
groups of isolates from a given region and/or time period.
[000110] In a further non-limiting embodiment, step-wise cycles may be
identified between
successive time points. In a further embodiment, specific conserved Replikin
sequences are
identified within the step-wise cycles.
[000111] An increase in virulence, morbidity, or mortality of a pathogen may
be determined
using the methods of the invention in any pathogen or infectious agent where a
concentration of
Replikins may be determined in the genome, a genome fragment, another nucleic
acid sequence,
a protein, a protein fragment, or other amino acid sequence from the pathogen.
A pathogen may
be malaria, West Nile virus, foot and mouth disease virus, porcine circovirus,
porcine respiratory
and reproductive syndrome virus, taura syndrome virus, white spot syndrome
virus, tomato leaf
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curl virus, bacillus anthracis, small pox virus, human immunodeficiency virus,
sindbis virus,
hepatitis virus, staphylococcus, legionella, human papilloma virus,
Helicobacter, Acetobacter,
Aerobacter, Brivebacterium, Clostridium, Erinia, Esheria, Klebsiealla,
Maemophilus,
Mycoplasma, Psuedomonas, Salmonella, Candida, Entamoeba, or any other form of
infectious
agent including viruses, bacteria, protozoa, fungi, or other infectious agent.
[000112] Any Replikin sequence, Replikin Peak Gene, or protein fragment
containing a
Replikin sequence or Replikin Peak Gene identified in a strain of pathogen
that is predicted to
have an increase in virulence, morbidity, or mortality may be isolated and/or
synthesized as a
diagnostic, therapeutic, or prophylactic agent to mitigate the predicted
outbreak of the pathogen.
[000113] A cycle of Replikin concentration or "Replikin cycle" of a
trypanosome may be
seen in Figure 1. Cycles of Replikin concentrations in West Nile virus, foot
and mouth disease
virus, and influenza virus may be seen in Figures 3-6, respectively. A
Replikin cycle is
identified by initially isolating at least four isolates or groups of isolates
from at least four time
points or time periods, for example, an isolate or group of isolates may be
obtained in 1999,
2001, 2002, and 2004, or may be obtained in January, May, September, and
December of a given
year. Isolates may be obtained from more than four time points or time periods
and precision of
a Replikin Cycle generally will improve with increases in the number of
isolates per time point
or time period and with increases in the number of time points or time
periods. The Replikin
Count of the genome or expressed proteins of each isolate is determined.
Replikin Count may be
determined in a Replikin Peak Gene, in the entire genome, in a particular gene
or gene segment,
or in a particular protein or protein fragment of each of the isolates. Mean
Replikin Count for a
given time point or given time period is determined if a plurality of isolates
has been obtained for
the given time point or given time period. Replikin Count may then be analyzed
per unit time.
A cycle in Replikin concentration is identified by four time points or time
periods, where the
Replikin Count at a second time point or time period is higher than at first
time point or time
period, the Replikin Count at a third time point or time period is lower than
at second time point
or time period, and Replikin Count at a fourth time point or time period is
higher than at the third
time point or time period; or where the Replikin Count at a second time point
or time period is
lower than at first time point or time period, the Replikin Count at a third
time point or time
period is higher than at second time point or time period, and Replikin Count
at a fourth time
point or time period is lower than at the third time point or time period.
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[000114] A peak in a Replikin cycle is identified within the cycle at a second
time point or
time period within a Replikin cycle, wherein the Replikin concentration at a
first time point or
time period sequentially preceding the second time point or time period is
lower than the
Replikin concentration at the second time point or time period, and the
Replikin concentration at
a third time point or time period sequentially following the second time point
or time period is
lower than the Replikin concentration at the second time point or time period.
One of skill in the
art will understand that because of the variability of biological systems, a
peak may include a
general region of a cycle that is generally higher than a sequentially
preceding region and
generally higher than a sequentially following region rather than an exact
time point or time
period.
[000115] A trough in a Replikin cycle is identified within the cycle is
identified within the
cycle at a second time point or time period within a Replikin cycle, wherein
the Replikin
concentration at a first time point or time period sequentially preceding the
second time point or
time period is higher than the Replikin concentration at the second time point
or time period, and
the Replikin concentration at a third time point or time period sequentially
following the second
time point or time period is higher than the Replikin concentration at the
second time point or
time period. Once again, one of skill in the art will recognize that troughs
may be identified as a
general region of a cycle that is generally lower than a sequentially
preceding region and
generally lower than a sequentially following region rather than an exact time
point or time
period.
[000116] Replikin peptides of the invention identified at a peak of the
Replikin cycle include
Replikin peptides identified at or near the peak of the Replikin cycles
including prior to and
subsequent to the precise point of the peak. A rising portion of a Replikin
cycle is any point at
which the trend of Replikin concentration in the Replikin cycle is increasing
from at least a first
time point or time period to at least a second time point or time period and
can include a peak.
As may be seen in Figures 1-8, an increase in virulence, morbidity, or
mortality may be predicted
following a rising portion or peak in a Replikin cycle.
[000117] In the past, it had been understood that outbreaks of pathogens
correlated with
increases in Replikin Count and that contractions of pathogenic populations
correlated with
decreases in Replikin Count. It was not understood, however, that cycles in
morbidity, mortality,
virulence or population expansion could be directly correlated with cycles in
Replikin Count.
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With the new data presented in the present application, the ordinary skilled
artisan will now
understand, and it is contemplated by the present invention, that entire
Replikin cycles from peak
to trough to peak to trough and/or from trough to peak to trough to peak
correlate with
pathogenic cycles in virulence, morbidity, mortality, and expansion into new
regions or hosts.
As such, the invention now provides methods of tracking and predicting tracks
of pathogens as
they increase in virulence, expand in population within a region or into a
region, or increase in
morbidity or mortality by monitoring changes in Replikin concentration. In the
past, it was not
possible until months after an event to predict or track the course of
pathogens as they increase in
virulence, expand in population within region or into a region, or increase in
morbidity or
mortality where epidemiological data was collected and analyzed post hoc.
Replikins analysis
provides the skilled artisan with information on population expansion, and
increases in virulence,
morbidity, and mortality months before or at the very beginning of an
outbreak. This
information is clearly important for the time needed to organize public health
responses,
including the testing and administration of specific vaccines. The importance
of prior
information concerning pathogenic outbreaks may be analogized to the savings
of life and
property that have resulted from advance warning of hurricanes since
information from weather
satellites has become available.
[000118] For example, in Figure 3 the present application provides data
demonstrating a
cycle of Replikin concentration and a cycle of West Nile virus human morbidity
that are
observed to correlate. In the past, it was understood that Replikin Count data
fluctuate from low
to high over time. This may be seen in 20th century data for the HINT and H3N2
influenza
strains. See Figures 7 and 8. But a correlation of cycles from peak to trough
and/or from trough
to peak was not possible with the earlier data in part because all of the
epidemiological data and
all of the genomic sequence data of the actual number of cases due to the
particular HINT strain
or H3N2 strain were not available or not recorded. Instead, as may be seen in
Figures 7 and 8,
the only data for HINT or H3N2 morbidity in the early- and mid-20th century
related to Replikin
Count was a record of epidemics or pandemics. As is now shown in Figure 3 (as
well as Figures
1, 4, 5, and 6), cycles of Replikin Count correlate with cycles in morbidity
over time and, at
times, over more than one cycle.
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An Expansion Index for Populations of Pathogens
[000119] The present invention also provides a method of predicting an
expansion of a strain
of pathogen by (1) determining a mean Replikin Count and a standard deviation
of the mean
Replikin Count for a plurality of isolates of a strain of pathogen for a first
time period in a first
geographic region; (2) determining a Replikin Count of at least one isolate of
the same or a
related strain of pathogen from a second time period and/or second geographic
region wherein
the second time period is different from the first time period and/or the
second geographic region
is different from the first geographic region; and (3) predicting an expansion
of the strain of
pathogen isolated in the second time period and/or second geographic region,
if the Replikin
Count of the at least one isolate is greater than one standard deviation of
the mean of the
Replikin Count of the plurality of isolates isolated in the first time period
and in the first
geographic region.
[000120] In the above-described method, at least one isolate of the same or
related strain of
pathogen from a second time period and/or second geographic region may be a
plurality of
isolates from the second time period and/or second geographic region. In this
case, the Replikin
Count of each isolate of the plurality of isolates from the second time period
and/or second
geographic region is compared separately to one standard deviation of the
mean. An expansion
of pathogen isolated in the second time period and/or second geographic region
may also be
predicted if the number of Replikin Counts of a plurality of isolates from the
second period
and/or second geographic region that is greater than one standard deviation of
the mean is greater
than the number of Replikin Counts of said plurality of isolates from the
second period and/or
second geographic region that is less than one standard deviation of the mean.
[000121] The method may also employ a ratio of the number of Replikin Counts
that are
greater than one standard deviation of the mean divided by the number of
Replikin Counts that
are less than one standard deviation of the mean. The ratio is called a
Replikin Count Expansion
Index (RCE Index). Another way to determine the RCE Index is to divide the
percent of
Replikin Counts in a plurality of isolates of influenza virus grouped by time
and/or region that
are higher than one standard deviation of the mean by the percent of Replikin
Counts that are
lower than one standard deviation of the mean. An RCE Index may be used to
quantify the
future risk of an outbreak of pathogen by tracking Replikin Counts in strains
of pathogen over
time.
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[000122] In determining a RCE Index, the mean Replikin Count of the plurality
of isolates
from the first time period and geographic region may be considered a control.
A control
population preferably has a relatively large number of isolates with a
relatively small variability
in the Replikin Count of the isolates but any population may be deemed a
control when a
comparison between the control and a related isolate or plurality of isolates
is desired. A control
may be related to the population that is being studied. For example, if an
infection in a bird
species, such as swans, is being studied, the control may be something closely
related, such as
chickens, wherein isolates from chickens may be relatively numerous (if
available) and relatively
stable (if possible) wherein stability in Replikin Count through the
population demonstrates a
level of equilibrium between the expansion and contraction of the strain or a
related strain of
influenza virus in chickens. A control may reflect a highest number of
isolates reported in a year
or in several years in a geographic area.
[000123] An expansion of a strain of pathogen may be determined using the
methods of the
invention in any pathogen or infectious agent where a concentration of
Replikins may be
determined in the genome, a genome fragment, another nucleic acid sequence, a
protein, a
protein fragment, or other amino acid sequence from the pathogen. A pathogen
may be malaria,
West Nile virus, foot and mouth disease virus, influenza virus, porcine
circovirus, porcine
respiratory and reproductive syndrome virus, taura syndrome virus, white spot
syndrome virus,
tomato leaf curl virus, bacillus anthracis, small pox virus, human
immunodeficiency virus,
sindbis virus, hepatitis virus, staphylococcus, legionella, or any other form
of infectious agent
including viruses, bacteria, protozoa, fungi, or other infectious agent.
[000124] Any Replikin sequence, Replikin Peak Gene, or protein fragment
containing a
Replikin sequence or Replikin Peak Gene identified in a strain of pathogen
that is predicted to
have an increase in virulence, morbidity, or mortality may be isolated and/or
synthesized as a
diagnostic, therapeutic, or prophylactic agent to mitigate the predicted
outbreak of the pathogen.
Diagnostics and Therapies Using Replikin Peptides Identified in Replikin
Cycles
[000125] The present invention further provides the opportunity to identify
Replikin
sequences (including nucleic acid sequences and peptide sequences) for
diagnostic, therapeutic,
or preventive purposes (such as the construction of vaccines and other
pharmaceuticals). The
present invention contemplates, for example, Replikin peptides identified
within a pathogen
where the pathogen is predicted to have an expanding population or a higher
virulence,
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morbidity, and/or mortality than another pathogen of the same or a related
species based on the
predictive methods of the invention. Replikin peptides identified in an
isolate of a pathogen,
wherein said isolate is isolated during a rising portion of a cycle in
Replikin concentration among
a plurality of isolates of the pathogen or is isolated at a peak in a cycle in
Replikin concentration
among a plurality of isolates of the pathogen, are useful for diagnostic,
therapeutic, and
preventive purposes. For example, a Replikin peptide identified in the genome
of an isolate
identified in a rising portion of a cycle in Replikin concentration or
identified at a peak in a cycle
in Replikin concentration is useful as a peptide to stimulate the immune
system of a human or
animal to produce an immune response against infection by the pathogen or to
produce
antibodies against a pathogen predicted to have higher virulence, morbidity,
and/or mortality.
One of ordinary skill in the art will recognize that antibodies against these
pathogens are useful
for diagnosing the more highly virulent or mortal disease in a subject or
useful as therapies
against the infection either as a prophylactic or after onset of the
infection.
[000126] Additionally, Replikin peptides identified during a rising portion in
Replikin
concentration in a Replikin cycle or identified at or near a peak in Replikin
concentration in a
Replikin cycle that are conserved during the rising portion of the Replikin
cycle are useful as
compounds for diagnostic, therapeutic, and preventive purposes. Conservation
of the Replikin
peptides during a rise in virulence, morbidity, and/or mortality provides
targets that are more
constant and likely more involved in the mechanisms of rapid replication that
provide the
predicted increase in virulence, morbidity, and/or mortality. As such, these
conserved Replikin
peptides are of use as compounds or in compositions for stimulating the immune
system of a
subject to produce an immune response, an antibody response, and/or a
protective effect in the
subject.
[000127] Replikin peptides identified and isolated using the methods of the
invention include
influenza peptides such as HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1),
KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2), KKNNAYPTIKRTYNNTNVEDLLIIWGIHH
(SEQ ID NO: 3), HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4),
HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH
(SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ
ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID
NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or
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HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12), West Nile virus
peptides such as KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14),
KLTSGHLK (SEQ ID NO: 15), or HNDKRADPAFVCK (SEQ ID NO: 16), and foot and mouth
disease peptides such as HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO:
18).
[000128] Identification of portions of a pathogen (such as Replikin Peak Genes
or Replikin
peptides) predicted to expand in population provide unique compounds for
diagnostics and
treatment of expanding pathogens, wherein the unique compounds would otherwise
not be
identifiable but for the methods of the invention and the compounds disclosed
herein.
[000129] The invention further contemplates use of the Replikin peptide as
immunogenic
compositions and contemplates the immunogenic compositions as vaccines,
including vaccines
that provide an immune response, vaccines that provide a Immoral immune
response, vaccines
that provide an antigenic immune response, and vaccines that provide a
protective effect. The
invention additionally contemplates an antibody to the Replikin peptides of
the invention.
[000130] High Replikin Counts and RPGs have been shown to be related to rapid
replication,
viral outbreaks, epidemics, morbidity and host mortality, for example, in
influenza virus strains,
including H5N1, in SARS coronavirus, in shrimp taura syndrome virus, and in
foot and mouth
disease virus. Replikin sequences identified at or near the peak of the
Replikin cycle or during a
rising portion of the Replikin cycle in a pathogen are appropriate peptides
for diagnostics,
vaccines, and other treatments.
[000131] Because Replikin sequences are chemically defined, the sequences may
be
synthesized by organic chemistry rather than biological techniques, and thus
are potentially more
specific, more reproducible and more reliable. The chemically defined Replikin
sequences
identified by Applicants are likewise potentially freer from adverse reactions
that are
characteristic of biologically derived vaccines and antibodies.
Mitigating and Treating Outbreaks of Pathogen with Cyclic Replikin Counts
[000132] One aspect of the present invention provides methods of preventing,
mitigating, or
treating pathogenic outbreaks predicted through analysis of cycles of Replikin
Counts or through
analysis of controls using mean Replikin Counts and standard deviation (e.g.,
Replikin Count
Expansion Index). For example, advance information concerning Replikin
peptides and Replikin
Peak Genes in expanding strains of a pathogen allows for the rapid production
of specific
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effective synthetic vaccines using one, or a combination, of Replikin peptides
or using Replikin
Peak Genes. Such synthetic vaccines have been demonstrated in rabbits,
chickens, and shrimp.
See, e.g., Examples 6 and 7 of U.S. Appln. Ser. No. 11/355,120, filed February
16, 2006 and
Example 2 of U.S. Appln. Ser. No. 12/108,458, filed April 23, 2008. For
example, a mixture of
Replikin peptides administered orally to shrimp provided up to a 91 %
protective effect for
shrimp challenged with taura syndrome virus. Taura syndrome virus is an often
lethal rapidly-
replicating pathogen that has a significant negative impact on the shrimp
industry.
[000133] Synthetic Replikin vaccines have also been demonstrated in the H5N1
strain of
influenza virus in chickens. For example, in a test of chickens administered a
mixture of twelve
H5N1 Replikin peptides from the hemagglutinin and pB1 gene areas intranasally,
intraocularly,
and by spray inhalation and challenged with low pathogenic H5N1 influenza
isolated from a
black duck in the state of North Carolina in the United States, a protective
effect was observed at
both the entry site of influenza (diminished antibody production in the mucus
was observed as
compared to a control) and at excretion sites of influenza (influenza virus
was not observed
excreted in feces or saliva from treated chickens as compared to a control).
See Example 10
below.
[000134] Administration of Replikin peptides in both shrimp and chickens
appears to have
provided a notable measure of mucosal immunity. For example, in Example 2 of
U.S. Appln.
Ser. No. 12/108,458, a mixture of Replikin peptides was administered by mouth
to shrimp later
challenged with taura syndrome virus. The 91 % protective effect of the
vaccine is expected to
have been a result, at least in part, of a mucosal immune-like responses in
the gut of the shrimp.
[000135] Likewise, in chickens, the administration of a mixture of Replikin
peptides provided
a protective effect against entry of the H5N1 virus. For example, as may be
seen in Example 10
below, three of six vaccinated chickens, when inoculated with H5N1 virus,
produced no
measurable amount of antibodies against H5N1 in their serum. Instead, the
virus was apparently
blocked by mucosal immunity from even entering the chickens' blood stream. For
those three
chickens in which a serum immune response was measured (that is, virus entered
the host and
was presented to antibody generating cells), the vaccine additionally provided
a protective effect
against replication of the virus in the chickens' system (no virus was
excreted in the feces or
saliva of the chickens). As such, mucosal immunity, in addition to other
immunities, is an
important aspect of the immunity imparted by Replikin-based vaccines.
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[000136] Cyclic increases in Replikin concentration in the genome can be a
mechanism of
expansion of a pathogen into a territory. The Replikin concentration in each
Replikin Peak Gene
of each Replikin cycle in an expanding population apparently may build on the
previous one.
Timely, repeated analyses of cyclic changes in a virus' Replikin structure is
useful to bring
current the targets for the chemical synthesis of Replikin vaccines having a
best fit for emerging
pathogens having increased virulence, morbidity, and/or lethality. These
strain-specific vaccines
may be manufactured in seven days as have been demonstrated with a 91 %
protection of shrimp
against the lethal taura syndrome virus. See, e.g., U.S. Appln. Ser. No.
12/108,458, filed April
23, 2008 (incorporated herein in its entirety by reference).
Replikin Cycles in Malaria
[000137] The present invention provides methods of predicting an expansion of
the
population of a trypanosome that causes malaria or an increase in the
virulence, morbidity,
and/or mortality of a trypanosome that causes malaria as compared to another
trypanosome of
the same species or a related species. An expanding population or increase in
virulence,
morbidity, and/or mortality of a trypanosome that causes malaria may be
predicted by identifying
a cycle of Replikin concentration among a plurality of isolates of the species
of trypanosome and
identifying a rising portion or peak in that cycle. An increase in virulence,
morbidity, and/or
mortality is predicted following the time point or time period when the rising
portion or peak is
identified. An expanding population may represent an increase in population in
a region or
expansion from one region into another region.
[000138] A further non-limiting embodiment of one aspect of the invention
provides a
method of predicting an increase in morbidity and mortality in malaria
comprising: (1)
determining the mean Replikin Count in a plurality of isolates of a malarial
trypanosome at a
plurality of successive time points; (2) comparing the mean Replikin Count at
at least four
successive time points and identifying at least one cycle of increasing mean
Replikin Counts
over the at least four time points; and (3) predicting an increase in
morbidity and/or mortality
following in time the increase in mean Replikin count in at least one of said
cycles. In a further
non-limiting embodiment, step-wise cycles are identified between successive
time points. In a
further non-limiting embodiment, specific conserved Replikin sequences are
identified within the
step-wise cycles. In a further non-limiting embodiment, Replikin sequences are
identified at the
peak of a step-wise cycle. The Replikin sequences identified at the peak of a
step-wise cycle are
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useful for developing a vaccine or therapeutic composition of an isolated or
synthesized Replikin
peptide for use in preventing or treating outbreaks of malaria with relatively
higher mortality.
[000139] Figure 1 illustrates cycling between 1986 and 2007 of annual mean
Replikin
concentration in the histidine rich protein of Plasmodium falciparum. P.
falciparum is a
trypanosome that is most commonly associated with malaria. Cycles are
observable with peaks
in 1987 and 1999. A new cycle appears to have begun between 2005 and 2007.
Publicly
available accession numbers at www.pubmed.com containing amino acid sequence
listings for P.
falciparum were queried using the automated F1uForecast software (Replikins,
Ltd., Boston,
MA). The software analyzed the Replikin Count of each available sequence
between 1986 and
2007. The area of the P. falciparum genome observed to have the highest
concentration of
continuous Replikin sequences per 100 amino acids was found to be the
histidine rich protein.
The histidine rich protein includes the knob-associated histidine rich
protein.
[000140] Analysis of the mean annual Replikin Count of the histidine rich
protein between
1986 and 2007 revealed cycles of Replikin Count. A first rising portion
followed by a
decreasing portion of the cycle was observed from 1986 to 1995. A second
rising portion
followed by a decreasing portion was observed from 1996 to 2005. A first peak
was identified in
1987 with a mean annual Replikin Count of 38.2 and standard deviation of
23.5. A second
peak was identified in 1999 with an even higher mean annual Replikin Count of
62.9 and
standard deviation of 62.9 (overlap of Replikin sequences within an amino
acid sequence
generates a Replikin Count of greater than 100 Replikin sequences per 100
amino acids in some
sequences). Both the 1987 peak and the 1999 peak were observed to be related
to higher human
mortality. Following the 1999 peak, mean annual Replikin Counts were observed
to fall to a low
of 7.4 in 2005 with a standard deviation of 6.5. Mortality rates likewise
fell between 2000 and
2005. A third malaria Replikin cycle appears to have begun in 2005 with the
observed mean
annual Replikin Count increasing from 7.4 6.5 in 2005 to 17.2 19 in 2007. The
beginning of a
new cycle provides a prediction that Replikin Count may continue to increase
along with an
increase in malaria mortality rate.
[000141] The cycling observable in Figure 1 has also been observed in viruses,
namely, the
H1N1, H2N2, H3N3, H5N1, H3N8, and H9N2 strains of influenza virus, in West
Nile virus, and
in foot and mouth disease virus. See Figures 1-8. Thus Replikin cycles are
observable in both
viruses and organisms. Similar correlations between Replikin Count and
mortality have also
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been shown in an influenza H5N1 cycle between 1997 and 2007. See, e.g., U.S.
Appln. Ser. No.
12/010,027, filed January 18, 2008 (Figure 8).
[000142] The data for Figure 1 are seen in Table 1 below. Mean annual Replikin
Count,
standard deviation, significance of annual mean Replikin Count to the lowest
annual mean
Replikin Count and to the previous annual mean Replikin Count, and number of
accession
numbers analyzed per annum is provided.
Table 1
P. falciparum Replikin Count
Number of
Mean Significance Accession
Replikin Standard (compared to (compared Significedc to
o Records for
Year Count Deviation lowest value) previous year) malaria isolates
1986 15.9 15.2 low>0.5 6
1987 38.2 23.5 low<0.005 <0.02 11
1988
1989 13.9 0 <0.005 1
1990 5.2 0 1
1991
1992 13 18.2 >0.5 <0.2 9
1993
1994
1995 2.4 0 <0.1 1
1996 8.7 1.2 <0.01 <0.01 3
1997
1998 24.1 16.7 <0.01 <0.04 7
1999 62.9 62.9 <0.2 <0.24 4
2000 33.3 24.7 <0.3 <0.4 3
2001
2002 18 29 >0.5 <0.3 13
2003 28.4 3 <0.001 <0.2 7
2004 17 0 <0.001 1
2005 7.4 6.5 <0.05 <0.02 5
2006
2007 17.2 19 >0.5 <0.2 8
[000143] As is seen in Figure 1 and Table 1 above and as also seen in Figure 2
and Table 6
below, changes in malaria virulence and mortality may be predicted by
identifying a peak within
an identified cycle in the Replikin concentration of isolates of a plurality
of the trypanosome and
predicting an increase in the virulence, morbidity, and/or mortality of a
trypanosome of the same
species isolated at a time point or time period subsequent to the time point
or time period of the
identified peak in the cycle of Replikin concentration. In contrast to Figures
3, 7, and 8 for West
Nile virus and influenza, morbidity data is not reflected in the analysis of
malaria in Figure 1 and
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is also not contained in Figure 2, which compares Replikin Count in the ATP-
ase protein of P.
falciparum to mortality. Use of mortality data and not morbidity data in
Figures 1 and 2 and
their related analysis and tables is based on the skilled artisan's
understanding that morbidity
data in malaria is generally unreliable while mortality data is considered
more reliable. While
the analysis of Figure 1 and the data in Figure 2 demonstrate a relationship
between Replikin
Count in P. falciparum and mortality, the skilled artisan will understand that
the relationship
would also be expected to extend to morbidity and general virulence in malaria
just as it has in
West Nile virus (see Figure 3), foot and mouth disease (see Figure 4), and
influenza (see Figures
5-8).
[000144] Cyclic increases in Replikin concentration in the genome can be a
mechanism of
expansion of an infectious organism into a territory. The Replikin
concentration in each Replikin
Peak Gene of each Replikin cycle apparently builds on the previous one. In
both the mosquito-
borne West Nile Virus and mosquito-borne malaria trypanosomes, this build-up
probably occurs
during winter seasons, dry seasons, or otherwise dormant periods. Timely,
repeated analyses of
cyclic changes in the organism's Replikin structure is useful to bring current
the targets for the
chemical synthesis of Replikin vaccines having a best fit for emerging
pathogens having
increased virulence, morbidity, and/or mortality. These strain-specific
vaccines may be
manufactured in seven days as has been demonstrate with a 91 % protection of
shrimp against the
lethal taura syndrome virus. See, e.g., U.S. Appln. Ser. No. 12/108,458, filed
April 23, 2008
(incorporated herein in its entirety by reference).
[000145] The Replikin cycle may be identified in any trypanosome that causes
malaria. For
example, it may be identified in the genome of a trypanosome, including P.
falciparum,
Plasmodium vivax, Plasmodium ovate, or Plasmodium malariae. The Replikin cycle
may
likewise be identified in the histidine rich protein or in the ATP-ase
protein, including in these
proteins in P. falciparum. The Replikin cycle may likewise be identified in a
Replikin Peak
Gene of a trypanosome that causes malaria.
[000146] Malaria trypanosomes have been found to have the highest Replikin
counts seen to
date in any infectious organisms-up to twenty times those in influenza and
West Nile Virus.
Consistent with these high counts, trypanosomes have one of the highest
replication rates in
nature. This property may account in part for the resistance of malaria to
previous attempts at
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vaccination. The discovery of the relation of Replikin sequences to rapid
replication offers a
new approach, and means, to inhibit rapid replication in malaria.
[000147] In the data analysis reported in Tables 1 and 5, as well as Figure 1,
Replikin
sequences were identified as conserved sequences in the histidine-rich protein
of malaria in the
rising portion and peak of the illustrated Replikin cycle. Such sequences are
useful as diagnostic
and therapeutic compounds for virulent malaria infections. The sequences are
useful in the
production of immunogenic compounds including vaccines and may be comprised in
immunogenic therapies including vaccines.
[000148] For example, Replikin peptides identified in the ABU43157 isolate in
2007 are
available as a diagnostic, therapeutic, or preventive compounds or
compositions of the invention
because they were identified in a rising portion of a Replikin cycle. See
Figure 1 and Table 5.
Replikin peptides identified in the 1999 isolate at accession number CAD49281
are likewise
Replikin peptides of the invention. The 1999 isolate is present at the peak of
a Replikin cycle, as
such, Replikin peptides identified in the isolate reported at CAD49281 may be
used as
immunogenic compounds. Additionally, the 1998 accession number XP001349534 is
identified
as from an isolate from a rising portion in a Replikin cycle. Replikin
peptides identified in
XP001349534 are likewise useful as immunogenic compounds or vaccines or for
diagnosis or
treatment of malaria. See Figure 1 and Table 5 for all accession numbers
discussed in this
paragraph.
Replikin Count Cycles in West Nile Virus
[000149] In a further aspect of the invention, an expanding population of West
Nile virus or
an increase in virulence, morbidity, or morality of West Nile virus may be
predicted by
identifying a cycle of Replikin concentration in isolates of West Nile virus
and predicting an
expanding population of virus or an increase in virulence, morbidity, and/or
mortality of West
Nile virus following a rising portion, or peak in the cycle of Replikin
concentration. An
expanding population may represent an increase in population in a region or
expansion from one
region into another region.
[000150] For example, using analysis of Replikin sequences in West Nile virus,
including, for
example, analysis of Replikin sequences in the envelope protein of West Nile
virus, a correlation
between virus biochemical cycles and virus virulence, morbidity, and/or
mortality cycles may be
identified and used to predict expansions in a virus population or increases
in virulence,
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morbidity, and/or mortality in a virus in a host population. A non-limiting
embodiment of the
aspect of the invention provides a method of predicting an increase in
morbidity in a viral disease
such as West Nile virus comprising: (1) determining the mean Replikin Count in
genomes of a
plurality of isolates of a virus at a plurality of successive time points; (2)
comparing the mean
Replikin Count at at least four successive time points and identifying at
least two peaks or two
troughs in the trend of Replikin Counts over the at least four time points;
and (3) predicting an
increase in morbidity following in time the increase in mean Replikin count
within said cycles.
In a further non-limiting embodiment, step-wise cycles are identified between
successive time
points. In a further embodiment, specific conserved Replikin sequences are
identified within the
step-wise cycles.
[000151] Table 2, below, provides data from analysis of envelope protein
sequences in West
Nile virus available at www.pubmed.com for isolates from 2000 through 2007.
The data, which
are illustrated in Figure 3, provide an example of cycling in mean annual
Replikin Count in a
virus wherein the cycle predicts morbidity. The data additionally further
support immunogenic
compounds, diagnostic compounds, and, among other things, vaccines 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
pathogenic diseases,
the correlation of Replikin Count with pathogenicity generally, and targeting
of the Replikin
structures for control of rapid replication and disease generally. See, e.g.,
U.S. Appln. Ser. No.
11/355,120, filed February 16, 2006 and U.S. Appln. Ser. No. 12/010,027, filed
January 18, 2008
(each incorporated herein by reference in their entirety).
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Table 2
Mean Annual Replikin Count in West Nile Virus Envelope Protein
Year No. of Mean S.D. Significance
Isolates per Replikin
year Count per
year
2000 4 2.9 0.1 low <0.001, rev <0.001
2001 130 3.6 2.0 low <0.02, rev <0.001
2002 18 4.7 1.5 low <0.001, rev <0.005
2003 94 5.3 1.5 low <0.001, rev <0.05
2004 55 4.2 1.7 low <0.001, rev <0.001
2005 125 4.3 1.8 low <0.001, rev >0.50
2006 312 6.0 1.3 low <0.001, rev <0.001
2007 (Incomplete) (Incomplete) (Incomplete) (Incomplete) low p<0.001,
27 4.6 1.2 rev p<0.001
2008 (Incomplete) (Incomplete) (Incomplete) (Incomplete) low p<0.002,
5.5 0.7 rev <0.04
[000152] In Figure 3 and Table 2, cycles in mean Replikin Count in isolates of
West Nile
virus 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
(available through
Replikins LLC, Boston, MA) (see, e.g., U.S. Appln. Ser. No. 11/116,203, filed
April 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.
[000153] Immunogenic compounds for therapeutic vaccines against West Nile
virus include,
for example, KIIQKAHK (SEQ ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK
(SEQ ID NO: 15), and HNDKRADPAFVCK (SEQ ID NO: 16). These Replikin peptide
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sequences are conserved within the step-wise cycles of West Nile virus in
Figure 3, which render
them of particular use for therapies against expanding West Nile virus
populations following the
cyclic peaks identified in Figure 3. The sequences may be administered to
animals or humans as
a vaccine. A vaccine may comprise a pharmaceutically acceptable carrier and/or
adjuvant. A
vaccine can be manufactured within seven days of the identification of
sequences, such as these,
that are conserved in step-wise cycles identified in the Replikin Count of a
pathogen such as
West Nile virus. The sequences may likewise be used for diagnostic purposes to
identify isolates
of the expanding population of West Nile virus.
Replikin Count Cycles in Foot and Mouth Disease Virus
[000154] In a further aspect of the invention, an expanding population of foot
and mouth
disease virus or an increase in virulence, morbidity, or mortality of West
Nile virus may be
predicted by identifying a cycle of Replikin concentration in isolates of foot
and mouth disease
virus and predicting an expanding population of virus or an increase in
virulence, morbidity,
and/or mortality of virus following a rising portion, or peak in the cycle of
Replikin
concentration. An expanding population may represent an increase in population
in a region or
expansion from one region into another region.
[000155] For example, using analysis of Replikin sequences in foot and mouth
disease virus,
including, for example, analysis of Replikin sequences in the VP 1 protein of
foot and mouth
disease virus, a correlation between virus biochemical cycles and virus
virulence, morbidity,
and/or mortality cycles may be identified and used to predict expansions in a
virus population or
increases in virulence, morbidity, and/or mortality in a virus in a host
population. A non-limiting
embodiment of the aspect of the invention provides a method of predicting an
increase in
morbidity in a viral disease such as foot and mouth disease virus comprising:
(1) determining
the mean Replikin Count in genomes of a plurality of isolates of a virus at a
plurality of
successive time points; (2) comparing the mean Replikin Count at at least four
successive time
points and identifying at least two peaks or two troughs in the trend of mean
Replikin Counts
over the at least four time points; and (4) predicting an increase in
virulence and/or morbidity
following in time an increase in mean Replikin count within a cycle. In a
further non-limiting
embodiment, step-wise cycles are identified between successive time points. In
a further
embodiment, specific conserved Replikin sequences are identified within the
step-wise cycles.
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[000156] Increased Replikin Counts provide advance warnings of Foot and Mouth
Disease
outbreaks and the basis of a conserved synthetic FMDV Vaccine. One aspect of
the invention
contemplates provision of advance warning of outbreaks of FMDV by identifying
cycles in the
Replikin Count of isolates of FMDV over time. As may be seen from the data in
Figure 4, in
2000, an outbreak of Foot and Mouth Disease Virus (Type 0) (FMDV) was
predicted by a peak
in annual mean Replikin Count. An outbreak in 2001-2002 was observed in the
United Kingdom
and in the Netherlands. In a new cycle beginning in 2005, the highest Replikin
counts in ten
years, observed in 2007 and 2008, were followed by severe FMDV outbreaks in
2008 and 2009
in the Middle East, Africa, India, China, and other Asian countries. Replikin
peptide structures
found to be conserved over decades are now the basis of a synthetic Replikins
vaccine for
FMDV. Replikin sequences identified as conserved within the Replikin cycles in
Figure 4
include HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18). These
sequences are also observed to be conserved over time in isolates of foot and
mouth disease type
A.
[000157] Figure 4 illustrates cycles of Replikin Count in Type 0 isolates of
FMDV. The
data illustrated in Figure 4 are contained in Table 3.
Table 3
Foot and Mouth Disease Virus Protein Replikin Cycles
Year Mean Replikin Count of Standard
Reported FMDV Isolates in Deviation
Listed Year
1999 0.9 0
2000 2 0.4
2001 1.4 0.8
2002 1.8 0.6
2003 1 0.2
2004 1.5 0.9
2005 0.9 0
2006 0.9 0
2007 2 0.8
2008 3.2 0.3
[000158] The data in Table 3 and Figure 4 illustrate that the annual Replikin
Counts (Mean
and Standard Deviation (SD)) in Foot and Mouth disease virus occurred in two
rising portions
and a decreasing portion. The first rising portion followed by the first
decreasing portion occurs
from 1999-2005 and the second rising portion occurs from 2005-2008. Increases
in Replikin
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Counts provided advance warning signals (p<O.001) prior to severe FMDV
outbreaks in the U.K.
and the Netherlands in 2001-2002, and in the Middle East, Africa, India, and
Asia in 2008-2009.
[000159] Replikin peptides (1) were identified and counted automatically, with
tests of
statistical significance of changes, using a software program
(ReplikinsForecastTM Replikins
LLC, Boston, MA) designed to analyze the protein sequences of any organism, in
this case
FMDV published in PubMed. When the history of each Replikin structure in the
virus was
tracked for its occurrence in each virus specimen in each of the years for
which virus sequence
data was published, conservation of Replikin structures for decades was found.
The structure of
these conserved Replikins is the basis of synthetic Replikins vaccines for
FMDV.
[000160] Replikin peptides conserved in FMDV over decades include HKQKIIAPAK
(SEQ
ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18). Sequences identified within
Replikin cycles
and as conserved within Replikin cycles are particularly useful for diagnostic
and therapeutic
purposes. For example, the sequences identified as new and/or conserved in
FMDV Replikin
cycles are useful for (1) designing and chemically synthesizing vaccines that
contain both older
conserved Replikins as well as newer ones to provide the most accurate and
maximal anti-
organism immune stimulating properties, (2) designing and chemically
synthesizing antibodies
that contain reactive sites against both older conserved Replikins and newer
ones to provide the
most accurate and maximal anti-organism immune protective properties, and (3)
designing and
chemically synthesizing compounds that contain reactive sites against both
older conserved
Replikins and newer ones to provide the most accurate and maximal anti-
organism protective
properties.
Predicting Expansion of Populations of Influenza Virus
[000161] One aspect of the present invention provides methods of predicting an
outbreak of
influenza by predicting an increase in the virulence, morbidity, and/or
lethality of a strain of
influenza virus or an expansion of the population of a strain of influenza
virus using a Replikin
Count Virus Expansion Index. In this aspect of the invention, an increase in
virulence,
morbidity, and/or lethality or an expansion of a strain of influenza virus is
predicted by (1)
determining a mean Replikin Count and a standard deviation from the mean
Replikin Count for a
plurality of isolates of a strain of influenza virus for a first time period
in a first geographic
region, (2) determining a Replikin Count of at least one isolate of the same
or a related strain of
influenza virus from a second time period and/or a second geographic region
different from the
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first time period and/or the second geographic region, and (3) predicting an
increase in virulence,
morbidity, and/or lethality or an expansion of the strain of influenza
isolated in the second time
period and/or second geographic region, if the Replikin Count of the at least
one isolate from a
second time period and/or a second geographic region is greater than one
standard deviation of
the mean of the Replikin Count of the plurality of isolates isolated in the
first time period in the
first geographic region.
[000162] In the above-described method, at least one isolate of the same or
related strain of
influenza virus from a second time period and/or second geographic region may
be a plurality of
isolates from the second time period and/or second geographic region. In this
case, the Replikin
Count of each isolate of the plurality of isolates from the second time period
and/or second
geographic region is compared separately to one standard deviation of the
mean.
[000163] An expansion of influenza isolated in the second time period and/or
second
geographic region may also be predicted if the number of Replikin Counts of a
plurality of
isolates from the second period and/or second geographic region that is
greater than one standard
deviation of the mean is greater than the number of Replikin Counts of said
plurality of isolates
from the second period and/or second geographic region that is less than one
standard deviation
of the mean.
[000164] The method may also employ a ratio of the number of Replikin Counts
that are
greater than one standard deviation of the mean divided by the number of
Replikin Counts that
are less than one standard deviation of the mean. The ratio is called a
Replikin Count Virus
Expansion Index (RCVE Index). Another way to determine the RCVE Index is to
divide the
percent of Replikin Counts in a plurality of isolates of influenza virus
grouped by time and/or
region that are higher than one standard deviation of the mean by the percent
of Replikin Counts
that are lower than one standard deviation of the mean. An RCVE Index may be
used to
quantify the future risk of an outbreak of influenza by tracking Replikin
Counts in strains of
influenza over time.
[000165] In determining a RCVE Index, the mean Replikin Count of the plurality
of isolates
from the first time period and first geographic region is considered a
control. A control
population preferably has a relatively large number of isolates with a
relatively small variability
in the Replikin Count of the isolates, but any population may be deemed a
control when a
comparison between the control and a related isolate or plurality of isolates
is desired. A control
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may be related to the population that is being studied. For example, if
influenza infection in a
bird species, such as swans, is being studied, the control may be something
closely related such
as chickens, wherein isolates from chickens may be relatively numerous (if
available) and
relatively stable (if possible) and wherein stability in Replikin Count
through the population
demonstrates a level of equilibrium between the expansion and contraction of
the strain or
related strain of influenza virus in chickens. A control may reflect a highest
number of isolates
reported in a year or in several years in a geographic area. As may be seen in
Figure 3, Influenza
B may be a model control during the 20th century for influenza strains because
both Replikin
Count and morbidity in all hosts are remarkably stable throughout some 40
years with a
relatively small standard deviation and no lethal outbreaks recorded. In
influenza B, Replikin
Count and replication rate appear to be just sufficient to balance losses for
steady survival of the
species. This is in contrast to H2N2, which disappeared at the end of the
century after dropping
Replikin Counts less than one standard deviation of the mean with no Replikin
Counts greater
than one standard deviation of the mean to balance the survival of the strain.
[000166] In determining an RCVE index, any measure of Replikin concentration
may be used
in influenza or in other pathogens. Replikin Count may reflect the
concentration of Replikin
peptides identified encoded in the genome of an isolate. Replikin Count may
also reflect the
concentration of Replikin peptides identified in the expressed proteins of an
isolate or in at least
one protein or protein fragment of an isolate. Replikin Count may also reflect
the concentration
of Replikin peptides identified in a Replikin Peak Gene of an isolate. The
Replikin Peak Gene of
an influenza virus may be any segment of the genome or of any expressed
protein or protein
fragment having the highest concentration of continuous and/or overlapping
Replikin peptides
identified.
[000167] In many influenza isolates the Replikin Peak Gene is identified in
the polymerase
area of the influenza virus genome. Within the polymerase area, the Replikin
Peak Gene is often
identified in the pB1 gene area. Replikin Counts within the pB1 gene may also
be used.
[000168] Any Replikin peptide, Replikin Peak Gene, protein, protein fragment,
or nucleic
acid sequence encoding any Replikin peptide, Replikin Peak Gene, protein, or
protein fragment
in an isolate predicted by the methods of the invention to be expanding may be
used for
diagnostic, therapeutic, and/or preventive purposes. Further, a vaccine may be
manufactured by
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identifying a portion of the structure or genome of an influenza isolate
predicted to expand in
population and using that portion in a vaccine composition.
[000169] Methods of the invention also provide methods of predicting a
decrease in
virulence, morbidity, and/or lethality of a strain of influenza and/or
predicting a contraction or
failure of a strain of influenza wherein a Replikin Count of at least one
isolate of a strain of
influenza from a second time period and/or second geographic region is less
than one standard
deviation of the mean of the Replikin Count of a plurality of isolates of
influenza from a first
time period and first geographic region. A decrease may also be predicted
where the number of
Replikin Counts of a plurality of isolates from a second period and/or a
second geographic
region that are greater than one standard deviation of the mean is less than
the number of
Replikin Counts less than one standard deviation of the mean. A decrease,
contraction, or failure
is predicted if the ratio of the Replikin Count Virus Expansion Index is less
than one.
[000170] When a population contains isolates with Replikin Counts above one
standard
deviation of the mean of a control and does not contain isolates with Replikin
Counts below one
standard deviation of the mean of the control, the ratio of the RCVE Index is
considered to have
a denominator of one to avoid an index of infinity.
[000171] In determining a Replikin Count Virus Expansion Index, Replikin
Counts from
Replikin Peak Genes may be analyzed from regions (such as all reporting
countries) in a given
time period (such as a year) for a range of species. Within a country in a
year, there may be a
range of values over a range of species. The ordinary skilled artisan may
select a mean Replikin
Count as a control from the range of values, a time, a region, a species, or
any combination
thereof (such as a time, a region, and a species, e.g., 2004, China, and
chicken). For example, in
Example 7 below, the mean Replikin Count of all H5N1 isolates from chickens in
China in 2004
was selected as an initial control against which Replikin Counts from swans in
China in 2004
were compared. When comparing a control to the Replikin Count of an individual
isolate or
related group of isolates, a control that shares some similarity with the
isolate or group of isolates
may be used. For example, a control of all isolates from chicken in China in
2004 may be
compared with other isolates from 2004. Likewise, a control of swans from 2005
in Japan may
be compared to future isolates from swans in Japan. The ordinary skilled
artisan will understand
when a control shares similarity with an isolate or group of related isolates
such that the control
may be used in comparison with the isolate or group of related isolates.
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[000172] When comparing the Replikin Count of an individual isolate or related
group of
isolates to a control, all Replikin Count values within the group of related
isolates that fall within
one standard deviation of the mean may be treated as a group. Additionally,
all values that fall
outside the range of one standard deviation from the mean may be treated as
two outlying
groups. A first group is the group of Replikin Counts that are greater than
the mean plus one
standard deviation. A second group is the group of Replikin Counts that are
less than the mean
minus one standard deviation. Because higher Replikin Counts are associated
with future
outbreaks or an expanding virus population and lower Replikin Counts are
associated with
cessation of outbreaks or decrease or failure of the virus population, the
ratio of the percent of
isolates having Replikin Counts above mean plus one standard deviation to the
percent of
isolates having Replikin Counts below the mean minus one standard deviation
provides a
quantitative index of the viability and expansion of the virus. The index
provides a snapshot of
current status of the virus population and the propensity for change in that
population. If the
ratio is greater than one, the RCVE Index predicts an expanding population. If
the ratio is less
than one, the RCVE Index predicts a contracting or failing virus population.
Mitigating and Treating Expanding Populations of Influenza Virus
[000173] One aspect of the present invention provides methods of preventing or
treating
outbreaks of influenza virus by predicting an expansion of a strain of
influenza virus using a
Replikin Count Virus Expansion Index and administering therapies comprising an
isolated or
synthesized portion of the structure or genome of the influenza virus
identified using the RCVE
Index to prevent, mitigate, or treat the outbreak of influenza virus. A
prediction of an outbreak
may be made by (1) determining a mean Replikin Count with standard deviation
for a group of
isolates of a strain of influenza isolated during a first time period in a
first geographic region, (2)
determining a Replikin Count of at least one isolate of the same strain of
influenza virus from a
second time period and/or second geographic region that is different from the
first time period
and/or is different from the second geographic region, and (3) predicting an
expansion of the
strain of influenza isolated in said second time period and/or second
geographic region if the
Replikin Count of the isolate from a second time period and/or second
geographic region is
greater than one standard deviation from the mean of the Replikin Count of the
plurality of
isolates isolated in the first time period and in the first geographic region.
An outbreak may be
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prevented, mitigated, or treated by administering a pharmaceutical compound
that includes all or
some portion of the structure or genome of the at least one isolate of
influenza virus.
[000174] The at least one isolate of influenza from a second time period
and/or geographic
region may be a plurality of isolates from the second time period and/or
second geographic
region wherein the Replikin Count of each isolate of the plurality of isolates
is compared
separately to one standard deviation from the mean. Additionally, an outbreak
of influenza may
be predicted if the number of Replikin Counts of the plurality of isolates
from a second period
and/or a second geographic region that is greater than one standard deviation
of the mean is
greater than the number of Replikin Counts less than one standard deviation of
the mean.
[000175] The portion of the structure or genome may be isolated from an
influenza isolate or
may be synthesized based on sequences or other structure elucidated from the
influenza isolate as
well understood by the ordinary skilled artisan. The structure may be a
protein or protein
fragment that comprises a Replikin peptide or that consists of a Replikin
peptide. The structure
may comprise or consist of a Replikin Peak Gene or a fragment of a Replikin
Peak Gene or may
consist of a Replikin peptide identified within a Replikin Peak Gene. The
structure may also be
a nucleic acid including but not limited to a nucleic acid encoding a Replikin
Peak Gene, a
Replikin peptide or plurality of Replikin peptides within a Replikin Peak
Gene, or a Replikin
peptide or plurality or Replikin peptides.
[000176] A peptide or mixture of peptides may be comprised in an immunogenic
compound
for influenza and may include at least one of HAQDILEKEHNGKLCSLKGVRPLILK (SEQ
ID
NO: 1), KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2),
KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3),
HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4),
HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5), KVRLQLRDNAKELGNGCFEFYH
(SEQ ID NO: 6), KDVMESMDKEEMEITTH (SEQ ID NO: 7), HFQRKRRVRDNMTKK (SEQ
ID NO: 8), KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9), HKRTIGKKKQRLNK (SEQ ID
NO: 10), HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); or
HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12).
Synchronous Replikin Cycles in H9N2 and H5N1 Strains of Influenza
[000177] Another aspect of the invention provides methods of predicting an
increase in the
virulence, morbidity, and/or lethality or an expansion of the population of an
isolate of a strain of
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influenza virus as compared to another isolate or group of isolates of the
same or a related strain.
Such an increase may be predicted by identifying a cycle of Replikin
concentration among a
plurality of isolates of influenza and identifying a peak in that cycle. An
increase is predicted
following the time point or time period when the peak is identified or
following a rising portion
of the cycle. An increase may likewise be predicted following the time point
or time period
when a peak is identified in two synchronous cycles wherein a first cycle is
the cycle of a strain
of influenza and the second cycle is a cycle of a different strain of
influenza. The increase is
predicted following the time period in which the peaks of the synchronous
cycles are identified
or in a rising portion identified in both synchronous cycles.
[000178] A cycle of Replikin concentration or "Replikin cycle" of H9N2 may be
seen in
Figure 5. A comparison of synchronized cycles of Replikin concentration in
H5N1 and H9N2
may be seen in Figure 6. The synchronized cycles in these two influenza
strains correspond to
and retrospectively predict H5N1 outbreaks in 1997, 2001, 2004, 2007 and the
present outbreak
in 2008 and 2009.
[000179] In Figure 5, the mean annual Replikin Count of the pBl gene area of
H9N2 is
shown in light gray columns with standard deviation shown in dark gray columns
above the
H9N2 annual mean Replikin Count. The standard deviation data emphasize the
extent of the
expanding Replikin Counts within the annual population. The number of poultry
flocks reported
in Israel with H9N2 infection is provided in white columns. In Figure 6, mean
annual Replikin
Count for H9N2 is again reported in light gray columns with standard deviation
reported above
in dark gray columns. Mean annual Replikin Count for H5N1 is reported in black
columns with
standard deviation reported in white columns above the H5N1 annual mean
Replikin Count.
Figure 6 visibly illustrates synchrony between the H9N2 and H5N1 Replikin
Cycles.
[000180] The data for Figures 5 and 6 are disclosed in Table 4 below. In Table
4, mean
annual Replikin Count with standard deviation are provided for all amino acid
sequences
publicly available at www.pubmed.com for H9N2 and H5N1 strains of influenza
isolated from
1993 through 2008. The number of poultry flocks reported to have H9N2
infections in Israel are
also disclosed for years 2000 through 2004 as a measure of outbreaks of H9N2.
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Table 4
Synchronous Replikin Cycles in H9N2 and H5N1
H9N2 pB1 Infected H5N1 pB1
gene area Poultry gene area
Mean Standard Flocks Mean Standard
Replikin Deviation Israel Replikin Deviation
Year Count H9N2 (x 1/2) Count H5N1
1993 1.9 0.0 1.9 0.0
1994 2.4 0.7 2.2 0.4
1995 1.8 0.2 1.8 0.3
1996 6.0 4.4
1997 2.3 0.6 2.5 0.4
1998 2.2 0.5 1.8 0.3
1999 3.3 3.4 1.8 0.4
2000 7.6 9.3 5.0 1.8 0.1
2001 8.7 8.9 4.5 6.3 7.7
2002 14.3 12.4 12.5 11.3 8.3
2003 8.5 9.5 48.5 14.6 6.6
2004 10.1 10.4 42.5 2.6 2.8
2005 17.5 10.5 3.9 4.1
2006 18.5 17.1 5.2 5.2
2007 23.5 15.4 18.6 3.0
2008 12.7 16.0
[000181] As illustrated in Figures 5 and 6 with data provided above in Table
4, the H9N2
strain of influenza, which commonly infects poultry and occasionally infects
humans, has been
found to have completed a second five year Replikins expansion cycle in which
H9N2 Replikin
Counts of Replikin peptides identified as encoded in the pB 1 region of the
influenza genome
reached levels twice those found in H5N1. As may be seen in the figures, H9N2
Replikin
Counts increased in 1996, one year before the H5N1 outbreak in Hong Kong in
1997. In 1999,
increasing Replikin Counts in the pB1 region of H9N2 also preceded increases
in Replikin
Counts in the pBl region of H5N1 as well as H5N1 outbreaks. As may be seen in
Figure 6, the
Replikin Cycles of H9N2 and H5N1 coincide and share a visible level of
synchrony. Further, as
may be seen from Figure 6, the Replikin Count level for H9N2 has, as of 2008,
increased in
concentration more than the Replikin Count level for H5N1. As such, while not
wishing to be
bound by theory, it is noteworthy that H9N2 strains of influenza and H5N1
strains of influenza
appear to have a synchronized cyclic precursor-competitor evolutionary
biochemical
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relationship. The data predict that H9N2 is an alternate candidate to H5N1 for
a future influenza
pandemic.
[000182] In Figures 5 and 6, each cycle of H9N2 or H5N1 is defined by Replikin
Counts
(number of Replikin peptides per 100 amino acids) of specific Replikin
peptides in the pB 1 gene
area. An increase in successive years followed by a decrease in successive
years is observable.
Figure 5 illustrates that increasing H9N2 Replikin Counts precede the
occurrence of increasing
numbers of H9N2 infections in poultry flocks. Figure 5 further demonstrates
that Replikin
Counts in H9N2 began to increase again in 1999, two years before a reported
increase of H9N2
outbreaks in poultry in the Middle East, including Israel. As may be seen in
Figure 6, following
the increase in H9N2, Replikin Counts began to increase in H5N1 in 2000 with
infections
beginning in 2000 and forward.
[000183] The H9N2 sequences analyzed and reported as mean Replikin Count in
Table 4 and
in Figures 5 and 6 include all those published on PubMed worldwide. A
principal portion of the
sequences are from influenza isolated in China and the Middle East.
[000184] Two Replikin Count expansion rising portions of cycles are seen in
Figure 6 with
visible synchrony. The first expansion rising portion of a cycle is observed
from 1999 to 2003.
The second expansion rising portion of a cycle is observed from 2004 to 2008.
In the second
rising portion, the maximum Replikin Counts for H9N2 were greater than those
in its first rising
portion and double the maximum Replikin Counts seen in H5N1. The maximum
Replikin
Counts observed for H9N2 are likewise double the maximum Replikin Counts
observed for any
other influenza strain so far analyzed. See, e.g., Figures 7 and 8.
[000185] Additionally, the standard deviations for H9N2 as illustrated in
Figures 5 and 6 are
clearly greater than the standard deviations for the H5N1 values, indicating
greater activity in
Replikin Count in H9N2. This observable up-regulation of H9N2 Replikin Peak
Gene area
through observable changes in Replikin Count is seen in advance of H9N2
outbreaks. A similar
trend is observable in Replikin Counts in West Nile Virus for viruses isolated
from 2000 through
2008. See Figure 3. Likewise, predictive cycles have been noted in malaria,
foot and mouth
disease and other influenza strains. See, id. Figures 1-6.
[000186] The data in Table 4 and Figures 5 and 6 predict that additional
increases in both
Replikin Counts and consequent H5N1 and H9N2 infections may be expected in a
coming third
Replikin Count cycle in H9N2 and H5N1. An outbreak of H5N1 in chickens in Hong
Kong in
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early December 2008 and a reported H9N2 infection in a child in Hong Kong in
late December
2008 substantiates the predictive capacity of the data. Other H5N1 outbreak
data from the
Assam, Meghalaya, and West Bengal regions of Indian in late 2008 further
substantiate the
prediction.
Vaccines, Treatments and Therapeutics
[000187] The observations of specific Replikins and their concentration in
malaria, West Nile
virus, foot and mouth disease virus, and influenza virus proteins provides
specific quantitative
early chemical correlates of outbreaks and increases in mortality and provides
for production and
timely administration of vaccines tailored specifically to treat the prevalent
emerging or re-
emerging strain virus in a particular region of the world. Synthesis of these
vaccines may be
accomplished in seven days or less, which allows for administration of
vaccines that are a best fit
for a particular virulent strain of virus or organisms including malarial
trypanosomes, West Nile
virus, foot and mouth disease virus, and influenza virus.
[000188] By analyzing the protein sequences of isolates of a virus or other
pathogen for the
presence, concentration and/or conservation of Replikins, pandemics,
epidemics, and other
changes in virulence and mortality can be predicted and treatments developed.
Furthermore, the
severity of such outbreaks can be significantly lessened by administering a
peptide vaccine based
on the Replikin sequences found to be most abundant or shown to be on the rise
in virus isolates
over a given time period, such as about one to about three years.
[000189] A peptide vaccine of the invention may include a single Replikin
peptide sequence
or may include a plurality of Replikin sequences observed in particular virus
strains. However, a
vaccine may include a conserved Replikin peptide(s) in combination with a new
Replikin(s)
peptide or may be based on new Replikin peptide sequences. The Replikin
peptides can be
synthesized by any method, including chemical synthesis or recombinant gene
technology, and
may include non-Replikin sequences, although vaccines based on peptides
containing only
Replikin sequences are preferred. Preferably, vaccine compositions of the
invention also contain
a pharmaceutically acceptable carrier and/or adjuvant. Among the Replikin
peptides for use in a
virus or pathogen vaccine are those Replikins observed to "re- emerge" after
an absence from the
amino acid sequence for one or more years.
[000190] The vaccines of the present invention can be administered alone or in
combination
with antiviral drugs, such as gancyclovir; interferon; interleukin; M2
inhibitors, such as,
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amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and
oseltamivir; and the
like, as well as with combinations of antiviral drugs.
[000191] The vaccine of the present invention may be administered to any
animal capable of
producing antibodies in an immune response. For example, the vaccine of the
present invention
may be administered to a rabbit, a chicken, a shrimp, a pig, or a human.
Because of the universal
nature of Replikin sequences, a vaccine of the invention may be directed at a
range of strains of a
virus or organism or a particular strain of virus or organism.
[000192] The Replikin peptides of the invention, alone or in various
combinations are
administered to a subject, in a non-limited embodiment by i.v., intramuscular
injection, by
mouth, or by spray inhalation, intranasal administration, or intraocular
administration. The
peptides are administered in order to stimulate the immune system of the
subject to produce
antibodies to the peptide. Generally the dosage of peptides is in the range of
from about 0.01 g
to about 500 mg, about 0.05 g to about 200 mg, or about 0.075 g to about 30
mg, from about
0.09 g to about 20 mg, from about 0.1 g to about 10 mg, from 10 g to about
1 mg, and from
about 50 g to about 500 g. The skilled practitioner can readily determine
the dosage and
number of dosages needed to produce an effective immune response.
[000193] In another aspect of the invention, isolated Replikin peptides may be
used to
generate antibodies, which may be used, for example to provide passive
immunity in an
individual or for diagnostics. See, e.g., U.S. Appln. Ser. No. 11/355,120,
filed February 16, 2006
and U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008 (each incorporated
herein by
reference in their entirety).
Example 1
Analysis of Replikin Count in Malaria to Predict Increased Mortality
[000194] Publicly available sequences of isolates of P. falciparum at
www.pubmed.com were
analyzed using proprietary search tool software (ReplikinForecastTM available
in the United
States from REPLIKINS LLC, Boston, MA) for years 1986 to 2007 to determine the
mean
Replikin Count for the histidine-rich protein of all isolates available in
each of those years.
Mean annual Replikin Counts for each year were then compared with changes in
mortality as
reported by the World Health Organization.
[000195] A list of the accession numbers analyzed for the presence and
concentration of
Replikin sequences is provided in Table 5 below. The mean Replikin Count for
each year is
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provided following the list of accession numbers from isolates in each
corresponding year.
Standard deviation and significance as compared to the mean Replikin Count of
the previous
year and of the lowest mean Replikin Count within the data set are also
provided along with the
mean Replikin Count for each year.
Table 5 - Malaria Annual Mean Replikin Count
Year PubMed Accession Number-Replikin Count No. of Mean S.D. Significance
Isolates Replikin
per year Count
per year
1986 ---`5_AA51639 25P09346 295P0522;7 25P0 5228 23=?,,-V/U()61," 6 15.9 15.2
low p>0.50
23 AAA29631 37
1987 P06719 30 7 P13817 268 AAA29630 268 P0522 9 236 11 38.2 23.5 low p<0.005,
CAA68268 307 AAA29629 295 AAA2962 ] 15 AAA29620 33 prey p<0.02
P13825 33 P14588 15 AAA' !3197 135
1988
1989 CAA01078 23 1 13.9 0.0 rev <0.005
1990 / k'74651 19 1 5.2 0.0 1990
1991
1992 C A49542 21 C1\A49548 23 C r-V 49547 23 cAA49546 23 9 13.0 18.2 low
p>0.50
C A.49545 23Cy-\-,k49543 23C_-V 4954 23 AA5_r 29 7 393.204 prev p<0.20
NP 001771 11
1993
1994
1995 CAK_3 8915 9 1 2.4 0.0 prev p<O.10
1996kAC;47454 23 AAC47453 23 C-AB01211 32 3 8.7 1.2 low p<0.01,
prev p<0.01
1997
1998 XP 001349534 295 AAC71810 295 XP 00 1349-1102 279 7 24.1 16.7 low p<0.10,
A.AC 71973 279 AAD40570 45 A_- 40569 31 AAD20952 52 rev <0.04
1999 CAD49281 1366 AAD23574 249 AAD31511 23 AAF14632 23 4 62.9 62.9 low
p<0.20,
rev <0.20
2000 k- 74261 290 AG01323 295 AAF74262 22 3 33.3 24.7 low p<0.30,
rev <0.40
2001
2002 XP 001351550 1366 'A125049 193X1?0013480 2 21. 13 18.0 29.0 low p>0.50,
' P0013 "35 485XP001350"35 874 / AN3598S 121 prev p<0.30
35448 485 AAN36415 874 XP 960846 25 E -\ A31610 25
CAD36995 25 XP 726238 110 EA.AU7803 110
2003 AAQ63567 87 AA063566 64 AAQ63565 63 AAQ63564 66 7 28.4 3.0 low p<0.001,
AA063563 66 AA063562 66 AA063561 67 rev <0.20
2004 XP 96-6219 193 1 17.0 0.0 rev <0.001
----- -------------- --------
2005 EDL47342 352 BD1t15776 150 XP 7 63898 7 AAW78557 234 5 7.4 6.5 low
p<0.05,
1=.AN31615 7 rev <0.02
2006
2007 kBU43157 23 BAP93906 2 ZP 02691628 36 XP 001617069 8 17.2 19.0 low
p>0.50,
352 XP 001615503 150 XP 001615499 369 XP 001639181 prev p<0.20
505 1D047118 505
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[000196] Analysis of the annual mean Replikin Count of the histidine rich
protein between
1986 and 2007 revealed cycles of Replikin Count. The beginning of a new cycle
provides a
prediction that Replikin Count may continue to increase along with an increase
in malaria
mortality rate. The data is graphically illustrated in Figure 1 and summarized
in Table 1 above.
[000197] Replikin peptides in an isolate identified at a peak or in a rising
portion of the
Replikin cycles revealed in Figure 1 are available as peptides of the
invention. For example, any
Replikin peptide identified in the ABU43157 accession number of an isolate
from 2007 is
available as a diagnostic, therapeutic, or preventive compound or composition
of the invention
because it is identified in a rising portion of a Replikin cycle. Replikin
peptides identified in the
1999 isolate reported at accession number CAD49281 are likewise available. See
Figure 1 and
Table 2. The 1999 isolate is present at the peak of a Replikin cycle, as such,
Replikin peptides
identified in the isolate reported at CAD49281 may be used as immunogenic
compounds.
Additionally, the 1998 accession number XP001349534 is identified from an
isolate in a rising
portion in a Replikin cycle. See Figure 1 and Table 5. Replikin peptides
identified in
ABU43157, CAD49281, and XP001349534, among others, are likewise useful as
immunogenic
compounds or vaccines or for diagnosis or treatment of malaria.
Example 2
Analysis of Replikin Count in Malaria ATP-ase to Predict Increased Mortality
[000198] Applicants analyzed publicly available sequences of the ATP-ase
enzyme of
isolates of P. falciparum at www.pubmed.com. The data is summarized below in
Table 6 and
illustrated in Figure 2. The data illustrate that mortality rates per 1000
clinical cases of malaria
in humans correlate with annual mean Replikin Count in sequences of the P.
falciparum ATP-
ase enzyme publicly available at www.pubmed.com. Replikin Counts of P.
falciparum ATP-ase
increased from 1997 to 1998 along with an increase in mortality per malaria
case from 1997 and
1998 to 1999. The Replikin Count of P. falciparum ATP-ase decreased from 1998
to 2006 along
with mortality rates from 1999 to 2005 (consistent mortality presently
available only through
2005). High malaria morbidity and mortality rates occurred in the late 1990s
and 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
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1999 to 2005. Mortality rates in Table 6 are recorded as declared by the World
Health
Organization. See www.who.int.
Table 6
Year Mean Standard Mortality Rate
Replikin Deviation per 1000
Count in P. Malaria Cases
falciparum
ATP-ase
1997 19 7.7 17
1998 19.4 16.6 17
1999 16.1 9.1 19
2000 11.2 10.5 16
2001 7.7 8.1 13
2002 12.7 9.9 10
2003 3.3 2.5 10
2004 4.2 4.6 9
2005 6.3 3.9 9
2006 3.4 2.6
2007 6.2 8.4
Example 3
Analysis of Replikin Count Cycles in West Nile Virus Predict Increased
Morbidity
[000199] Envelope protein sequences from isolates of West Nile virus isolated
between 2000
and 2008 that were publicly available at www.pubmed.com were analyzed for
Replikin
sequences and a mean annual Replikin Count was determined. The data are
contained in Table 7
below and illustrated in Figure 3.
[000200] Figure 3 illustrates cycling of mean annual Replikin Count in West
Nile virus in
correlation with cycling of West Nile virus morbidity. Cycles are detectable
because of
repeating conserved virus structures and continuity of the Replikin phenomenon
through time.
The mean annual Replikin Count of the Envelope Protein of WNV (black), and
standard
deviation, is compared to the annual number of human cases in the U.S. per CDC
reports (gray).
[000201] 2000 to 2003: The standard deviation of the mean of the Replikin
Count of the
envelope protein increases markedly from 2000 to 2001 (p<0.001). This change
has been
observed in all common strains of influenza virus (not the same virus genus as
WNV) to signal
rapid replication and expansion of the range of the Replikin Count, thus virus
population
expands with Replikin Count and precedes virus outbreak. The increase in the
mean Replikin
Count from 2000 to 2003 appears to accompany, or precede, the increase in the
number of
human WNV cases recorded independently and published by the Center for Disease
Control
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(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 Plasmodiumfalciparum (malaria) to human
morbidity, and to
mortality rate in shrimp with shrimp taura syndrome virus. Since the
relationship has already
been demonstrated in several species, including crustaceans, horses, and
humans, it appears to be
a broadly distributed general principle. 2004 to 2007: In 2004 and 2005, there
was a decrease
from 2003 in both the Replikin Count and the number of human cases of WNV. In
2006, there
was an increase in the Replikin Count followed by an increase in 2007 of the
number of human
cases.
[000202] In Figure 3, cycles of Replikin concentration and cycles of WNV human
morbidity
may be observed to correlate. Until the present data, it was not understood
that cycles within a
particular strain of pathogen actually continued from a peak to a trough to
another peak to
another trough. Instead, in the past, it was understood that an increase in
Replikin concentration
correlated with outbreaks and a decrease in Replikin concentration correlated
with retraction.
With these new data, however, it is now understood and contemplated by the
invention that
entire Replikin cycles from peak to trough to peak to trough and/or from
trough to peak to trough
to peak correlate with cycles in virulence, morbidity, and mortality. The
invention now provides
methods of tracking pathogens as they increase in virulence, expand in
population within a
region or into a region, or increase in morbidity or mortality by monitoring
changes in Replikin
concentration.
[000203] The rising numbers for both the Replikin Count and the number of
cases in the
second rising portion of the cycle, 2004-2008, when compared to the first
rising portion of the
cycle, suggests an increased or `improved' infective efficiency accompanying
an increased
Replikin Count in the second rising portion, compared to the first. The drop
in efficacy of the
virus is probably due to the generation of resistance in the host; the
subsequent rise in infectivity
in the second rising portion of the cycle is related to the appearance of new
Replikins identified
in WNV. Once again, the close relationship of Replikins to infectivity is
demonstrated; both
literally rise and fall together.
[000204] 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
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temporarily ended in 1998 by the complete culling of chickens in Hong Kong.
The H5N1
clinical `sub-cycle' resumed in 2000, continued to the present, and was
predicted prospectively
each year by the Replikin Count. In this case, occurring mostly in East Asian
countries, H5N1
was not as subject to exact epidemiological reports by the WHO of morbidity
and mortality as in
the case of West Nile Virus in the U.S. as here presented, since the CDC keeps
much more
accurate surveillance records of the morbidity and mortality.
[000205] 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.
[000206] Figure 3 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 Boston, MA).
[000207] Accession numbers, number of isolates, mean Replikin Count, standard
deviation
and significance for accession numbers available for West Nile virus envelope
protein from
www.pubmed.com are contained below in Table 7. Specific conserved Replikin
sequences
identified within the step-wise cycles of West Nile virus in Figure 3 include,
KIIQKAHK (SEQ
ID NO: 13), HLKCRVKMEK (SEQ ID NO: 14), KLTSGHLK (SEQ ID NO: 15), and
HNDKRADPAFVCK (SEQ ID NO: 16). The accession numbers in which these sequences
are
conserved are listed in Example 6 of U.S. Appln. Ser. No. 12/108,458, filed
April 23, 2008,
which is incorporated herein by reference in its entirety.
Table 7
West Nile Virus Envelope Protein Replikin Count Cycles
Year PubMed Accession Number No. of Mean S.D. Significance
Replikin Count West Nile Virus Envelope Protein Isolates per Replikin
year Count per
year
2000 BR19638 102 AAK06624 97 AAG02039 98 AAG02038 97 4 2.9 0.1 low p<0.001,
prey p<0.001
2001 A-AM70028 28 AAL07765 6 AAL07764 6 AAL07763 6 AAL07762 6 130 3.6 2.0 low
p<0.02, prey
L07761 6 AAL14222 30 AAL14221 30 AAL14220 30 AAL14219 p<0.001
30 AAL14218 30 AAL14217 30 AAL14216 30 AAL14215 30
A-AK58104 30 AAK58103 31 AAK58102 30 AAK58101 30
A-AK58100 30 AAK58099 31 AAK58098 30 AAK58097 30
A-AK58096 30 AAK52303 30 AAK52302 30 AAK52301 30
A-AK52300 30 AAK62766 32 AAK62765 32 AAK62764 32
[AAK62763 32 AAK62762 32 AAK62761 32 AAK62760 32
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A-AK62759 32 AAK62758 32 AAK62757 32 AAK62756 32
K91592 20 ABR19637 111 AAM81753 97 AAM81752 97
A-AM81751 97 AAM81750 97 AAM81749 97 AAK67141 7
A-AK67140 7 AAK67139 7 AAK67138 7 AAK67137 7 AAK67136 7
A-AK67135 7 AAK67134 7 AAK67133 7 AAK67132 7 AAK67131 7
A-AK67130 7 AAK67129 7 AAK67128 7 AAK67127 7 AAK67126 7
A-AK67125 7 AAK67124 3 AAK67123 7 AAK67122 7 AAK67121 7
A-AK67120 7 AAK67119 7 AAK67118 7 AAK67117 7 AAK67116 7
A-AK67115 7 AAK67114 7 AAK67113 7 AAK67112 7 AAK67111 7
A-AK67110 7 AAK67109 7 AAK67108 7 AAK67107 7 AAK67106 7
A-AK67105 7 AAK67104 7 AAK67103 7 AAK67102 7 AAK67101 7
A-AK67100 7 AAK67099 7 AAK67098 7 AAK67097 7 AAK67096 7
A-AK67095 7 AAK67094 7 AAK67093 7 AAK67092 7 AAK67091 7
A-AK67090 7 AAK67089 7 AAK67088 7 AAK67087 7 AAK67086 7
A-AK67085 7 AAK67084 7 AAK67083 7 AAK67082 7 AAK67081 7
A-AK67080 7 AAK67079 7 AAK67078 7 AAK67077 7 AAK67076 7
A-AK67075 7 AAK67074 7 AAK67073 7 AAK67072 7 AAK67071 7
A-AK67070 5 AAK67069 7 AAK67068 7 AAK67067 7 AAK67066 7
A-AK67065 7 AAK67064 7 AAL87748 19 AAL87747 18 AAL87746
19 AAL87745 18 AAL37596 18 AAM21944 24 AAM21941 32
2002 M09856 6 AAM09855 6 AAM09854 6 AA026579 30 AA026578 18 4.7 1.5 low
p<0.001,
30 AAN77484 3 AAN85090 97 AA073303 36 AA073302 36 prey p<0.005
A-A073301 36 AA073300 36 AA073299 36 AA073298 36
A-A073297 36 AA073296 36 AA073295 36 AAL87234 96
CAD60131 96
2003 P20887 96 AAR10793 6 AAR10784 6 AAR17575 32 AAR17574 94 5.3 1.5 low
p<0.001,
32 AAR17573 32 AAR17572 32 AAR17571 32 AAR17570 32 prey p<0.05
R17569 32 AAR17568 32 AAR17567 32 AAR17566 32
R17565 32 AAR17564 32 AAR17563 32 AAR17562 32
R17561 32 AAR17560 32 AAR17559 32 AAR17558 32
R17557 32 AAR17556 32 AAR17555 32 AAR17554 32
R17553 32 AAR17552 32 AAR17551 32 AAR17550 32
R17549 32 AAR17548 32 AAR17547 32 AAR17546 32
R17545 32 AAR17544 32 AAR17543 32 AAR17542 32
A-AQ87608 16 AAQ87607 16 AAQ87606 14 AAR10804 6 AAR10803
6 AAR10802 6 AAR10801 6 AAR10800 6 AAR10799 6 AAR10798 6
A-AR10797 6 AAR10796 6 AAR10795 6 AAR10794 6 AAR10792 6
A-AR10791 6 AAR10790 6 AAR10789 6 AAR10788 6 AAR10787 6
A-AR10786 6 AAR10785 6 AAR10783 6 AAR10782 6 AAR10781 6
A-AR10780 6 AAQ88403 10 AAQ88402 10 AAX99361 97 AAR84198
36 AAQ55854 97 AAR14153 36 AAR84614 95 AAR06948 36
R06947 36 AAR06946 36 AAR06945 36 AAR06944 36
R06943 36 AAR06942 36 AAR06941 36 AAR06940 36
R06939 36 AAR06938 36 AAR06937 36 AAR06936 35
R06935 36 AAR06934 36 AAR06933 36 AAR06932 36
A-AR06931 36 AAQ00999 100 AAQ00998 97 AAP22087 97
[AAP22086 97 AAP22089 97 AAP22088 96
2004 T11553 32 AAT11552 32 AAT11551 32 AAT11550 32 AAT11549 55 4.2 1.7 low
p<0.001,
32 AAT11548 32 AAT11547 32 AAT11546 32 AAT11545 32 prey p<0.001
T11544 32 AAT11543 32 AAT11542 32 AAT11541 32 AAT11540
32 AAT11539 32 AAT11538 32 AAT11537 32 AAT11536 32
T11535 32 AAT11534 28 AAS75296 6 AAS75295 6 AAS75294 6
S75293 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
BV82765 97 AAZ91684 106 AAW56064 97 AAW56066 97
W56065 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 P_001527880 32 ABC 18309 8 ABC 18308 9 ABC02196 3 125 4.3 1.8 low
p<0.001,
Y67877 9 AAY67876 11 AAY67875 11 AAY67874 8 AAY67873 prey p>0.50
8 AAY67872 8 AAY67871 8 AAY67870 8 AAY67869 8 AAY67868 8
Y67867 8 AAY67866 8 AAY57985 8 ABB01532 97 ABC40712
100 YP001527877 97 ABBO 1533 101 ABA62343 97 AAY32590 36
Y32589 36 YP_001527879 4 AAY55949 97 AAY29684 6
Y29685 6 AAY29683 6 AAY29682 6 AAY29681 6 AAY29680 6
Y29679 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
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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
BA43046 36 ABA43045 36 ABA43044 36 ABA43043 36
BA43042 36 ABA43041 36 ABA43040 36 ABA43039 36
BA43038 36 ABA43037 36 ABA43036 36 ABA43035 36
BA43034 36 ABA43033 37 ABA43032 37 ABA43031 36
BA43030 37 ABA43029 37 ABA43028 36 ABA43027 36
BA43026 36 ABA43025 36 ABA43024 36 ABA43023 36
BA43022 36 ABA43021 36 ABA43020 36 ABA43019 36
BA43018 36 ABA43017 36 ABA43016 36 ABA43015 36
BA43014 36 ABA43013 36 ABA43012 36 ABA43011 36
BA43010 36 ABA43009 34 ABA43008 36 ABA43007 36
BA43006 36 ABA43005 36 ABA43004 36 ABA43003 36
BA54595 97 ABA54594 97 ABA54593 97 ABA54592 97
BA54591 97 ABA54590 97 ABA54589 97 ABA54588 97
BA54587 97 ABA54586 97 ABA54585 98 ABA54584 97
BA54583 105 ABA54582 97 ABA54581 93 ABA54580 97
BA54579 97 ABA54578 97 ABA54577 97 ABA54576 97
BA54575 97 AAY54162 97
2006 BI81406 32 ABI81405 32 ABI81404 32 ABI81403 32 ABI81402 32 312 6.0 1.3
low p<0.001,
BI81401 32 ABI81400 32 ABI81399 32 ABI81398 32 ABI81397 32 prey p<0.001
BI81396 32 ABI81395 32 ABI81394 32 ABI81393 32 ABI81392 32
BI81391 32 ABI81390 32 ABI81389 32 ABI81388 32 ABI81387 32
BI81386 32 ABI81385 32 ABI81384 32 ABI81383 32 ABI81382 32
BI81381 32 ABI81380 32 ABI81379 32 ABI81378 32 ABI81377 32
BI81376 32 ABI81375 32 ABI81374 32 ABI81373 32 ABI81372 32
BI81371 32 ABI81370 32 ABI81369 32 ABI81368 32 ABI81367 32
BI81366 32 ABI81365 32 ABI81364 32 ABI81363 32 ABI81362 32
BI81361 32 ABI81360 32 ABI81359 32 ABI81358 32 ABI81357 32
BI81356 32 ABI81355 32 ABI81354 32 ABI81353 32 ABI81351 32
BI81350 32 ABI81349 32 ABI81348 32 ABI81347 32 ABI81346 32
BI81345 32 ABI81344 32 ABI81343 32 ABI81342 32 ABI81341 32
BI81340 32 ABI81339 32 ABI81338 32 ABI81337 32 ABI81336 32
BI81335 32 ABI81334 32 ABI81333 32 ABI81332 32 ABI81331 32
BI81330 32 ABI81329 32 ABI81328 32 ABI81327 32 ABI81326 32
BI81325 32 ABI81324 32 ABI81323 32 ABI81322 32 ABI81321 34
BI81320 32 ABI81319 32 ABI81318 32 ABI81317 32 ABI81316 32
BI81315 32 ABI81314 32 ABI81313 32 ABI81312 32 ABI81311 32
BI81310 32 ABI81309 32 ABI81308 32 ABI81307 32 ABI81306 32
BI81305 32 ABI81304 32 ABI81303 32 ABI81302 32 ABI81301 32
BI81300 32 ABI81299 32 ABI81298 32 ABI81297 32 ABI81296 32
BI81295 32 ABI81294 32 ABI81293 32 ABI81292 32 ABI81291 32
BI81290 32 ABI81289 32 ABI81288 32 ABI81287 32 ABI81286 32
BI81285 32 ABI81284 32 ABI81283 32 ABI81282 32 ABI81281 32
BI81280 32 ABI81279 32 ABI81278 32 ABI81277 32 ABI81276 32
BI81275 32 ABI81274 32 ABI81273 32 ABI81272 32 ABI81271 32
BI81270 32 ABI81269 32 ABI81268 32 ABI81267 32 ABI81266 32
BI81265 32 ABI81264 32 ABI81263 32 ABI81262 32 ABI81261 32
BI81260 32 ABI81259 32 ABI81258 32 ABI81257 32 ABI81256 32
BI81255 32 ABI81254 32 ABI81253 32 ABI81252 32 ABI81251 32
BI81250 32 ABI81249 32 ABI81248 32 ABI81247 32 ABI81246 32
BI81245 32 ABI81244 32 ABI81243 32 ABI81242 32 ABI81241 32
BI81240 32 ABI81239 32 ABI81238 32 ABI81237 32 ABI81236 32
BI81235 32 ABI81234 32 ABI81233 32 ABI81232 32 ABI81231 32
BI81230 32 ABI81229 32 ABI81228 32 ABJ90133 32 ABJ90132 32
BJ90131 32 ABJ90130 32 ABJ90129 32 ABJ90128 32 ABJ90127 32
BJ90126 32 ABJ90125 32 ABJ90124 32 ABJ90123 32 ABJ90122 32
BJ90121 32 ABJ90120 32 ABJ90119 32 ABJ90118 32 ABJ90117 32
BJ90116 32 ABJ90115 32 ABJ90114 32 ABJ90113 32 ABJ90112 32
BJ90111 32 ABJ90110 32 ABJ90109 32 ABJ90108 32 ABJ90107 32
BJ90106 32 ABJ90105 32 ABJ90104 32 ABJ90103 32 ABJ90102 32
BJ90101 32 ABJ90100 32 ABJ90099 32 ABJ90098 32 ABJ90097 32
BJ90096 32 ABJ90095 32 ABJ90094 32 ABJ90093 32 ABJ90092 32
BJ90091 32 ABJ90090 32 ABJ90089 32 ABJ90088 32 ABJ90087 32
BJ90086 32 ABJ90085 32 ABJ90084 32 ABJ90083 32 ABJ90082 32
BJ90081 32 ABJ90080 32 ABJ90079 32 ABJ90078 32 ABJ90077 32
BJ90076 32 ABJ90075 32 ABJ90074 32 ABJ90073 32 ABJ90072 32
BJ90071 32 ABJ90070 32 ABJ90069 32 ABJ90068 32 ABJ90067 32
BJ90066 32 CAL49454 98 AB197486 99 ABG36517 36 ABG81344
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92 ABG81343 97 ABG81342 97 ABG81341 97 ABG81340 99
BG76816 41 ABG76815 43 ABG76814 43 ABG76813 43
BG76812 43 ABG76811 43 ABG76810 43 ABG76809 43
BG76808 43 ABG76807 43 ABG76806 43 ABG76805 43
BG76804 43 ABG76803 43 ABG76802 43 ABG76801 43
BG76800 43 ABG76799 43 ABG76798 43 ABG76797 43
BG76796 43 ABG76795 43 AB126622 40 AB126621 40 ABD19642
97 ABD19641 97 ABD19640 97 ABD19513 97 ABD19512 96
BD19511 97 ABD19510 97 ABD85083 98 ABD85082 93
BD85081 97 ABD85080 97 ABD85078 97 ABD85077 97
BD85076 97 ABD85075 97 ABD85074 99 ABD85073 97
BD85072 99 ABD85070 97 ABD85069 96 ABD85068 97
BD85067 97 ABD85066 95 ABD85065 97 ABD85064 97
BD67762 97 ABD67761 97 ABD67760 97 ABD67759 97
BD67758 97 ABD67757 97
2007 BR19639 111 ABV22897 97 ABU54838 97 ABU52997 98 (Incomplete) (Incomplete)
(Incomplete) (Incomplete)
BQ52692 97 AB069610 36 AB069609 36 AB069608 36 27 4.6 1.2 low p<0.001,
B069607 36 AB069606 36 AB069605 36 AB069604 36 prey p<0.001
B069603 36 AB069602 36 AB069601 36 AB069600 36
B069599 36 AB069598 36 AB069597 36 AB069596 36
B069595 36 AB069594 36 AB069593 36 AB069592 36
BU41789 114 CAM91200 97 ABR10608 56
2008 BZ10682 21 ABZ10681 29 ABZ10680 29 ABZ10679 29 ABZ10678 (Incomplete)
(Incomplete) (Incomplete) (Incomplete)
29 5 5.5 0.7 low p<0.002,
prey p<0.04
Example 4
Analysis of Replikin Count Cycles in Foot and Mouth Virus to Predict Increased
Morbidity
[000208] All protein sequences from isolates taken between 1999 and 2008 that
were
publicly available at www.pubmed.com were analyzed for Replikin sequences and
a mean
annual Replikin Count was determined. The data are contained in Table 3 above
and illustrated
in Figure 4. Figure 4 illustrates cycling of mean annual Replikin Count in
foot and mouth
disease virus type O. The peaks in the cycles correlate with outbreaks in the
U.K and the
Netherlands in 2001-2002 and in the Middle East and Asia in 2008-2009. The
cycles illustrated
in Figure 4 are detectable because of repeating conserved virus structures and
continuity of the
Replikin phenomenon through time. In a new cycle beginning in 2005, the
highest Counts in ten
years was observed (2007-2008), which was followed by severe FMDV outbreaks in
2008 and
2009 in the Middle East, Africa, India, China, and other Asian countries.
[000209] Figure 4 shows that the annual Replikin Counts (Mean and Standard
Deviation
(SD)) occurred with two rising portions and a decreasing portion. The first
rising portion
followed by the first decreasing portion occurred from, 1999-2005 and the
second rising portion
occurred in 2005-2008. Increases in Replikin Counts provided advance warning
signals with
p<0.001 prior to the 2001-2002 and 2008-2009 severe outbreaks.
[000210] To provide the data in Figure 4, Replikin peptides were identified
and counted
automatically in sequences available at www.pubmed.com using the
ReplikinsForecastTM
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software (Replikin LLC Boston, MA) designed to analyze protein sequences of
any organism.
Statistical analysis was likewise accomplished using the software. When the
history of each
Replikin structure in the virus was tracked for its occurrence in each virus
specimen in each of
the years for which virus sequence data was published, conservation of certain
Replikin
structures was observed over decades. The structure of these conserved
Replikin peptides is the
basis of synthetic Replikin vaccines for FMDV.
[000211] The following Replikin peptide sequences were identified for
vaccines:
HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18). These sequences
have been observed to be conserved in the Replikin cycles illustrated in
Figure 4 and, as taught
by the invention, are vaccines for predicted outbreaks of foot and mouth
disease virus.
[000212] The two above-listed conserved Replikin peptides have been identified
and tracked
annually in publicly available sequences in foot and mouth disease virus at
www.pubmed.com
from 1934 through 2008. The sequence HKQKIIAPAK (SEQ ID NO: 17) is observed to
be
conserved 100% of the time in the publicly available sequences from isolates
from 1934 through
2008. The sequence HKQKIVAPVK (SEQ ID NO: 18) is observed also to be conserved
in
100% of isolates from 1934 through 2007 with the exception of two
substitutions, namely a
valine at residue 6 in the peptide and a valine at residue 9 in the peptide.
[000213] Table 8 provides the accession numbers at www.pubmed.com wherein
sequence
HKQKIIAPAK (SEQ ID NO: 17) and HKQKIVAPVK (SEQ ID NO: 18) were conserved over
time. The residue at which the peptide begins in the sequence disclosed in the
accession number
is noted.
Table 8
FMDV Conserved Sequences
Year Accession Numbers in which Accession Numbers in which
hkqkiiapak (SEQ ID NO: 17) are hkqkivapvk (SEQ ID NO: 18) are
conserved conserved
1934 ACC63172 position 201 , ACC63171 position ACC63139 position 201 ACC63138
position 201
201 , ACC63169 position 201 , ACC63168 , ACC63137 position 201 , ACC63130
position
position 201 , ACC63167 position 201 , 201 ACC63129 position 201 , ACC63128
ACC63165 position 200, ACC63164 position position 201 A CC6312 7 position 201
,
201 , ACC63162 position 200, ACC63160 ACC63133 position 201 , ACC63132
position 201
position 201 , ACC63159 position 201 , , ACC63131 position 201
ACC63158 position 200 , ACC63155 position
200, ACC63154 position 201 , ACC63153
position 201 , ACC63152 position 201 ,
ACC63151 position 200 , ACC63150 position
200_, ACC63149 position 201 , ACC63148
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position 201 , ACC63147 position 200 ,
ACC63146 position 201 , ACC63145 position
200, ACC63144 position 200 , ACC63143
position 200 , ACC63142 position 201 ,
ACC63140 position 201
1955 CAB62583 position 926 .
1958 CAA10475 position 131 .
1962 CAC22210 position 202 AAP81678 position 153
AAP81677 position 153 AAP81676 position 153
AAP81675 position 153 AAP81674 position 153
ABA46701 position 201 ABA46700 position 201
ABA46699 position 201 ABA46698 position 201
ABA46697position 201 ABA46696 position 201
ABA46695 position 201 ABA46693 position 201
ABA46692 position 201 ABA46691 position 201
ABA46690 position 201 ABA46689 position 201
ABA46688 position 201 ABA46687 position 201
ABA46686 position 201 ABA46685 position 201
ABA46684 position 201 ABA46683 position 201
ABA46682 position 201 ABA46681 position 201
ABA46679 position 201 ABA46678 position 201
ABA46677 position 201 ABA46675 position 201
ABA46674 position 201 ABA46673 position 201
ABA46672 position 201 ABA46671 position 201
ABA46670 position 201 ABA46669 position 201
ABA46668 position 201 ABA46666 position 201
ABA46664 position 201 ABA46663 position 201
ABA46662 position 201 ABA46661 position 201
ABA46660 position 201 ABA46659 position 201
ABA46658position 201 ABA46657position 201
ABA46655 position 201 ABA46654 position 201
ABA46653 position 201 ABA46652 position 201
ABA46651 position 201 ABA46650 position 201
ABA46649 position 201 ABA46648 position 201
ABA46647 position 201 ABA46644 position 201
ABA46643 position 201 ABA46642 position 201
ABA46641 position 201 ABA46640 position 201
ABA46639 position 201 ABA46638 position 201
ABA46637 position 201 ABA46614 position 201
ABA46613 position 201 ABA46612 position 201
ABA46611 position 201 ABA46610 position 201
ABA46609 position 201 ABA46606 position 201
ABA46605 position 201 ABA46604 position 201
ABA46603 position 201 ABA46602 position 201
ABA46601 position 201 ABA46600 position 201
ABA46597position 201 ABA46596 position 201
ABA46594 position 201 ABA46591 position 201
ABA46590 position 201 ABA46589 position 201
ABA46588position 201 ABA46586 position 201
ABA46585 position 201 ABA46583 position 201
ABA46582 position 201 ABA46581 position 201
ABA46580 position 201 ABA46579 position 201
ABA46578 position 201 ABA46576 position 201
ABA46574 position 201 ABA46573 position 201
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ABA46571 position 201 ABA46570 position 201
ABA46569 position 201 ABA46568 position 201
ABA46566 position 201 ABA46565 position 201
ABA46563 position 201 ABA46561 position 201
ABA46560 position 201 ABA46542 position 201
ABA46541 position 201 ABA46539 position 201
ABA46538 position 201 ABA46537 position 201
ABA46536 position 201 ABA46535 position 201
ABA46534 position 201 ABA46533 position 201
ABA46532 position 201 ABA46531 position 201
ABA46559 position 201 ABA46540 position 201
1964 CAB62582 position 640
1968 CAC48168 position 201
1969 CAB62584 position 724.
1971 CAC48169 position 201
1972 ABL75440 position 40 , ABL75439 position 43 , CAC22304 position 202
ABL75437 position 43 , ABL75435 position 43 ,
ABL75434 position 43 , ABL75433 position 43 ,
ABL75432 position 43 , ABL75431 position 43 ,
ABL75427 position 43 , ABL75424 position 43 ,
ABL75423 position 43 , ABL75422 position 43 ,
ABL75421 position 43 , ABP82766 position 200
, ABP82765 position 200 , ABP82764 position
201 , ABP82763 position 201, ABP82762
position 201 , ABP82759 position 200 ,
ABP82757 position 200, ABP82756 position
201 , ABP82755 position 201 , ABP82754
position 201 , ABP82753 position 201 ,
ABP82752 position 201 , ABP82751 position
201 , ABP82750 position 201 , ABP82749
position 201 , ABP82748 position 201 ,
ABP82747 position 201 , ABP82746 position
201 , ABP82744 position 201 .
1974 CAC22211 position 202 AAK69575 position 153
, AAR85362 position 153 AAR22955 position 153
, AAR22953 position 153
1975 AAK69576 position 153 , CAC20174 position 201
AAR85363 position 153 , AAG35653 position
724
1976 CAC34727 position 201 AAR22952 position 153 , AAR22933 position 153 ,
AAR22932 position 153.
1977 AAD26458 position 58 , AAD26457 position 58 AAR22963 position 153 ,
AAR22950 position 153 ,
, AAD26456 position 58 , AAD26455 position CAC48179 position 201 .
48, AAD26454 position 58 , AAD26452
position 58 , AAD26451 position 58 ,
AAD26450 position 58, AAD26449 position 58
, AAD26448 position 52 , AAD26447 position
48, AAD26446 position 58 , AAD26445
position 58 , AAD26443 position 58 ,
AAD26442 position 58, AAD26438 position 58
, AAD26437 position 58 , AAD26436 position
58 , AAD26435 position 58 , AAD26434
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position 58 , AAD26433 position 53 ,
AAD26432 position 58 , AAF75833 position
725
1978 ABA46745 position 201 ABA46744 position 201 ,
ABA46743 position 201 , ABA46742 position 201 ,
ABA46740 position 201 AAR22930 position 153.
1979 CAC22173 position 43 , JA 88330 position 153 ,
AA088328 position 153 , AA088327 position 153
AA088325 position 153, AA088324 position
153, AA088323 position 153, AA088322
position 153 , AA088321 position 153 ,
AA088320 position 153 , AA088319 position 153
AA088318 position 153, AA 088317 position
153, AA088316 position 153, AA088315
position 153 , AA088314 position 153 ,
AA088313 position 153 , AA088312 position 153
, AAG28368 position 43, AAG28367 position 43,
AAG28366 position 43 , AAG28362 position 43 ,
AAG28357 position 43 , AAG28356 position 43 ,
AAG28355 position 43 , AAG28354 position 43 ,
AAG28353 position 43 , AAG28352 position 43 ,
AAG28348 position 43.
1980 AAR22962 position 153 , AAR22959 position 153 ,
AAR22941 position 153
1981 CAC27325 position 201 AAR22951 position 153
1982 AAA42596 position 190 , P03309 position 190. CAC20178 position 201 ,
AAZ31359 position 201 ,
AAZ31358 position 201 , JAZ31357 position 201
AAZ31356position 201 , JAZ31354 position 201
AAZ31353 position 201 , AAZ31352 position 201 ,
AAZ31350 position 201 , AAZ31349 position 201 ,
AAZ31348 position 201 , JAZ31347 position 201
AAZ31346position 201 , JAZ31345 position 201
AAZ31344 position 201 , AAZ31343 position 201 ,
AAZ31342 position 201 .
1983 AAR22960 position 153 , AAR22938 position 153 ,
AAR22937 position 153
1984 ABZ80842 position 201 , CAA00589 position
190.
1985 AAA42601 position 201 , AAA42598 position CAC22326 position 90.
157, AAA42597 position 200 , AAA42595
position 198 , AAA42594 position 201
1986 AAB93439 position 71 , ABZ80846 position AAR22954 position 153.
202, AAA42664 position 225 .
1987 AAB93449 position 93 , AAB05766 position AAK62003 position 43.
201 , AAB05764 position 201 , AAB05763
position 201 , AAB05762 position 201 ,
AAB93450 position 69, AAA42604 position
183 , AAA42614 position 75, AAA42603
position 183 , AAA42602 position 180 ,
CAC27328 position 201 , CAC27326 position
201.
1988 AAK69568 position 153 , AAK69567 position 153
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1989 CAC22174 position 90 , AAR22961 position 153 ,
AAK62024 position 69.
1990 CAC48172 position 201 , CAC48170 position CAC22178 position 43 , CAC22327
position 58
201.
1991 AAA42666 position 708, CAC48173 position CAC22175 position 43 , CAC22328
position 62
201
1992 CAC48176 position 201 CAC22176 position 43 , CAC22240 position 85,
CAC48182 position 201 .
1993 CAC22179 position 43 , CAC40792 position 201 ,
CAC40789 position 201 , CAC40796 position 102
1994 CAC22180 position 76 , CAC22233 position 62,
CAC22227 position 60 , CAC22215 position 47,
CAC22208 position 82 , CAC22201 position 43,
CAC22167 position 43 , AAK62012 position 43,
CAC40794 position 102 , CAC40790 position 201
, CAC40795 position 102, CAC40797 position
201.
1995 CAC22231 position 152 , CAC22216position 44,
CAC22171 position 103, AAK62022 position 69
1996 AAB05765 position 201 . CAC22194 position 127 , CAC51235 position 201
AAR22945 position 153 , AAR22942 position 153
AAK62005 position 69
1997 CAC51273 position 201 , CAC51268 position 201
, CAC51249 position 201, CAC51236 position
201, AAL05257 position 43 , AAL05249 position
43 AAL05248 position 85 , AAL05247 position
62 AAL05246 position 76 , AAL05245 position
43 AAL05243 position 56 , AAL05242 position
AAL05236 position 43 , AAL05235 position
AAL05234 position 43 , AAL05233 position
AAL05232 position 43 , AAL05231 position
AAL05230 position 43 , AAL05229 position
AAL05228 position 43 , AAL0522 7 position
AAL05226 position 43 , AAL05225 position
AAL05223 position 43 , AAL05222 position
AAL05221 position 43 , AAL05220 position
122 AAL05219 position 43 , AAL05218 position
52 AAL05217 position 43 , AAL05216 position
66 AAL05214 position 43 , AAL05213 position
AAL05211 position 58 , AAL05207 position
AAL05206 position 62 , AAL05205 position
AAL05196 position 64.
1998 AAL73360 position 113 . CAC22229 position 201 , ABI16250 position 201 ,
ABI16249 position 201 , ABI16248 position 201 ,
ABIJ 6247 position 201 , ABIJ 6246 position 201 ,
ABI16245 position 201 , ABI16244 position 201 ,
ABI16242 position 201 , ABI16241 position 201 ,
ABI16240 position 201 , ABI16239 position 201 ,
ABI16238 position 201 , ABIJ 6237 position 201 ,
ABI16236 position 201 , ABI16235 position 201 ,
ABI16234 position 201 , ABI16233 position 201 ,
ABI16232 position 201 , ABI16231 position 201 ,
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ABI16230 position 201 , AB116229 position 201 ,
AB116228 position 201 , JBI16227 position 201
CAC51269 position 201 , AAR85364 position 153
, AAR22957 position 153 , AAL05256 position 43 ,
AAL05255 position 43, AAL05254 position 43 ,
AAL05253 position 43, AAL05250 position 43 ,
AAL05244 position 43, AAL05241 position 43 ,
AAL05240 position 43, AAL05238 position 43 ,
AAL05237 position 45, AAL05212 position 43.
1999 CAC22228 position 100 , CAC22200 position 100
AAG43385 position 43, CAC51332 position 143
CAC51270 position 175, CAC51255 position
201, CAC51318 position 201, CAC51247
position 201 , CAC51246 position 201,
CAC51245 position 201 , CAD62370 position 925
, CAD62369 position 925, CAC20187 position
201, AAR22956 position 153 , AAR22940 position
153, ACD44908 position 201 , ACD44906
position 201 , AAF06146 position 43 , AAD41912
position 81 , AAD41131 position 81, AAL05251
position 43 , AAL05215 position 43 , AAL05210
position 43 , AAL05209 position 43 , AAL05208
position 43 , AAL05204 position 43 , AAL05203
position 45 , AAL05202 position 43 , AAL05201
position 43 , AAL05200 position 43 , AAL05199
position 43 , AAL05198 position 43 , AAL05197
position 70 , AAL05195 position 59 , AAL05194
position 58 , AAL05193 position 43 , AAL05192
position 43 AAL05191 position 43.
2000 ABF18566 position 43 , ABF18562 position 43 , CAC22209 position 201 ,
AAL09392 position 153 ,
ABF18557 position 43 , ABF18555 position 43 , AAL09391 position 153 , AAK69397
position 153,
ABF18553 position 43 , ABF18552 position 43 , ABF18551 position 43 , ABF18550
position 43 ,
ABL60850 position 201 , ABL60849 position ABF18549 position 43 , ABF18548
position 43 ,
201 , ABL60848 position 201 , ABL60847 CAC51275 position 201, CAC51271
position 201
position 201 , ABL60845 position 201 , , CAC51267 position 201, CAC51264
position
ABL60844 position 201 , ABL60843 position 201, CAC51263 position 201, CAC51261
201 , ABL60842 position 201 , ABL60841 position 201 , CAC51258 position 201,
position 201 , ABL60840 position 201 , CAC51257 position 201 , BAC06475
position 925
ABL60839 position 201 , ABL60838 position , AAG27038 position 153, AAG27037
position
201 , ABL60837 position 201 , ABL60836 153, AAR22931 position 153 , ACD44909
position 201 , ABL60835 position 201 , position 201 , ABA46733 position 201 ,
ABA46732
ABL60834 position 201 , ABL60833 position position 201 , ABA46731 position 201
, ABA46730
201 , ABL60832 position 201. position 201 , ABA46729 position 201, ABA46728
position 201 , ABA46727position 201 , ABA46726
position 201 , ABA46725 position 201 , ABA46724
position 201 , ABA46723 position 201 , ABA46722
position 201 , ABA46721 position 201 , ABA46720
position 201 , ABA46719 position 201 , ABA46717
position 200, ABA46716position 201 , ABA46715
position 201 , ABA46714 position 201 , ABA46713
position 201 , ABA46712 position 201 , ABA46711
position 201 , ABA46709 position 201 , ABA46708
position 201 , ABA46706position 201 , ABA46705
position 201 , ABA46704 position 201 , BAB18050
position 201 , ABV53920 position 201 .
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2001 ABY75530 position 78, ABY75529 position 78 CAD62373 position 925 ,
AAK92375 position 925
, ABY75528 position 78, ABY75527 position , ACD44910 position 201 , CAC35464
position
76, ABY75526 position 78-, ABY75524 201, CAC35463 position 201, CAC35462
position 78 , ABY75523 position 78 , position 201 , CAC35461 position 201,
ABY75522 position 78, ABY75521 position 78 CAG23917 position 925, CAC86575
position 925
, ABY75520 position 78, ABY75519 position
L8_, ABZ80836 position 201 .
2002 ABZ80844 position 201 , ABZ80835 position AAR07959 position 153, AAM62134
position 201
201.
2003 ABZ80845 position 201 , AAR00255 position AA093493 position 925, AAR07963
position 153
80, ABR13023 position 201 , ABR13022 , AAR07962 position 153 , AAR07961
position 153
position 201 , ABR13021 position 201 , , AAR07960 position 153 , AAR07965
position 153
ABR13020 position 201 , ACD44915 position 201 , ACD44914 position
201, ACD44913 position 201 , ACD44912
position 201 , ACD44911 position 201 ,
ACD44903 position 191 , ACD44902 position 188
ACD44898 position 192 , ACD44897 position
187, AAR07964 position 153 , ABR13026 position
201, ABR13025 position 201 , ABR13024 position
201
2005 AAY56402 position 81 , CAJ51050 position 201 ABD14417 position 201 ,
ABC55721 position 43,
, CAJ51049 position 201 , CAJ51047 position CAJ51080 position 201, CAJ51079
position 201 ,
201 , CAJ51046 position 201 , CAJ51045 CAJ51078 position 201 , CAJ51077
position 201 ,
position 201 , CAJ51043 position 201 , CAJ51076 position 201 , CAJ51075
position 201 .
CAJ51042 position 201 , CAJ51041 position
201 , CAJ51040 position 201 , CAJ51039
position 201 .
2006 ACD44924 position 200, ACD44923 position ACD44919 position 201 , ACD44916
position 201
200, ACD44922 position 200, ABG77560 , ABG77563 position 197, ABG77564
position 30
osition 219 , ABG77557 _position 126 .
2007 ABR1 8732 position 185 , ABN70732 position ABY75534 position 286 ,
ABY75533 position 97
171 , ABN70731 position 217.
2008 ACI96104 position 201
Example 5
Analysis of Replikin Count Cycles in West Nile Virus to Predict Entry into
Geographical
Regions
[000214] As discussed above in Example 3, Applicants analyzed the Replikin
concentration
of West Nile virus envelope protein isolates publicly available in accession
numbers of
www.pubmed.com. As seen in Figure 3, the cycles of mean annual Replikin
concentration in the
envelope proteins of the isolates are related to cycles of morbidity in the
United States.
Additionally, the cycles of mean annual Replikin concentration are related to
step-wise
geographical expansion into the United States from the first known infection
of West Nile virus
in the state of New York in 1998.
[000215] For example, as mean annual Replikin concentration increased between
2000 and
2003, West Nile virus morbidity expanded initially from New York and certain
contiguous states
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in 2000, to the Northeast and Southeast in 2001, to most states except the
Mountain states and
Northwest in 2002, and to all states but the Northwest in 2003. See, e.g.,
annual maps available
from the CDC at
http://www.cdc.gov/ncidod/dvbid/westnile/surv&control.htm#maps. When the
mean annual Replikin concentration began to fall in 2004, West Nile Virus was
present in all
continental U. S. states but with a much lower rate of morbidity. In 2005,
West Nile virus
infections were observed to retreat from certain parts of the U.S. and
infections were not
observed in Washington State, northern New England, or West Virginia. However,
as annual
mean Replikin concentrations began to increase again in 2006, West Nile virus
morbidity again
spread to all states except northern New England.
[000216] A review of the progression of West Nile virus across the United
States from its
first observation in New York reveals that monitoring changes in Replikin
concentration
provides evidence of geographic expansion of West Nile Virus. An aspect of the
invention,
therefore, is the prediction of an expansion into a geographic region or
contraction from a
geographic region based on a determination of the progression of mean annual
Replikin
concentrations in a graph of a cycle or series of cycles of Replikin
concentration including
observed step-wise cycles. For example, a peak in Replikin concentration in a
cycle of Replikin
concentration of a plurality of isolates from a given region provides evidence
of expansion
beyond the geographical area of that region into other contiguous or nearby
geographical areas.
Furthermore a second, still higher, peak provides even greater evidence of a
pathogen that is
poised for expansion.
Example 6
Analysis of Replikin Count Cycles in Malaria to Predict Entry into
Geographical Regions
[000217] The phenomenon of geographical expansion also applies to malaria and
other
pathogens. Analysis of the Replikin concentration of a Replikin Peak Gene,
histidine-rich
protein, or ATP-ase of P. falciparum demonstrates that Replikin concentration
cycles may
provide a prediction of an expansion of P. falciparum mortality and/or
morbidity. For example,
if a Replikin concentration cycle based on isolates from a particular region
demonstrates a
prolonged rise in mean annual Replikin Count or a peak following a rise in
mean annual Replikin
Count, the significant rise or peak predicts an expansion of the mortality
rate or morbidity rate of
that isolate into contiguous or nearby regions that until the significant rise
or peak in Replikin
Count did not experience the mortality rate or morbidity rate of the
particular region.
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[000218] For example, a cycle of Replikin concentration is established in the
Sahel region of
Africa with two peaks at years 2 and 7. The second peak at year 7 is
significantly higher than the
first peak at year 2 with a p value of 0.01. The Sahel region between years 0
and 7 has
experienced a higher rate of mortality than more southerly regions. Based on
the higher peak at
year 7, it is predicted that the mortality from malaria will increase in the
region contiguous to the
south of the Sahel. A plurality of Replikin sequences are isolated from year 7
isolates. Replikins
that have been conserved between years 0 and 7 are selected as vaccines for
malaria in the Sahel
and contiguous regions to the south. Replikins that are new in year 7 are
likewise selected as
vaccines. A mixture of these Replikin sequences is combined with a
pharmaceutically
acceptable carrier and/or adjuvant and administered to a subject to produce an
immune response
to treat and/or protect against malaria predicted to have a higher mortality
rate following the dry
season in year 8 in the Sahel and in its contiguous regions to the south.
Example 7
Replikin Count Virus Expansion Index in Same and Related Influenza Strains
over Time
[000219] Applicants analyzed all amino acid sequences of the pB l gene area of
isolates of
H5N1 strains of influenza virus publicly available at www.pubmed.com for
specimens isolated
between 2004 and 2008. Isolates were grouped by species of bird within
countries for each year
in which sequences were available.
[000220] The concentration of continuous and overlapping Replikin peptides in
the pB 1 gene
area was determined for each isolate (the Replikin Count of the Replikin Peak
Gene). Within
each year in each country a mean Replikin Count with standard deviation was
determined.
China was found to have the largest number of isolates for each year from 2004
to 2008 and the
mean Replikin Count (with standard deviation) of all H5N1 isolates from
chicken in China in
each year was chosen as a control against which other Replikin Counts would be
determined
(China was chosen as a control because of a limited variability in Replikin
Count among a very
large number of isolates available for analysis).
[000221] The Replikin Count for each individual isolate in a given country in
a given year
was compared to one standard deviation from the mean Replikin Count for all
isolates from
chicken in China in that year. Within each country, the number of Replikin
Counts greater than
one standard deviation of the mean and the number of Replikin Counts less than
one standard
deviation of the mean were determined. For each country in each year, the
percent of Replikin
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Counts greater than one standard deviation of the mean was then divided by the
percent of
Replikin Counts less than one standard deviation of the mean to provide a
ratio, or Replikin
Count Virus Expansion (RCVE) Index. In countries having an RCVE Index of
greater than one,
an expansion of H5Nl was predicted for the following year or years. In
countries having a
RCVE Index of less than one, a contraction or viral failure was predicted for
the following year
or years.
[000222] Five sets of RCVE Indices are calculated and reported below as
examples for the
ordinary skilled artisan. The ordinary skilled artisan will understand how to
repeat the predictive
methods for data from any region, time, or species and will understand from
the disclosure
herein how to practice methods of prevention, mitigation, and treatment for
outbreaks predicted
by the RCVE Indices including therapeutic compounds identified in isolates
predicted to be
expanding in population.
[000223] In Tables 9-13 below, individual Replikin Counts that are above the
reported
standard deviation of the mean of the control are bolded. Individual Replikin
Counts that are
below the reported standard deviation of the mean of the control are
italicized and bolded. The
RCVE Index ratio is reported for each group of isolates as compared to the
control.
[000224] In Table 9, Replikin Counts for individual H5N 1 isolates from swans
in China for
2004 are compared to a control of the annual mean Replikin Count for all
chicken H5N1 isolates
from China in 2004.
Table 9
H5N1 Replikin Counts 2004
Control
(all H5N1 isolates
Individual Swans (China) chickens in China)
2.0, 2.4, 2.4, 2.0, 2.4, 2.0, 3.8, 2.0
Mean Annual RC = 2.3 Mean Annual RC = 2.3 SD 1.1
no. of isolates = 533
percent of isolates above
(Mean + SD) = 12.5
percent of isolated below
(Mean - SD) = 0 (equals 1 if in
denominator of RCVE)
RCVE Index = 12.5/1 = 12.5
[000225] The RCVE Index for swans in China in 2004 is 12.5/0. Because zero is
set as 1
when it is in the denominator, the index returns a ratio of 12.5, which
predicts an expanding
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population. This predicted expansion is seen below in Table 11 in an expanding
population in
swans in China in 2006.
[000226] In Table 10, Replikin Counts for individual isolates from swans in
Mongolia,
Russia, and Japan in 2005 are compared to a control of the annual mean
Replikin Count for all
H5N1 chicken isolates from China in 2005.
Table 10
H5N1 Replikin Counts 2005
Control
Mongolia Russia Japan (all H5N1 isolates from
(Individual Swans) (Individual Swans) (Individual Swans) chickens in China)
3.6, 3.7, 3.7, 3.7, 3.7, 3.3, 2.0, 3.3, 2.0, 3.3, 0 Mean Annual RC = 2.5
LSD 1.0
2.4 3.7,1.2, 2.0, 1.7, 2.0, 1.8 no. of isolates = 362
3.7, 3.1, 1.7, 1.8, 7.1,
1.2,3.1, 7.1,2.1, 3.1,
1.2,1.2,0.4,1.2,2.3,
1.8, 1.8, 1.7,1.2
percent of isolates percent of isolates percent of isolates
above (Mean + SD) = above (Mean + SD) = 0 above (Mean + SD) = 0
9/29=31% percent of isolated percent of isolated
percent of isolated below (Mean - SD) = 0 below (Mean - SD) = 0
below (Mean - SD) =
7/29=24.1 %
RCVE Index = No RCVE Index No RCVE Index
31.0%/24.1%= 1.3
[000227] The RCVE Index for swans in Mongolia in 2005 is 1.3, which predicts
an
expansion of the H5N1 population in Mongolia, because the RCVE Index is
greater than 1. This
predicted expansion from Mongolia is seen below in European countries, such as
Sweden and
Denmark, known to be in the flight path for swans and other birds from
Mongolia.
[000228] In Table 11, Replikin Counts for individual isolates from a variety
of bird species in
eight different countries are compared to a control of the annual mean
Replikin Count for all
H5N1 chicken isolates from China in 2006. In Denmark, duck, swan, and falcon
isolates are
reported. In Czech Republic, turkey and falcon isolates are reported. All
other non-control
isolates are from swans.
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Table 11
H5N1 Replikin Counts 2006
Control
(all H5N1
isolates
Denmark from
Sweden (Duck)(swan)(Falc Germany Slovenia Scotland Czech Mongolia China
chicken
(Swans) (Swans) (Swans) (Swans) Turk Falc (Swans) (Swans) in China)
2.0 2.4 2.4 3.8 3.6 4.3 3.6 3.7 Mean
2.5 2.4 3.6 2.4 2.4 4.3 2.4 2.0 Annual
3.4 3.6, 3.7 2.0 2.4 4.3 2.0 3.7 RC =
3.4 3.6, 3.7 2.0 22.2 4.3 1.8 2.0 2.7 f
3.4 3.9 2.4 17.8 2.4, 2.2 17.8 2.5 SD 0.8
2.5 3.9 2.0 2.4, 2.2 3.7 3.7 no. of
17.8 2.4 2.4 2.4, 2.2 0.4 2.0 isolates
3.9 2.4 2.0 2.4,2.2 1.8 2.5 =576
0.9 2.0, 2.1 2.1 2.0
1.8 2.4 1.2 2.5
1.2 2.4, 2.0, 2.0 2.4
1.1 1.2 1.7
1.7 1.2
1.7, 1.8, 1.8
0.4, 0.4, 1.2
RCVE RCVE RCVE RCVE RCVE RCVE RCVE RCVE
Index = Index = Index = Index = Index = Index = Index = Index =
16.7/33.3 25/33.3 = 12.5 60 50 33.3 23/46 = 30
= 0.50 0.75 0.50
[000229] The RCVE Index predicts expansion in Germany, Slovenia, Scotland,
Czech
Republic, and China. The Index predicts contraction or failure in Sweden,
Denmark, and
Mongolia. It is noteworthy that the index predicts contraction or failure of
the H5N1 influenza
population in swans in Mongolia in 2006 while in 2005 the index of 1.3
predicted expansion. In
2007, as predicted in 2006, no H5N1 isolates were reported in Mongolia. See
Table 12 below.
[000230] In Table 12, Replikin Counts for individual isolates from swans in
Japan for 2007
are compared to a control of the annual mean Replikin Count for all chicken
H5N1 isolates from
China in 2007.
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Table 12
H5N1 Replikin Counts 2007
Control
(all H5N1 isolates
Individual Swans (Japan) chicken in China)
2.0, 4.2,3.8,16.7
Mean Annual RC = 6.7 Mean Annual RC = 2.7 SD 0.8
no. of isolates = 112
percent of isolates above
(Mean + SD) = 50
percent of isolated below
(Mean - SD) = 0 (equals 1 if in
denominator of RCVE)
RCVE Index = 50/1 = 50
[000231] The RCVE Index for swans in Japan in 2007 is 50/0. Because zero is
set as 1 when
it is in the denominator, the index returns a ratio of 50, which predicts an
expanding population.
So despite a small sample size, the index predicts expansion, which is seen
below in Table 13 in
an expanding population in swans in Japan.
[000232] In Table 13, Replikin Counts for individual isolates from swans in
Japan for 2008
are compared to a control of the annual mean Replikin Count for all chicken
H5N1 isolates from
China in 2008. Only 3 isolates from chicken in 2008 were reported and
available for analysis.
Table 13
H5N1 Replikin Counts 2008
Control
(all H5N1 isolates
Individual Swans (Japan) chicken in China)
3.7, 3.7, 3.7, 3.7, 3.8, 3.8, 3.8, 3.8, 3.8,
3.8, 3.8, 3.8, 4.5, 17.8, 17.8, 17.8,
2.4, 1.8, 2.4, 1.8,2.2, 2.1, 2.7, 2.4, 1.8,
1.2, 1.7, 1.7, 2.4, 1.8, 2.7, 2.1, 1.2,
0. 8, 0.4,0.8, 0.4, 0.8, 0.4, 0.8, 0.4, 0.8
Mean Annual RC = 6.7 Mean Annual RC = 2.6 SD 0.9
no. of isolates = 3
percent of isolates above
(Mean + SD) = 38.1
percent of isolated below
Mean - SD = 21.4
RCVE Index = 38.1/21.4 = 1.8
[000233] The RCVE Index for swans in Japan in 2008 is 1.8, which predicts
future expansion
of influenza in swans in Japan.
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[000234] The RCVE Indices as described above may be practiced by one of
ordinary skill in
the art as a measure of the current survival and expansion status or
contracting/failing status of a
population of pathogen engaged in an outbreak. The ordinary skilled artisan
may isolate in silico
the Replikin Peak Gene, may measure the Replikin Count of the Replikin Peak
Gene, and may
compare the Replikin Count data of related strains of virus in other
geographic regions in the
same and previous time periods to understand the severity of the outbreak, the
direction of the
outbreak, and the attendant risk to neighboring geographic regions. Like
identifying and
tracking a hurricane, the appreciable advantage to the ordinary skilled
artisan is time to develop
therapies and to institute public health measures known now or hereafter such
as isolation and
culling of poultry, vaccination, and other measures. The methods disclosed
herein further
provide the ordinary skilled artisan with time to manufacture the synthetic
Replikin vaccines
disclosed herein.
Example 8
Replikin Count Expansion Index in Same and Related Malarial Strains over Time
[000235] All publicly available sequences of the histidine rich protein gene
of isolates P.
falciparum from 2004 through 2008 are analyzed for Replikin concentration.
Isolates are
grouped by region.
[000236] Within each year and in each region a mean Replikin Count with
standard deviation
is determined. The region having the largest number of isolates or the least
variability among
Replikin Count in isolates (or both) for each year from 2004 to 2008 is chosen
as a control
against which other Replikin Counts are analyzed. The Replikin Count for each
individual
isolate in a given region in a given year is compared to one standard
deviation from the mean
Replikin Count for all isolates from the control region. Within each region,
the number of
Replikin Counts greater than one standard deviation of the mean and the number
of Replikin
Counts less than one standard deviation of the mean is determined. For each
region in each year,
the percent of Replikin Counts greater than one standard deviation of the mean
is then divided by
the percent of Replikin Counts less than one standard deviation of the mean to
provide a ratio, or
Replikin Count Expansion (RCE) Index. In regions having an RCE Index of
greater than one, an
expansion of malaria is predicted for the following year or years. In regions
having an RCE
Index of less than one, a contraction of malaria is predicted for the
following year or years.
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[000237] In regions wherein malaria is predicted to expand, a Replikin Peak
Gene is
identified in an isolate having a Replikin Count that is higher than the mean
Replikin Count for
the region. The Replikin Peak Gene and/or a Replikin peptide (or plurality of
Replikin peptides)
within the Replikin Peak Gene is selected as an immunogenic compound for
diagnostic and/or
therapeutic purposes. A vaccine against the expanding population is
manufactured comprising
the immunogenic compound. The vaccine is administered to mitigate the
expanding malarial
population.
Example 9
Replikin Count Virus Expansion Index in Same and Related Foot and Mouth
Disease Virus
Strains over Time
[000238] All publicly available sequences of the VPl gene of isolates of Foot
and Mouth
Disease Virus Type 0 from 2004 through 2008 are analyzed for Replikin
concentration. Isolates
are grouped by region.
[000239] Within each year and in each region, a mean Replikin Count with
standard
deviation is determined. The region having the largest number of isolates or
the least variability
among Replikin Count in isolates (or both) for each year from 2000 to 2008 is
chosen as a
control against which other Replikin Counts are analyzed. The Replikin Count
for each
individual isolate in a given region in a given year is compared to one
standard deviation from
the mean Replikin Count for all isolates from the control region. Within each
region, the number
of Replikin Counts greater than one standard deviation of the mean and the
number of Replikin
Counts less than one standard deviation of the mean is determined. For each
region in each year,
the percent of Replikin Counts greater than one standard deviation of the mean
is then divided by
the percent of Replikin Counts less than one standard deviation of the mean to
provide a ratio, or
Replikin Count Virus Expansion (RCVE) Index. In regions having a RCVE Index of
greater
than one, an expansion of foot and mouth disease is predicted for the
following year or years. In
regions having a RCVE Index of less than one, a contraction of foot and mouth
disease is
predicted for the following year or years.
[000240] In regions wherein foot and mouth disease is predicted to expand, a
Replikin Peak
Gene is identified in an isolate having a Replikin Count that is higher than
the mean Replikin
Count for the region. The Replikin Peak Gene and/or a Replikin peptide (or
plurality of Replikin
peptides) within the Replikin Peak Gene is selected as an immunogenic compound
for diagnostic
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and/or therapeutic purposes. A vaccine against the expanding population is
manufactured
comprising the immunogenic compound. The vaccine is administered to mitigate
the expanding
foot and mouth disease virus population.
Example 10
Synthetic Replikin Vaccines Block H5N1 in Chickens
[000241] A synthetic Replikin vaccine containing an approximately equal-parts-
by-weight
mixture of twelve H5N1 Replikin peptides was tested in chickens against a low
pathogenic strain
of H5N1 isolated from a black duck in North Carolina, USA. Low-Path H5N1
strains infect
migratory birds and impair health and productivity of commercial flocks of
U.S. chickens,
usually with little mortality in the commercial flocks. These Low-Path H5N1
strains are very
closely related in virus structure to their more lethal High-Path H5N1
relatives in Eurasia. A
mutation from a Low-Path to a High-Path strain has so far not been observed
but mutations of
this type over time may be expected by one of skill in the art.
[000242] The tested vaccine was engineered to block both the entry site of
H5N1 virus and
the replication site of those H5N1 viruses that manage to enter into host
cells. As such, the
vaccine is called the TWO-PUNCH vaccine. As demonstrated below, evidence from
the
described test of the TWO-PUNCH vaccine in chickens suggests that both
mechanisms for
which the vaccine was designed were effective: (1) virus entry into inoculated
chickens was
diminished by immunity from the vaccine and (2) virus replication within
infected cells was
sufficiently limited by immunity from the vaccine to block excretion of the
virus in feces of
tested birds.
[000243] The TWO-PUNCH Replikins Vaccine is based on influenza Replikin
peptides
shared between influenza strains and conserved for decades within influenza
strains. The
vaccine was engineered as a mixture of twelve Replikin peptides identified as
expressed from the
genome of H5N1 virus. Six of the Replikin peptides are synthesized according
to sequences
isolated from the hemagglutinin protein of H5N1, which is involved in
attachment and entry of
influenza virus into a cell. Six of the Replikin peptides are synthesized
according to sequences
isolated from the pBl gene area of H5N1, which has been identified as involved
in replication of
influenza virus in a host cell.
[000244] The following six Replikin sequences contained in the vaccine were
isolated from
the hemagglutinin protein:
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(1) HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1);
(2) KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2);
(3) KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3);
(4) HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4);
(5) HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5); and
(6) KVRLQLRDNAKELGNGCFEFYH (SEQ ID NO: 6).
[000245] The following six Replikin sequences contained in the vaccine were
isolated from
the pB l gene area:
(1) KDVMESMDKEEMEITTH (SEQ ID NO: 7);
(2) HFQRKRRVRDNMTKK (SEQ ID NO: 8);
(3) KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9);
(4) HKRTIGKKKQRLNK (SEQ ID NO: 10);
(5) HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11); and
(6) HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12).
[000246] The vaccine comprises an approximate equal-parts-by-weight mixture of
the twelve
peptides. The following peptide amounts were combined to create an initial
mixture of the
vaccine:
HAQDILEKEHNGKLCSLKGVRPLILK (SEQ ID NO: 1) 239.6 mg
KEHNGKLCSLKGVRPLILK (SEQ ID NO: 2) 200.8 mg
KKNNAYPTIKRTYNNTNVEDLLIIWGIHH (SEQ ID NO: 3) 213.0 mg
HHSNEQGSGYAADKESTQKAIDGITNK (SEQ ID NO: 4) 135.6 mg
HDSNVKNLYDKVRLQLRDNAK (SEQ ID NO: 5) 170.8 mg
KVRLQLRDNAKELGNGCFEFYH )SEQ ID NO: 6) 188.3 mg
KDVMESMDKEEMEITTH (SEQ ID NO: 7) 161.9 mg
HFQRKRRVRDNMTKK (SEQ ID NO: 8) 138.3 mg
KKWSHKRTIGKKKQRLNK (SEQ ID NO: 9) 217.8 mg
HKRTIGKKKQRLNK (SEQ ID NO: 10) 178.0 mg
HEGIQAGVDRFYRTCKLVGINMSKKK (SEQ ID NO: 11) 159.2 mg
HSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEK (SEQ ID NO: 12)233.8 mg
The total amount of the mixture was 2237.1 mg.
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[000247] The peptide mixture was then divided into three equal parts for
administration of
the vaccine on three different days (days 1, 7, and 28). After dissolution
with water, the three
equal parts were administered to individual birds in two groups of 20 birds
each for a total
administration at each day of 40 birds. The total amount of active peptide
ingredient
administered to each bird at the time of administration (either intranasally
and intraocularly or
via spray inhalation) was about 18.6 mg per bird per administration.
[000248] The vaccine solution was administered to chickens intranasally at a
first
administration on day 1 after hatch, intraocularly at a second administration
on day 7 after hatch,
and via fine spray inhalation at a third administration on day 14 after hatch.
[000249] Chickens on the first day of life were separated into four groups
with twenty
chickens per group. The first group was a control group not vaccinated and not
challenged with
Low-Path H5N1. The second group was vaccinated and not challenged with Low-
Path H5N1.
The third group was vaccinated and subsequently challenged with Low-Path H5N1.
The fourth
group was not vaccinated and was challenged with Low-Path H5N1.
[000250] For those chickens that were vaccinated, the synthetic H5N1 Replikins
Vaccine was
administered intranasally on day 1 after hatch, administered intraocularly on
day 7 after hatch,
and administered by fine spray inhalation on day 14 after hatch. The groups of
challenged
chickens were than challenged with Low-Path H5N1 virus on day 28 of the life
of the chicken.
Serum from selected chickens was analyzed in all groups for antibodies against
the H5N1 virus
on days 7, 14, and 21 following challenge. PCR for virus fecal excretion was
also analyzed for
all groups.
[000251] Unvaccinated control chickens demonstrated both an expected high
virus entry (as
indicated by a high titer of antibodies against H5N1) and an expected high
virus replication (as
indicated by high fecal and salival excretion of the virus detected by PCR).
In contrast, the
vaccinated chickens demonstrated lower virus entry (as indicated by a low
titer of antibodies
against H5N1 or by the observation of no antibodies against H5N1 in serum) and
an absence of
fecal or saliva excretion of virus indicating low or no virus replication in
the vaccinated
chickens. The data suggest, therefore, that the virus was partially blocked on
entry by the
chickens' immune response to the vaccine and the limited amount of virus that
did enter the
chicken's system was blocked from sufficient replication in the chickens' host
cells to excrete
virus in the feces or saliva.
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[000252] The data in Table 14 below provide the numbers of chickens tested in
each of the
four groups (Negative Control, Vaccinated, Vaccinated and Challenged with Low-
Path H5N1,
and Challenged with Low-Path H5N1 (not vaccinated)) on a particular test day
and the numbers
of chickens in which production of antibodies to H5N1 was detected with a
serum titer.
Table 14
Serum Antibody Test of Low-Path H5N1 Challenge of Vaccinated Chickens
Day 7 Day 14 Day 21
(Chickens Producing (Chickens Producing (Chickens Producing
GROUP Antibody to H5N1) Antibody to H5N1) Antibody to H5N1)
Negative Control 0 of 7 0 of 7 0 of 7
Vaccinated 0 of 7 6 of 6 0 of 5
Vaccinated and
Challenged with Low-
Path H5N1 1 of 7 3 of 6 2 of 7
Challenged with Low-
Path H5N1 4 of 7 7 of 9 3 of 9
[000253] The data in Table 15 below provide the number of chickens tested for
H5N1 virus
in their saliva and feces in each of the four groups (Negative Control,
Vaccinated, Vaccinated
and Challenged with Low-Path H5N1, and Challenged with Low-Path H5N1 (not
vaccinated))
on a particular test day and the numbers of chickens in which H5N1 was
detected in their feces
and saliva based on PCR analysis.
Table 15
PCR Test for Excreted H5N1 Virus from Low-Path H5N1 Challenge of Chickens
Day 7 Day 14 Day 21
(Chickens Producing (Chickens Producing (Chickens Producing
GROUP Antibody to H5N1) Antibody to H5N1) Antibody to H5N1)
Negative Control 0 of 10 0 of 7 0 of 7
Vaccinated 0 of 10 0 of 7 0 of 7
Vaccinated and
Challenged with Low-
Path H5N1 0 of 7 0 of 7 0 of 7
Challenged with Low-
Path H5N1 3 of 7 2 of 9 1 of 7
[000254] The data in Tables 14 and 15, demonstrate the effectiveness of the
double-
protective mechanism of the TWO-PUNCH vaccine. First, while several non-
vaccinated
chickens challenged with H5N1 excreted virus in their feces and saliva, no
vaccinated chickens
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challenged with H5N1 excreted virus in their feces or saliva. See Table 15.
These data
demonstrate that the vaccine provided a protective effect against replication
of the virus.
Second, while four of seven unvaccinated chickens challenged with H5N1 were
producing serum
antibodies against H5N1 on day 7, seven of nine unvaccinated chickens
challenged with H5N1
were producing serum antibodies against H5N1 on day 14, and three of nine
unvaccinated
chickens challenged with H5N1 were producing serum antibodies against H5N1 on
day 28, only
one of seven vaccinated and challenged chickens was producing serum antibodies
against H5N1
on day 7, only three of six vaccinated and challenged chickens were producing
serum antibodies
against H5N 1 on day 14, and only two of seven vaccinated and challenged
chickens were
producing serum antibodies against H5N1 on day 21. See Table 14. These data
demonstrate that
for some of the vaccinated chickens, the H5N1 virus challenge was stopped
prior to entry into
the chicken's system (likely by antibodies produced at the mucus membranes).
These data
further demonstrate that for those vaccinated and challenged chickens in which
the virus entered
the system (resulting in production of serum antibodies), the virus was
nonetheless not excreted
in feces or saliva.
[000255] As may be seen from the data in Table 14, almost all of the non-
vaccinated
challenged birds seroconverted (producing detectable antibody). This
demonstrates infection of
the non-vaccinated birds. On the other hand, only some of the vaccinated
challenged birds
seroconverted. Further, for those vaccinated birds that did seroconvert, the
antibody titers were
low. Additionally, the negative control group had no seroconversion. These
data demonstrate a
protective effect of the vaccine on the birds.
[000256] Additionally, Table 15 demonstrates the absence of detectable
influenza in the feces
and saliva of vaccinated birds. That viral excretion was blocked by this
influenza Replikins
vaccine is particularly significant because it is generally acknowledged that
the maintenance of
reservoirs of H5N1 virus in flocks of migratory birds and domestic chickens in
both Asia and the
U.S. (and the regional spread of H5N1 virus from these reservoirs) is
dependent on viral
excretions picked up by neighboring chickens and birds. Regardless of the
level of lethality of a
strain of H5N1 virus, absent excretion of virus, there is expected to be no
spread of the virus.
[000257] As such, data observed from administration of the TWO-PUNCH Replikin
peptide
vaccine in chickens demonstrates the efficacy of the vaccine as (1) a barrier
to entry of the virus,
(2) a block of replication of the virus, and (3) a block of fecal spread of
the virus.
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